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This is to certify that the dissertation entitled IDENTIFICATION AND CHARACTERIZATION OF MAKER'S DISEASE VIRUS UNIQUE SHORT REGION GENES AND THEIR PRODUCTS presented by MW has been accepted towards fulfillment of the requirements for PhD- degree in m WM Major professor Date M— MS U i: an Affirmative Action/Equal Opportunity Institution 042771 LIBRARY Mlchlgan State Unlverslty -“ PLACER RENE" Boxwmnovombchoekoufiomywnoord. TOAVOIDFINESMunonorbdor-ddodm. DATE DUE DATE DUE DATE DUE J J MSUlAn A o I a WWW momma-duo" m1 IDENTIFICATION AND CHARACTERIZATION OF MAREK’S DISEASE VIRUS UNIQUE SHORT REGION GENES AND THEIR PRODUCTS By Peter Bnmovslds A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1992 ABSTRACT IDENTIFICATION AND CHARACTERIZATION OF MAREK’S DISEASE VIRUS UNIQUE SHORT REGION GENES AND THEIR PRODUCTS BY Peter Brunovslds Because of its lymphotropic properties, Marelt's disease virus (MDV) has been classified as a gammaherpesvims. However, its genome structure most closely resembles that of the alphaherpesvirus prototype, herpes simplex virus (RSV). To examine the nature of this discrepancy, an analysis of MDV’s unique short (U s) region was undertaken. Alphaherpesvirus Us regions exhibit significant evolutionary divergence, encoding genes specific to members of this phylogenetically related herpesvirus lineage. This includes a cluster of glycoprotein genes possibly conserved in MDV. Attempts to identify such genes of potential importance to pathogenesis and immunoprotection, led to the complete nucleotide sequence determination of MDV’s 11,286 base pair Us region (pathogenic GA strain). Sequence analysis identified 7 alphaherpesvirus related genes, including MDV counterparts of HSV U81, -2, and -3; U86, -7, and -8 (glycoproteins gD, 91, and gE, respectively) and USlO. In addition, three MDV-specific ORFs and a novel fowlpox virus-related ORF were identified. These results confirm and extend upon recently published data which demonstrate a closer phylogenetic relationship between MDV and alphaherpesviruses. MDV Us region polypeptides were analyzed with monospecific, polyclonal antisera generated from a panel of 16 difierent bacterially—expressed apE fusion protein immunogens representing 9 of the 11 ORFs. The resulting antibodies were found to immunoprecipitate 6 of the 7 alphaherpesvirus-related MDV homologs from infected avian cell cultures. MDV USl was found to express an unusual 2'1 .24-kDa late class cytoplasmic phosphoprotein, in contrast to its larger 68-kDa immediate-early nuclear phosphoprotein counterpart, HSV ICPZZ. U82 and -10 were identified as 30- and 24-kDa polypeptides, respectively. Antisera directed against three difi‘erent regions of the protein ldnase-related U83 gene were all found to precipitate a 47,49-kDa doublet; one of these was found to specifically react with a 68-kDa cellular protein as well. Like other gI/gE-related products, MDV’s counterparts were found to coprecipitate together. Antibodies reactive with the QD epitopes of three different bacterially-expressed fusion proteins failed to precipitate gD from avian cell cultures. Together, these results raise a number of interesting questions which highlight the potential importance of MDV Us region genes in determining many of MDV’s unusual biological properties. More precise information on genetic relatedness awaits resolution of methods to measure nucleotide sequences of the DNA of herpesviruses. Andre]. Nahmias, 1972. Nucleotide sequence data are necessarily the basis of the taxonomy of the family Herpesviridae. The central issue is the identification of the correlates which must be culled from such data for a truly useful taxonomy. Herpesvirus Study Group, International Committee on Taxonomy of Viruses, 1992. To my dear Lelda. without whose love and support this could not have been possible... To my wife’s parents, Maiga and Anseldis, whose emotional and material support carried us through many dificult times... and To my mother, Raita and late father, Talis for supporting my education and fostering an intellectual curiosity from the earliest of days. iv ACKNOWLEDGEMENTS My deepest thanks go to my major professor, Dr. Leland P. Velicer for his support, guidance, patience and for providing a positive working environment. I am particularly grateful for the many things I have learned from him and the kind and respectful way in which he has treated not only myself, but everyone else I have ever seen. I would also like to thank the members of my guidance committee, Dre. 8. Conrad. I. Dodgson, R. Schwartz and R. Silva for their guidance, their time and their helpful suggestions. Special thanks is extended to the people who I have worked with day in and day out-especially Xinbin Chen, Quentin McCallum and Ruth Stringer. I am particularly thankful for their helpful discussions, technical assistance, companionship and the professional way in which they have conducted themselves. Finally, I would like to Hsing—Iien Kung and Dan Jones for sharing unpublished sequence data and to Steve Spatz and Paul Coussens for their many helpful discussions and for helping make scientific conferences a particularly enjoyable experience. TABLE OF CONTENTS List of Tables vii List of Figures viii Chapter I: Literature Review 1 Introduction to herpesviruses 1 Introduction to Marek’s disease virus 3 Herpesvirus classification 12 Evolution of herpesviruses 28 Characterization and significance of the alphaherpesvirus 8 region 46 References 79 Chapter II: Structural characterization of the Marek’s fisease (MDV) unique short region: presence of alphaherpesvirus-homologous, fowlpox virus-homologous and MDV-specific genes 101 Abstract 102 Introduction 104 Materials and Methods 107 Results 1 10 Discussion 133 References 143 Chapter III: Marek’s disease vinis expresses an unusual 27-kDa late class cytoplasmic phosphoprotein protein homologous to the 68-kDa herpes simplex l immediate-early nuclear phosphoprotein, ICPZZ 148 Abstract 149 Introduction 151 Materials and Methods 154 Results 158 Discussion 177 References 185 Chapter N: Analysis of Marek’s disease virus unique short region polypeptides with antisera to inducible, bacterially-expressed fusion proteins 190 Abstract 191 Introduction 192 Materials and Methods 195 Results 198 Discussion 217 References 226 Chapter V: Summary and Conclusions - 231 vi LIST OF TABLES Table Chapter I l. The two parallel nomenclatures of the RSV IE gene products. Chapter II 1. Summary of MDV Us ORF data 2. Pairwise comparison of MDV and alphaherpesvirus 8 region homologs. Chapter N l. Polypeptides and glycoproteins identified in this study. vii Pace 120 122 218 LIST OF FIGURES Figure 9’95”.” Chapter I Map positions of MDV and HVT genes. Sequence arrangements in the 6 groups of herpesvirus genomes. Conserved blocks of sequence between HCMV, EBV, VZV, and HSV-1. Comparison of MDV and alphaherpesvirus 8 region genes. Map of HSV immediate-early mRNAs. Chapter II Map location of area sequenced and organization of MDV Us ORI-‘s. Nucleotide and predicted amino acid sequences. Homology between MDV SORF2 and FPV CRT-'4. Comparison of alphaherpesvirus Us homologies. Comparison of MDV and alphaherpesvirus 8 region genes. Chapter III Localization of the 1.9 kb MDV Eco RI-O transcript Northern blot analysis of the 1.9 kb EcoRl transcript from MDV-infected cells. 81 nuclease protection analysis of the 1.9 kb Eco RI-O transcript. Identification, characterization and subcellular localization of the MDV U81 polypeptide, pp27,24. viii Page 21 32 37 54 112 115 124 127 128 137 160 162 164 168 Temporal regulation of the MDV U81 homolog. pp27,24 processing kinetics and characterization. Homology and divergence of U81 (ICP22) homologs. Chapter N Map of MDV regions fused to £1125 in this study. Immunoprecipitation/SDS-PAGE analysis of the polypeptide encoded by MDV U810. Immunoprecipitation/SDS-PAGE analysis of the polypeptide encoded by MDV U82. Immunoprecipitation/SDS-PAGE analysis of the polypeptide encoded by MDV US3. Immunoprecipitation/SDS-PAGE analysis of the glycoproteins encoded by MDV U87 (g1) and U88 (gE). Immune-reactivity of trpE-gD-directed antibodies. ix 171 175 183 200 202 205 207 211 214 Chapterl theratunRsvlew I. Introduction to Imps-virtues. A. rhetorical upsets. Although the concept of herpes as a disease has existed for at least 25 centuries, its meaning, nomenclature and description has changed considerably (Wildy, 1973). Modern views of herpes were largely derived from the definitions and work of Wilan and Bateman (1814). Herpes simplex was shown to be an infectious agent in 1873, herpes zoster (varicella zoster) in 1909. Iozsef Marek was the first to recognize Marek’s disease in domestic fowl, although he could not ascertain the cause (Marek. 1907). Despite the clinical observations associated with these agents, a conceptual understanding of viruses was largely unknown prior to 1957. The introduction of negative staining in 1959 marked the the turning point in virus clusification (thdy, 1973). This technique enabled the full exploitation of electron microscopy to reveal fine difierences in virus particles. In conjunction with biochemical virological techniques for distinguishing DNA from RNA, morphological criteria for virus taxonomy soon took hold. A unifying description of herpesviruses was soon developed. All were found to share a characteristic morphology conforming to the following description: ‘Viruses of eukaryotes with linear, double-stranded DNA genomes of more than 80x106 mol. wt. which are replicated in the nucleus of infected cells, assembled into 100 nm diam. icosahedral capsids composed of 162 prismatic capsomeres which are enclosed in glycoprotein and lipid (ether-sensitive) envelopes to give the normally infectious extracellular form of the virus’ (Homes and Watson, 1977). Beyond 1 this general description, however, exists a large family of viruses with an extraordinary range of biological potentials. B. Clnracterlsticl and diversity of herpesviruses. More than 80 distinct herpesviruses have been isolated. Herpesviruses have been isolated from virtually every species examined. This includes hosts as diverse as fish, amphibians, reptiles, birds, and numerous mammals (Nahmias, 1972). A single host may be inhabited by multiple herpesviruses with uniquely difierent patterns of infection and pathogenesis. Herpesviruses exhibit greatly variable tissue tropisms, but are generally restricted in nature to a particular host or a few closely related species. Once infected, a host will usually harbor the virus for life. This aspect of herpesvirus biology, latency, is one of its most distinguishing features. Under this condition, the virus can periodically reactivate and infect new hosts. In its natural host, beyond the newborn age, herpesviruses are generally quite harmless; however, newborn or imrnunocompromised individuals generally exhibit a wide range of pathological manifestations. Despite their often inocuous nature, herpesviruses can be extremely dangerous when crossing their normal host-species barrier. Together, these aspects reflect a long evolutionary history characterized by the selection for isolates which live in relative harmony with their hosts. 0. The human herpesviruses. Because of their clinical significance to man, human herpesviruses have been the most studied. Presently, there are seven distinct herpesviruses identified in humans; herpes simplex vinis types 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and human herpesvirus types 6 and 7 (HEN-6, HI-IV-7). Considering their often inocuous nature, it shouldn't be a surprise if more were identified in the future. HHV-7 wasn’t recognized until 1990 (Frenlnel et al., 1990). Most adults are seropositive for several of these herpesviruses. In many cases, human herpesvirus infections are subclinical in nature. However, in neonates or immunocompromised adults viremias are frequently observed and ofien associated with deadly consequences. Infections in immunocompromised adults highlight the ability of herpesvimses to persist in a latent state, generally held in check by the host’s immune system. Immunosuppressive therapies for cancer or bone marrow transplantations often provide an opportunity for these viruses to reactivate and cause a severe, acute infection. In noncompromised hosts over one month of age, viremias are generally considered to be absent. However, this view may have to be reconsidered, viremias appear to occur rather routinely in irnrnunocompetent children with varicella (Ozaki et al., 1986). 11. Introduction to We dbease virus. A. General pathogenic characteristics. Marek’s disease virus (MDV) is a strongly cell-associated, acutely transforming herpesvirus which can rapidly (3 weeks) induce T-cell lymphomas in chickens (for a recent review, see Calnek and Witter, 1991). While Marek's disease (MD) is largely regarded as a useful natural host model for oncogenesis, other studies highlight the importance of MDV as a useful natural host model for atherosclerosis (Fabricant, 1985) and the Guillain-Barre syndrome (Pepose et al., 1981). Various isolates have been reported to cause lymphoproliferative neural lesions, peripheral nerve demyelination, blindness. and paralysis. B. MD as a mochl for armor lmrmmlty studies. Because of the protection afforded against MDV by vaccination (Churchill et al., 1969; Okazaki et al., 1970), the MD system has long been considered as a useful natural host model for tumor immunity studies. MD tumors can be prevented by vaccination with cell-culture-attenuated pathogenic stains and serologically-related naturally apathogenic stains, including herpesvirus of turkeys (INT). Although the mechanism for immunoprotection is far from clear, studies have implicated both anti-viral- and anti-tumor immunity (Powell and Rowell, 1977). C. Serotypes and pathotypes neoclated with the MD system. On the basis of immunological criteria serologically-related stains of MDV- and WT have ben assigned to three serotype groups: serotype 1, represent pathogenic stains of MDV and their cell-culture-attenuated variants; serotype 2, represent naturally apathogenic MDVs; and serotype 3, INT. These three serotypes can be distinguished by their reactivity with serotype-specific monoclonal antibodies (Lee et al., 1983). In addition, these serotypes exhibit uniquely distinct restiction digestion patterns. Taken together, the three ‘MDV’ serotypes represent antigenically-related, yet phylogenetically-distinct virus entities. The three MDV serotypes are characterized by a range of pathotypes according to their virulence and pathogenicity in susceptible chicks. These include highly pathogenic (acute) stains, causing a high incidence of visceral and neural lymphomatosis; moderately pathogenic (classic) stains, causing primarily neural lesions, often at a lower incidence; and mildly pathogenic or nonpathogenlc stains causing no gross lesions (Payne, 1982). For many years MDVs were primarily known to induce a classical, neuropathologic disease. Begirming with the late 1950’s, more vinilent, oncogenic stains began to replace the less virulent, neuropathologic stains as the predominant field isolates responsible for disease. Thus, an evolutionary pattern for virulence has been recognized, characterized by selective pressures for continually increased virulence (Calnek and Witter, 1991). This has led to a further difierentiation of serotype 1 isolates according to their pathogenicity in both vaccinated and unvaccinated chicks (Witter, 1983). The so-called very virulent (WMDV) isolates (e.g. Mdl l, RBlB) represent highly oncogenic serotype 1 stains that are often refractory to taditional HVT vaccines (e.g. less than 77% protection). These stains are largely responsible for vaccine failures in the field. In contast, better protection has been achieved against virulent (vMDV) serotype 1 stains (greater than 77% protection). These are represented by common oncogenic stains, such as GA, IM, and HPRS-16. As a group, We are more pathogenic and viscerotopic than vMDVs. One stategy for protection against vaDV stains is based on the observation that a combination of vaccine viruses provide better protection than the individual stains alone (Witter, 1982). This phenomenon has been referred to as protective synergism Further studies have indicated that serotype 2- and 3 combinations provide better protection than serotype 1 (attenuated) and 3, or 1- and 2 cembinations (Witter, 1987). Ideally, a vaccine virus should be attenuated for pathogenicity, yet able to replicate eficiently in birds. Since cell- culture-attenuation often leads to decreased replication in birds, an optimal balance between attenuation and in vivo replication must be achieved. This can be accomplished by alternating MDV passages between both cell-cultures and birds. The former selects for more attenuated stains, the latter for more pathogenic, better replicating stains. Recent stategies have employed this approach to develop vaccine stains derived from vaDV isolates in order to achieve better protection against their more pathogenic relatives (Witter, 1991). D. Natural tansmission. MDV has been considered as the only known oncogenic vinis with a highly eficient mode of horizontal tansmission in nature (Klein, 1972). This tansmission is mediated by the maturation and release of virus from infected feather follicle epithelial (FPE) cells, usually by an air-borne route involving poulty house dust and chicken dander. Like papillomaviruses, which are dependent on highly difi'erentiated epithelial cells for expression of stuctural proteins and formation of virions, production of fully enveloped MDV virions can only take place in highly difierentiated epithelial cells (Calnek et al., 1970; Naserian and Witter, 1970). Although the resulting virions are fully infectious in a cell-free form, release of these virions from FFE cells has never been observed. Based on the evidence below (Carozza et al., 1973), a compelling case can be made for cell-associated spread of MDV in horizontal tansmission. Herein appears to lie the secret to MDV's eficient tansmission. In contest to cell-free virus from poultry dust sediment, which loses its infectivity in less than two weeks, intact poulty dust carrying desquarnated MDV-containing FFE cells retains its infectivity for at least 205 days at ambient room temperatures (and much longer at lower temperatures) (Carozza et al., 1973). Unlike many other herpesviruses, MDV cannot be tansmitted in a vertical fashion. 8. Host range. Natural infection with MDV is known to primarily afiect members of the genus Callus, although it has been described in other birds of the Galliformes order, including turkey and quail (Biggs, 1985). MDV has been isolated from naturally infected quail with lymphoproliferative tumors afiecting many visceral organs; however, infected quail usually lack the peripheral nerve lesions characteristic of MD in chickens (Imai et al., 1990). A milder infection has been noted in jungle fowl, both feral and zoological (Biggs, 1985). Despite possible questions regarding the natural host from which MDV evolved throughout most of it existence, chickens are clearly the most important ‘natural host’ in an economic sense. P. Growth d MDV in rite. 1. Cell cultures. Unlike many other herpesviruses, especially other alphaherpesviruses, MDV has a very limited host range in vitro. Growth is primarily resticted to primary avian cell cultures. The major dificulty in working with MDV is it slowly progressing, tightly cell-associated nature. This precludes staightforward plaque purification of virus, as well as the establishment of one-step growth conditions for efiective temporal gene regulation studies. Cell culture systems for producing enveloped, cell-free virus are currently unavailable. To obtain sufficient quantities of material with which to work, it is necessary to passage infected cells onto uninfected cell monolayers. Even though cell-free infectious virus can be purified from FFE cells, titers are at best limited to 104 PFU/ml. Based on optimal growth properties, serotype 1 stains are usually grown in duck embryo fibroblast (DEF) cultures; serotypes 2- and 3 are most often grown in chick embryo fibroblast (CEF) cultures. Chick kidney- and lymphocyte (Calnek et al., 1982) cultures have been used as well. The differential growth characteristics in primary cultures can be used to difierentiate between pathogenic and non-pathogenic serotypes (Cho, 1976a, 1976b). Furthermore, in contest to MDV, which normally fails to grow in continuous cell lines, INT is distinguished by it ability to grow in the quail I fibroblast cell line, ores (Cho, 1981). 2. Lymphoblastold cell lines. Various T-lymphoblastoid cell lines have been established from MD tumors. Most of these are considered producer cell lines (e.g. MSB- 1), which can be induced to produce vinis following co-cultivation onto fibroblast- or kidney cell monolayers or following inoculation in birds. Non- producer lines (e.g. MDCC-RPl) are those from which virus cannot be rescued. The latter generally fail to produce antigens detectable with conventional anti- MDV seras. However, teatnent with 5-iodo-2-deoxyuridine (IUDR) can lead to the detection of a phosphorylated polypeptide (pp38) using a pp38-specific monoclonal antibody. Producer cell lines can be further characterized as expression or non-expression cell lines. The former contain a high proportion of cells that spontaneously produce immunologically detectable antigens; the latter contain few such cells and generally require IUDR teatnent in order to produce detectable levels of antigen. Two groups have recently succeeded in establishing lymphoblastoid cell lines following in vitro infection (Ikuta et al., 1987; Calnek and Schat, 1991). G. Sequential events in pathogenesis. In antibody-free, genetically susceptible chickens infected with oncogenic stains of MDV, a sequential pattern of event has been recognized which account for the establishment of lymphomas and death. This sequence involves four phases: (1) an early cytolytic infection; (2) a latent infection; (3) immunosuppression in conjunction with a secondary cytolytic infection, and (4) oncogenic tansforrnation (Calnek, 1986). 1. Early cytolytlc infection. MDV generally gains enty into chickens by way of the respiratory tact. This is probably the only place in which cell-free enty of virus can occur. From here the virus makes its way into the lymphoid system, where an early necrotizing infection is detectable as early as 1-2 days postinfection (DPI). In lymphoid- and other tissues (except the FEE), infections are cell-associated and productive-restictive; few or no enveloped virions are produced. Lymphoid infections involve the three major lymphoid organs (e.g. the spleen, thymus and bursa of Pabricius) and result in cell death and atophy to the bursa and thymus; consequently, a temporary immunosuppression develops. The infection peaks at 4-6 DPI and subsides by 6-7 DPI after which viral antigens become temporarily undetectable. Bursa-derived B-cells are the predominant target for this early phase of the infection. 2. Latent infection. The onset of an immune response beginning at 5-7 DPI coincides with two important event. A drop-ofi in the cytolytic infection occurs and a cell-mediated response leads to the activation of T-cells which begin to express the la or class II MHC antigen. The activated T-cells then become permissive for MDV, probably as a result of their interaction with the cytolytically-infected B cells. The MDV-infected Ia-bearing (class II MHC) T- cells represent the primary target for the ensuing latent infection. Little or no gene expression occurs during this phase. These latently-infected cells are thought to persist throughout the life of the bird (Witter et al., 1971) and can be activated into lytic growth by cocultivation onto primary fibroblast cell monolayers. Cytolytically- and/or latently-infected lymphoid cells are thought to seed the various epithelial tissues prior to a secondary cytolytic infection which begins after the 2nd or 3rd week post-infection. 3. hummesuppresslon and secondary cytolytic Motion. It is thought that irnmunocompetence is required for the establishment and maintenance of latency (Buscaglia et al., 1988). This appears to be mediated by a latency-maintenance factor (LMF) found in the conditioned media of latently-infected cells (Buscaglia and Calnek, 1988). For reasons that are not yet known, some birds undergo an irnrnunosuppression which leads to a release from latency. A secondary cytolytic infection follows, not only in epithelial tissues (such as the FEE), but in the lymphoid cells as well. Although chickens genetically susceptible- or resistant to tansformation by MDV are both 10 subject to the early cytolytic infection, late cytolytic lymphoid infections are generally resticted to genetically-suscepu'ble birds. Whatever the reason, an amplification of infected T-lymphocytes result; this large pool of infected cells ultimately serves as the reservoir for oncogenic tansfonnation which follows. 4. Oncogenic tansforrnation. Lymphomas can develop as early as three week post- infection. There 't no clear consensus regarding the type of T-cell subset permissive for tansforrnation. Most cell lines derived from normally occurring tumors have been found to be CD4+ CD8-. However, 45% of the cell lines derived tom experimentally-infected local lesions were found to be CD4- CD8+, 3496 CD4- CD8-, and only 21% CD4+ CD8— (Schat et al., 1991). On the other hand, all have been observed to express Ia antigen, CD3 and/or TCR. Thus, activation of T-cells appears to be a consequence of MDV infection and a prerequisite for tansforrnation. Tumor induction is determined by the complex interplay of a number of factors, including (i) innate oncogenic and imrnunosuppressive potential associated with a given MDV isolate, and (ii) difierences in genetic resistance dictated by factors such as age, sex and host genotype (Calnek and Witter, 1991). Genetic resistance to MD appears to be contolled by MHC-dependent or -independent mechanisms. Non-MHC-contolled resistance in line 6 chickens has been characterized by a reduced susceptibility to virus. This could be attibuted to T-cell targets with fewer vinis receptors, receptors of lower afinity for virus, or fewer target cells with appropriate receptors (Gallatin and Longnecker, 1979). At the present time, nothing is known about the genes responsible for tansformation. Most tanscriptional activity appears to reside in the inverted repeat regions, particularly the inverted repeat long (IRL) region. However, the 11 possible involvement of unique short (Us) region can not yet be ruled out (Schat et al., 1989). Various studies (Bradley et al., 1989a; Chen and Velicer, 1991) have focused on MDV tanscription near an area of the 1R1, containing a 132-bp repeat element whose copy number is significantly increased in attenuated MDV stains (Maotani et al., 1986; Silva and Witter, 1985). One group has reported that the l32-bp expansion causes the premature termination of a putative tumorigenicity-related tanscript (Bradley et al., 1989b) which contains two small open reading frames (Iwata et al., 1992). The significance of the above findings are not yet clear. The putative tumorigenicity-associated tanscript are similarly expressed during lytic infections as well; such expression may be incompatible with a tansformed state that requires repression of functions associated with lytic infections. In addition, a number of these studies have relied on iodo-deoxyuridine (IUdR) to artificially induce viral gene expression in MD tumor cell lines; this raises questions concerning the relevance of the accompanying gene expression with regard to tansformation. While a great deal of attention has focused on changes in gene expression concommitant with attenuation, rather than affecting the expression of tumorigenicity-associated genes, the changes may simply reflect in vito selection pressures that have altered the infectivity of oncogenic stains for T-cells (Schat et al., 1985). Nevertheless, further studies involving the IR], region are clearly warranted, inasmuch as IRL-specific gene expression has been consistently observed in natural tumors and tumor cell lines (Schat et al., 1989; Sugaya et al., 1990). Antisense oligonucleotides specific for MDV 1R1, sequences have recently been reported to arrest the growth of an MDV-tansformed cell line (Kawamura et al., 1991). Two groups have recently mapped and sequenced a gene upstearn of the l32-bp repeat (Cui et al., 1991; Chen and Velicer et al., 12 1992) which was found to express a 38-kDa phosphoprotein previously detected in tumor cell lines using a monoclonal antibody (Ikuta et al., 1986; Nakajima et al., 1987). Jones et al. (1992) have recently identified a gene located downsteam of the 132-bp repeat which is abundantly expressed in tumor cells. Sequence analysis of this gene, designated as meg; led to the identification of N-terminal proline, basic, and leucine zipper regions with the same spacing and characteristics shared by members of the jun/foe family. Together with a large proline rich region containing a novel repeat motif rich in proline, serine, threonine, and acidic amino acids (as) at its C-terminal end, these motifs suggest a role in tanscriptional activation. III. Herpeevlrus classification. A. Early attempts. By comparing B virus with pseudorabies vinis, Sabin (1934) provided the first evidence for biologic- and serologic relatedness between two herpesviruses. (Wildy, 1973). By 1952 the herpesvirus group grew to include (in addition to B virus and pseudorabies) herpes simplex, varicella, and zoster. At the time it was not yet known that the latter two were one and the same. Using morphological criteria, an increasing number of herpesviruses began to be identified in the early 1960’s. In one of the first attempt to classify herpesvinises, Melnick et a1. (1964) proposed a herpesvirus classification scheme based on in vito growth properties. Group A included vinises readily released from cells in active form; Qoup B included avidly cell-associated viruses releasing little, if any, cell-free virus. The former was represented (among others) by herpes simplex virus, B virus, and pseudorabies virus; the latter, varicella-zoster- and cytomegalo- viruses ofman. 13 B. Early views of MDV and the development of taxonomic criteria. Since the 1960’s, classification schemes have had an important impact on the way we view herpesvimses. In 1969, soon after MDV was determined to be the causative agent of lymphoid tumors in MD, studies of its DNA composition and in vito growth properties led Lee et a1. (1969) to suggest the possibility that MDV was a cytomegalovirus. Even to this day such an example illustates that taxonomy is an evolving process, largely dictated by the technology and popular views of it day. At that time, proportional analyses of guanosine and cytosine (G + C) content was a popular endeavor. This reflected its recently recognized value (in other systems) as an indicator of gross sequence relationships (Sueoka, 1961). Of disappointnent to taxonomist was the subsequent observation that herpesviruses possessed an unprecedented range of mean nucleotide compositions lacking any sort of consistency with other objective criteria, such as serological relatedness or similarities in host range (Honess and Watson, 1977). Beginning with the 1970’s, other objective criteria began to gain attention. One of these was DNA-DNA hybridization. It is somewhat of an irony that one of the major proponent of the current biologically-based classification system (B. Roizman), as early as 1972, led a study to examine whether vinises associated with neoplasia share common physical and genetic properties differentiating them from other herpesvinises. Its basis rested on the premise that ‘superficial' aspect of biological properties are ultimately determined by ‘the information content of the virus” (Bachenheirner, 1972). Interestingly enough, this question was addressed by focusing on the comparative genetic properties of MDV and HSV. Although HSV-1 and «2 were found to exhibit 40% homology by DNA-DNA hybridization, the extent of homology between MDV and HSV-1 or HSV-2 was found to be < 1-2% respectively. Despite what may 14 privately have been a disappointnent to Roizman (considering all the discussions about herpesviruses and cancer at that time, see Roizman, 1972 for instance), as a consolation, the study did find that G+C compositions among herpesviruses associated with neoplasia did not differ significantly from those of other herpesviruses. These result appeared to represent a foreshadowing of event to come. Not long before the 1971 ‘Oncogenesis and Herpesvinlses’ symposium held in Cambridge, England (at which the above result were presented), Epstein had reported the discovery and in vito growth adaptation of a herpes- type virus (EB virus) found in Burkitt’s lymphoma tumor cells (Epstein et al., 1964) which was later found to be antigenically distinct from HSV, VZV, and HCMV (Hurnmeler et al., 1966). Poor growth properties and a topism for lymphocytes appears to have raised interest in a possible relation between this new virus and MDV. Using a ‘state-of-the-art’ DNA-DNA hybridization protocol, zur Hansen and colleagues (1970) were unable to demonstate significant homology between MDV tumor DNA (or HSV-and human cytomegalovirus- infected cell lines) and the DNA present in the Burkitt’s lymphoma lines. Cortidering the stingent hybridization conditions of that day, and the difierences in homology between herpesviruses at the nucleic acid level that we recognize today, such a result (and that of Bachenheirner et a1, 1972, above) should not be surprising. Nevertheless, other investigators continued to search for a serological relationship between MDV and EBV (Ono et al., 1970; Kato et al., 1972; Ross et al., 1972). It is a little ironic that the latter study did in fact demonstate a significant serological relationship between MDV and PRV. C. Concerted attempts to name and classify herpesviruses. In spite of attempt to characterize and classify herpesviruses by objective criteria which employ serologic approaches (cross-neutalization, 15 complement fixation, immunodifiusion, irnmunofluorescence, particle agglutination) and comparative ‘high-resolution’ polyacrylarnide gel electophoresis (Honess and Watson, 1977), methodological limitations precluded an accurate and definitive assessment of phylogenetic relatedness. Th't led to a more subjective approach based on the recognition of biologically- shared properties. Such an approach began to take form in 1977, six years following the establishment (in 1971) of a Herpesvirus Study Group appointed by the International Commission for the Nomenclature of V'lnlses (ICNV) to make recommendations concerning the nomenclature and classification of herpesviruses. l. Nomenclature. ° The problem of nomenclature was first discussed at the 1971 Oncogenesis and Herpesviruses symposium (Pereira, 1972). Although it was deemed too early to ty to classify herpesvinlses, it was apparent that a unifying nomenclature system was needed to ward ofl the confusion that was likely to result from the rapid identification of new herpesvirus members. B. Roizman stated that the current nomenclature was unsatisfactory, since herpesviruses were being named at random according to the host species they infect, the disease they produce or their discoverers. This brought forth a proposal illustating some of the nauseating and somewhat humorous aspect occasionally associated with vinls nomenclature issues. The outlined proposal (Pereira, 1972) suggested a Latinized binomial nomenclature. MDV and HVT would be Herpesvirus galli 1 subspecies l and 2, respectively. It was suggested that the latinized forms would be used only for formal occasions, but that for conunon usage, anglicized forms could be adopted. People were informed to keep in mind the rules of the ICNV, which specified (among others) that neither personal, nor nonsense names could be 16 used, nor could the code of bacterial nomenclature be applied to viruses. One problem, voiced by Peter Wildy, was that it would be unwise to use Latin names as part of a temporary classification as such names tend to become permanent, thus causing potential dificulties in the future. Two years later, in 1973, the Herpes Study Group agreed on a non- Latinised, Anglican-based nomenclature system (Roizman et al., 1973). Thus, MDVandHVTnowbecame phasianidherpesvinls2and turkeyherpesvirus 1, respectively. B virus became Cercopithecid herpesvirus 1. The currently established nomenclature also recognizes a set of formal and conunon names. It is probably not a surprise that the common names continue to dominate the current literature. 2. Herpesvirus subclassificatlon. In 1977, the Herpesvirus Study Group of the ICNV proposed a provisional classification of herpesvinlses based on biological properties. The most often used classification, based on Melnick’s cell-associatedness scheme, was afictively done away with, since it was argued that cell-associatedness appeared to be in conflict with known in vivo properties of the virus. Moreover, others had criticized this system as being overly subjective, inasmuch as herpesviruses exhibited a spectum of ‘efficiency of behaviour in tissue culture’, with no clear break in the gradation of eficiency observed (Plummer, 1967). For the 1977 proposal, it was argued that even though phasianid herpesvirus 2 (MDV) was stongly cell-associated in cell culture its spread in nature occurred by dissemination of infectious virus rather than infected cells. As pointed out in II.D., the latter is actually more representative of the tuth. This new proposal outlined a basic framework for assigning members of the Herpesviridae into three subfamily groups: Alphaherpesvin’nae, Betaherpesvin'nae, and Gammaherpesvin’nae. These were ordered on the basis 17 of the historical precedence of their prototypes, herpes simplex 1, human cytornegalovirus, and Epstein-Barr vinls, respectively. Each of these three subfamilies were characterized by a set of biologically-shared properties characterized by factors such as host range, cell topism, duration of reproductive cycle, cytopathology in cell cultures, and latent infections. Alphaherpesviruses were characterized as possessing a variable host range, from very wide to very narrow; highly cytopathic, with a short replicative cycle; and frequently latent in ganglia. Betaherpesvinlses were characterized by a narrow in vivo host range, usually resticted to growth in fibroblast in vito; a slow reproductive cycle; slowly progressing foci in cell cultures with enlargement of infected cells (in vivo and in vito); inclusion bodies present in both the nucleus and cytoplasm; and latent infections frequently in salivary glands and/or other tissues. Garnmaherpesviruses were characterized by a narrow host range in vivo, usually limited to the same order as the host it naturally infect; replication in lymphoblastoid cells; some growing lytically in epithelioid and fibroblastoid cells; a variable cytopathology and variable- duration replication cycle; a particular specificity for B- or T-lyrnphocytes with infecu'ons frequently arrested at a prelytic stage, with persistence and a minimum level of gene expression, or at a postlytic stage, causing death of the cell without production of complete virions; latent infections frequently residing in the lymphoid tissues. Four years later, the above proposal was formally outlined in more precise detail (Roizman et al., 1981). However, the classification criteria described above were left unchanged. Also presented (at a time when sequence information was only beginning to be generated) was the rationale for this proposal. Biological properties were chosen as the dominant criterion, primarily because of practical considerations. In spite of their subjective nature, 18 biological properties were emphasized, since they could be most readily established following the isolation of a new herpesvinls. Phylogenetic relationships, based on genome stucture, sequence homology, and/or serological relationships were to assume a secondary role in further classifying subfamily members into genera. Because many of these relationships had been fairly well established over many years of work, it would appear that an inherent bias was already present when biological criteria were developed. D. Discrepancies between biologic- and phylogenetic properties in helps-vin- classification. The current biologically-based herpesvinls classification system (Roizman et al., 1981) initially gained it stength from the fact that biologic properties could be readily established following isolation of new herpesvinmes. This classification system has turned out to be generally consistent with genetic data that have accumulated over the past ten years or so. However, recent years have seen a downturn in the number of new herpesviruses characterized. More importantly, a number of discrepancies with this system have begun to emerge. l. MDV. Because of similar biological properties, especially their lymphotopism, MDV and HVT have been classified as gammaherpesviruses (Roizman et al., 1981). Members of this subfamily include, among others, EBV, a human B-lymphotopic herpesvinls, and herpesvirus saimiri (HVS), a T- lymphotopic herpesvirus of new world monkeys and lower vertebrates. Despite certain stuctural diflerences between EBV and HVS, the genomes of these and other gammaherpesviruses encode serologically-related proteins and share a conunon organization of coding sequences which differs from that of the neurotopic alphaherpesviruses (Davison and Taylor, 1987; Efstathiou et al., 19 1990; Gompels et al., 1988; Nicholas et al., 1992b). This latter subfamily includes (among others) the human herpesviruses HSV and VZV, porcine pseudorabies virus (PRV), bovine herpesvirus (BHV) and equine herpesvirus (EHV). In contest to other gamrnaherpesviruses, MDV and HVT have genome stuctures closely resembling that of the alphaherpesvinlses (Cebrian et al., 1982; Pukuchi et al., 1986; Igarashi et al., 1987). The latter possess similar genome stuctures consisting of covalently joined long (L) and short (8) component. The 8 component comprise a unique segment (Us) flanked by a pair of extensive inverted repeat regions (i.e. IRs/TRs or Rs). L component contain a unique segment (0;) that may or may not be flanked by similarly extensive repeat regions (i.e. TRL/IRL). While MDV, HVT and HSV contain extensive UL-flanking repeat sequences, VZV, PRV. BHV, and EHV do not. Thus, the genome stuctures of MDV and HVT most closely resemble that of HSV (Cebrian et al., 1982). Buckrnaster et al., (1988) have recently reported that the gammaherpesvinises MDV and HVT bear greater genetic similarity to alphaherpesviruses than garnmaherpesviruses. This was based on analyses of numerous, randomly isolated MDV and HVT clones at the predicted a level; not only were individual sequences found to exhibit greater relatedness to genes of HSV/VZV than EBV, but the MDV/HVT genomes were found to be generally collinear with VZV, at least with respect to the U1, region (Figure 1). Alphaherpesvinis Us- and other 8 region genes originate from an area specific to members of this taxonomic subfamily, arguably their most divergent coding region. Two groups have recently confirmed the earlier proposal of Buckmaster (1988) by demonstating extensive colinearity of alphaherpesvirus-homologous genes in the Us regions of MDV (Brunovskis and Velicer, 1992a; Ross et al., 20 .82 Jo as usages—n 52m damn 05mm an. to Eggnog severance 05 .3253 “82:32 noon to: no: con—32 econ someone 05 some some 5 “one masseuse“ E 5 some 33262 0232250.? 9.: one seen we 05 to encased dose 05 0539? use: canon 95. A3 5 gone as 2-... 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T we. __.._ i ___.V_J_ c\ o_ 0.0 a Z < O.— _ LZme Um 1 352—50.. <20 an casein—Sam _ O.— ommci £32m EBEQ m:_v=_n.m: 829.69. . . . cameo 3:83:32 as: refills 1.511.”: 0 5 “ £in mmZmO E DE >92 .mO mZOEmOm mm: 22 1991). Together these result show that, in spite of their biological properties, MDV and HVT bear a much closer phylogenetic relationhip to alphaherpesvinises than garnmaherpesviruses. 2. Human herpesvinn 8. Human herpesvirus 6 (KIN-6) is a novel herpesvirus independently isolated by several groups from patient with lymphocytic disorders. HHV-6 was initially named human B lymphotopic vinls because it was found to infect B lymphocytes (Salahuddin et al., 1986). However, recent studies have shown that it is primarily topic for CD4+ T-lymphocytes (Takahashi et al., 1989) where it may act as a cofactor in AIDS (Ensoli et al., 1989). Because of it lymphotopisrn, Lopez et al., (1988) suggested a provisional gammaherpesvinls classification. Genetic evidence based on a 6- base-pair (GGTTA)n repeat sequence identity between MDV and HIV-6 was further interpreted as warranting such a classification (Kishi et al., 1988). In contast to these proposals, recent sequence analysis has established a much closer phylogenetic relationship between HIV-6 and betaherpesviruses, characterized by it prototype human cytomegalovirus (Lawrence et al., 1990). 3. Charms! cabh virus. Channel catfish vinls (CCV) is a relatively uncharacterized herpesvirus with an unusual genome stucture packaged in a virion that is morphologically indistinguishable fi'om those infecting higher vertebrates (Wolf and Darlington, 1971). However, in contast to its alphaherpesvirus classification, sequence analysis of the 134,226-bp CCV genome has shown that ' its genetic properties are entirely distinct from those of either of the three herpesvirus subfamilies (Davison, 1992). Even though CCV possesses several characteristics common to herpesviruses, this new data suggest that CCV is representative of an entirely novel herpesvirus (sub) family. 23 E. Problems with the biologically-bued classification system. 1. Theoretical implication. The above discrepancies reflect a problem that may be more than simply academic. To it credit, the current biologically-based classification system has been remarkably consistent with the genetic data. It has been recently described as simple, fortuitously appropriate and defective (Roizman, 1990b). A further criticism relates to is subjectivity and potential to adveme afiect our view of herpesviruses. Herpesvinis biology is exceedingly complex, perhaps more so than originally thought. Since MDV has been regarded a gammaherpesvirus, much of the previous work interpreting MDV's properties has proceeded by analogy with the association between EBV and B cells (Wen et al., 1988, for example). MDV actually has little in common with gammaherpesviruses other than it potential to latently infect and tansforrn lymphocytes. Because of its closer relatedness to alphaherpesvinlses, it would appear that MDV’s seemingly divergent biological properties are unlikely to be determined by macromolecular stuctures homologous to those of gammaherpesviruses (Lawrence et al., 1990). This suggest a renewed emphasis focusing on phylogenetic difierences between MDV and other alphaherpesvinlses as an approach for examining the molecular basis of the divergent biological properties. One of the chief dificulties with the current classification system is maintaining objectivity in the face of biological distinctions which oversirnplify our view of herpesviruses. These distinctions largely originate as a function of our inability to study the effect of herpesvinlses in vivo, together with a limited availability of suitable culture systems for manipulating and studying these viruses in vito. Biological properties that were intended to classify 24 herpesviruses in a scientifically-beneficial manner are wrought with a number of inconsistencies and paradoxes. Many of these have only recently come to light. 2. Epitheliotoplsm. Epithelial cells are a critically important tissue for any herpesvirus replication stategy. However, relatively little is known about factors responsible for limiting or promoting growth in these cells. Despite all the emphasis on EBV's lymphotopic association with B-cells in Burkitt's lymphoma, the predominant malignancy associated with EBV is actually an epithelial malignancy, nasopharyngeal carcinoma. Although EBV and MDV are commonly labeled as 'lymphotopic' herpesviruses, oropharyngeal epithelial- and feather follicle epithelial cells, respectively, are responsible for the production and horizontal tansmission of fully enveloped infectious virions (Calnek et al., 1970; Nazerian and Witter, 1970; Sixbey et al., 1984). The relatively scarce molecular details about MDV and EBV growth in these cells is largely a reflection of technical limitations which have taditionally hindered in vito studies in epithelial cells. 3- Lmhotrophn. Although alpha- and betaherpesviruses are generally not referred to as ‘lymphotopic herpesviruses’ (in contast to the gammaherpesvinlses), lyrnphotopism is probably common to all herpesvinises. 1t clinical significance has been cited for betaherpesvirus- (e.g. HCMV; Saltzman et al., 1988) and alphaherpesvirus infections, including those of HSV (Nahmias and Roizman, 1973), VZV (Gross, 1982), equine herpesvirus (El-1V; Bryans, 1969; Scott et al., 1983), bovine herpesvirus (BHV; Nyaga and McKercher, 1980), pseudorabies virus (PRV; Wang et al., 1988; Wittmann and Rziha, 1989) and feline herpesvinis (Fl-N; Tham and Studdert, 1987). 25 Lymphotopism among alphaherpesviruses is often overlooked, despite it importance to pathogenesis and mortality. Upon further comparison with other alphaherpesviruses, a number of stiking parallels are noted in comparison to MDV infections. With respect to T cell topism, MDV and HSV are similar; replication of each is resticted to activated, Ia-bearing T cells (Braun et al., 1984; Calnek, 1986). In the case of MDV, these represent the target cells for tansformation (see II.G.). HSV-2- and VZV infections, involving neonatal and irrununocompromised host often result in severe systemic infections, characterized by widespread blood-bome dissemination of virus to multiple organs, often resulting in death (Gross, 1982; Nahmias and Roizman, 1973). Such infections bear a remarkable resemblance to the early event associated with MD pathogenesis. Both involve biphasic patterns of vinis replication in lymphoid cells characterized by the development of a primary viremia which serves to disseminate vinls to various organs. What follows is a brief period in which virus temporarily disappears from the blood in conjunction with the beginning of virus replication in various organs. Often a massive, secondary viremia follows and multiple lesions involving many organs are observed, ultimately leading to death. In both cases, overall viremia levels appear to directly correlate with death. The consequences of blood-borne MDV infections, particularly in young, genetically susceptible, antibody-free chickens often result in an early mortality syndrome leading to death, usually between 1.5 and 3 week post-inoculation (Witter et al., 1980). In such cases, tumor formation is absent; principal lesions are severe atophy of the bursa and thymus, destuction of lymphoid cells, encephalitis, and an occasional focal necrosis of spleen and liver. As is the case with MDV, contol of these blood- borne infections is largely influenced by imrnunocompetence and age (Calnek and Witter, 1991; Nahmias and Roizman, 1973). It will be interesting to see 26 whether the comparatively looser restictions against severe pathogenesis by MDV in older birds (compared to HSV/VZV) are determined by intinsic genetic properties of MDV or alternatively reflect fundamental difierences associated with these immune systems. 4. Latency and nemotoptm. It is becoming increasingly more dificult to define persistent virus infections as stictly ‘latent' or ‘productive’. Such infections can either be productive or latent depending not only on the cell type infected, but on the ‘activation’ state of the cell (Ahmed and Stevens, 1990). Increasing lines of evidence indicate that generalizations regarding sites for virus persistence may be premature. There are indications that herpesviruses from all three subfamilies (e.g. EBV, CMV, PRV) may latently persist in both lymphoid and epithelial cells (Schrier et al., 1985; Stevens, 1989; Wittmann and Rziha, 1989). Recent evidence indicates that the alphaherpesvinis, EI-N-l can establish latent infections in T-lymphocytes (Welch et al., 1992), lending support to an earlier proposal characterizing EI-N-l as a T-lymphotopic herpesvinis (Scott et al., 1983). Although alphaherpesviruses, are often distinguished by their ability to undergo latent infections in neural tissues, recent evidence suggest that in contast to HSV, VZV establishes latent infections in satellite and perhaps other nonneuronal cells (Green et al., 1988). The ‘1ymphotopic’ MDV also appears to go latent in neural tissues and like VZV this appears to involve some of the same nonneuronal tissues, namely the nonrnyelinated Schwann cells and satellite cells (Pepose et al., 1981). We pattern of gene expression in nonneuronal nervous tissues appears to be entirely distinct from that observed in neurons latently infected by HSV (Croen et al., 1988). Together, the above findings suggest that latency may take on many forms; its nature reflecting the 27 difierent requirement and contols which limit productive or semi-productive infections, depending on the given cell type involved. 5. Further biological parallels: pathogenic manifesttiorl attributed to MDV and alphaherpesviruses. The ‘neurotopic alphaherpesvinlses’ are characterized by a range of ocular and neurological manifestations. For example, HSV-1 is known to be a leading infectious cause of blindness and encephalitis. Despite all the attention emphasizing MDV’s lymphotopic/oncogenic properties, MD is often regarded more as a neuropathological disease. Classical MD (also called fowl paralysis) is characterized by neural lesions resulting in peripheral nerve demyelination. Certain pathogenic MDV isolates are particularly distinguished by a marked propensity to cause reversible encephalitis (e.g. tansient paralysis; Kenzy et al., 1973) or blindness (Picken et al., 1991). While HSV-induced encephalitis is often mediated by a neurogenic pattern of infection, hematogenous spread has been clearly established, particularly with regard to HSV-2 infections involving neonatal and immunocompromised individuals (Nahmias and Roizman, 1973). The previously described association between VZV infections and the Landry-Guillain-Barre syndrome (Sanders et al., 1987) is of interest in light of the fact that MDV has been considered a useful animal model system for this neurological condition (Pepose et al., 1981; Hughes, 1990). Finally, accumulating evidence has established a common etiological link involving both MDV and HSV in the pathogenesis of atherosclerosis (reviewed by Fabricant, 1986; Hajjar, 1991). P. Fixture tends in brpesvlrus clarification. With the onset of recombinant DNA approaches to rapidly analyze genome stucture and genetic organization (Buckmaster et al., 1988; Davison, 1992; Ettathiou et al., 1990; Gompels et al., 1988; Lawrence et al., 1990), 28 coupled with ever-expanding databases containing a wide range of nucleic- and predicted a sequences representing all three herpesvirus subfamilies, a phylogenetically-based classification approach appears much more feasible at this time. Such a change would be desirable not only because of the problems and discrepancies associated with a biologically-based classification system, but because of the fact that phylogenetic relationships provide the most useful tools for addressing the genetic nature of phenotypic difierences. A move towards a phylogenetically—based classification system would not require reclassification of most herpesviruses; it would simply place genetic relatedness as the dominant criteria for classifying herpesviruses. N. Evolution of herpesviruses. A. Introduction: Biological observation and hypotteses. It has often been said that the evolution of viruses is the evolution of their host. Since herpesvinlses are characterized by the presence of an envelope acquired by budding from nuclear and/or Golgi body membranes, bacterial host appear unlikely to have played a role in the evolution of herpesvinlses. Nevertheless, the widespread occurrence of herpesviruses in many varieties of eukaryotes is suggestive of a long evolutionary history. To gain further insight about a putative progenitor herpesvinls, N ahmias (1972) has suggested a concerted attempt to identify herpesvinls in primitive eukaryotic life forms. A major theoretical challenge is understanding the balance between intinsic vitally-encoded (pathogenic) influences and those attibutable to an immune system not adapted for dealing with such ordinarily harmless pathogens. To better appreciate this, it is necessary to consider some of the peculiarities of herpesvinises. In most cases, herpesviruses are relatively harmless in their natural host (beyond the newborn age). However, natural 29 tansmission across normal host-species barriers can have devastating consequences. This is best exemplified by B virus and herpesvims saimiri, two simian herpesviruses common to old- and new world monkeys, respectively. The former is almost invariably fatal to humans and various experimentally- infected host; the latter is harmless to squirrel monkeys, but highly oncogenic and fatal to owl monkeys (Barahona et al., 1974). In fact, isolation of herpesvimses from fatally-infected animals beyond the newborn age is generally an indication that another animal species was the source of the virus (Nahmias, 1972). Together with a long-lasting latent association with their host, these peculiarities suggest that herpesviruses have co-evolved with their host so as to cause little disturbance. According to the Theobald Smith doctine (1934), these characteristics indicate that herpesvinlses are ancient, well-adapted parasites (Wildy, 1973). By preserving the host, while at the same time allowing the survival of the virus and its ultimate dissemination to further host, often many years later, herpesvinises have an important edge over many other virus groups. To explain the nature of this phenomenon, Nahmias (1972) has postulated the existence of a host factor ‘X’ which, when present, keeps the vinls from harming its host. Because of the severity of infections in newborn- or immunocompromised individuals (non-human species as well), Nalunias has suggested that the ‘X factor’ is probably associated with the host’s immune system. Such a proposal appears consistent with recent MDV studies suggesting that latency is maintained by the presence of a so-called latency- maintenance factor (th1?) present in conditioned media (Buscaglia and Calnek, 1988), which is known to be rich in cytokines, including the lymphokines interleukin-2 and gamma-interferon. 30 B. Observations and hypotheses from genetic studies. 1. Gemtlc relationships among herpesviruses. l. Basis for genome structtn'e diversity. Herpesvirus genomes commonly contain a unique long region associated with various types of direct, inverted and/or internal repeat structures. These repeat structures vary in number, arrangement, and composition. Figure 2 illustrates five of the genome structures that have been presently identified (Roizman, 1990b). Genome structures do not necessarily reflect phylogenetic relationships. HSV and HCMV each have group B genome structures that can similarly isomerize. Moreover, HCMV also contain functionally homologous 3 sequences that can substitute as a cleavage/packaging signal for HSV-l (Spaete and Mocarski, 1985). Conversely, closely-related viruses, such as PRV and HSV-l , differ in their genome structures more than distantly-related viruses, HCMV and HSV-1 (Figure 2). Differences in organization of herpesvims repeat regions are thought to be a reflection of the replication process, which appears to be intimately associated with the propensity to undergo recombination (lioness, 1984; Weber et al., 1988; Dutch et al., 1992). One consequence of the herpesvirus replication process is that internally reiterated sequences can mediate reciprocol recombination with their homologous repeat sequences elsewhere, resulting in the production of distinct isomers with unique regions inverted relative to one another. Located in the inverted- and terminal repeats of HSV, VZV, and HCMV are the so-called a sequences, which contain cis-acting signals for cleavage of viral concatamers and packaging of their unit-length genomes (Mocarsld and Roizman, 1982; Spaete and Mocarski, 1985; Varrnuza and Smiley, 1985). Originally it was suggested that a sequences mediate the inversion process; however, recent studies indicate that HSV-l inversion/isomerization can be 31 589 .gfiom 52m .Eofio£ 55.535 v .5 «53:00 <20 3» «is not: :5 85:09:00 5:2 pa :9? 55 :5: 5.35:5 m 955 05 :— 05 5 45:09:50 «5% 05 .5 :on3:o:o 05 E 555% gore—smog oz: 558 <29 55 .5 «5:: .9555 :95“ on? >9: 50:25:00 5:2 05 :9552 5:2 05 2 2520: «:95 55:09:00 :93 o5 .Q 955 5 €35 m one Q m955 5 550:5 55 go 3:205:50 0:9 55> >9: 355:2 some be 303825: mo 59:5: 9E. 55:2 :5: 5 menu: 355:5: “.9825: 0.5 50:05am 5:55: 05 .m 955 :— .oontomoo :53 5: 25: m 955 mo 550:55 05 E 30555: 358.59 .3550: Ame :9? :5 33 5:2 95 «5:053 099:: 05 555553 .3955 >3 oo5:oE :ouflfifio octet: :w E “5:35 Eu 35050» no o5 a: 55.52 05. .o no “55535 5:050» m :05an >55 0:0 mm: 35:5: .550 e5. d as “55535 5:250» .593— a o. :5: a 5:253 .5 5500 : 53:00 355:2 0:0 £55555 0:: «o 53:00 9m: 5.3 m 955 no 3:5» 05. d 955 go 355: Amp. :5 EV 35:5: :5 5525 98 .0 955 mo 55.5: 3:52.: :8 vm 2 E Boone: d 955 5.“ 93.6 98 $2.6 Beans: 55:5: :5: pg :3 no “555:3: 93 pa 55550: no :39? 98 gov @3895: BE. .9552 0555-535 5 33:: 52552 5:: 3:035: 05 595$“: ougoson 05 5 .9295 955090: 38:» ooh o5 .2468 d 558? 595: .E :35» «among .996 3:? hamfioumnm Ami 35:3 E595: .900V 5850 5:55 on... .3 pea—9:05 o5 m o5 .m .D .0 .m d 5:5:5 9E. .gagsgofisfigoufifigsea dunno: F _=====_====_TI|II_III|..IIIIIII.IIJ_=====_= m EEC NE...— m5E0w. 9.80 mmEOZmU mbémmmmmm m0 mgOmnv m E E mBZMSMO—AE MOZMDOMm 33 mediated by repeat sequences idependant of the a sequences (Weber, 1988). The repeat sequences themselves appear to be inherently recombinogenic (Homes, 1984; Weber et al., 1990); this property applies to the a-sequence structure itself, apparently accounting for its own ability to undergo recombination and expansion (Umene, 1991). Recent evidence has shown that 1 sequences promote isomerization twice as readily as unrelated sequences, similar in size. More importantly, recombination between a sequences was found to occur at approximawa the same time as replication (Dutch et al., 1992), lending further support to the idea that replication and recombination are closely linked (lioness, 1984; Weber et al., 1988; Weber et al., 1990). Together these features characterize the nature of herpesvims replication-associated recombination, which is likely responsible for much of the structural- and genetic organizational divergence which characterize members of the herpesvirus family. These aspects are particularly highlighted by the identification of PRV- and HCMV variants with extra pairs of inverted repeats which facilitate the generation of four- (Lomniczi et al., 1987) or eight genome isomers (Takekoshi et al., 1987). The four-isomer PRV group B (also called class B, type B or class 3) structures have been postulated to arise from a double-crossover event between two inversely oriented concatameric group D (also called class D, type D, or class 2) molecules (Lu et al., 1989). Under certain growth conditions PRV variants evolve to acquire group B structures which confer a selective growth advantage in some types of cells, but a selective disadvantage in others (Lornniczi et al., 1987; Reilly et al., 1991). These mutants were found to have acquired alternate cleavage/encapsidation sites by the juxtaposition of terminally-located sequences next to the internal inverted repeat (Rail et al., 1991); their subsequent cleavage has been directly linked to the inversion of the 34 L component (Kupershmidt et al., 1992). Based on additional factors, these results have been interpreted to suggest that the emergence of viral populations with group B genomes represents an adaptation to the prevailing conditions in a given host or target tissue (Rall et al., 1991). An analogous process was postulated to account for the evolutionary divergence of HSV and VZV 8 regions from a common progenitor (Whitton and Clements, 1984a; Davison and McGeoch, 1986). Such a process is thought to have involved a series of homologous, semi-homologous and/or non-recipricol cross-over events that can potentially lead to the: (i) loss, gain, and/or diploidization of sequences; (ii) creation of new open reading frames (ORFs) and; (iii) recruitment (and substitution) of promoter and/or regulatory elements. This provides rationale for the observation that VZV lacks six HSV-l Us region homologs (U82, U84, U88, USS, U811, U812) and has two others (U51 and USlO) diploidized and relocalized to the adjoining repeat regions (Davison and McGeoch, 1986). Support for this proposal has been gained from HSV-l and —2 studies demonstrating that expansion and contraction of le/‘I'Rs- and Us regions can indeed occur (Brown and Harland, 1987; Umene, 1986). An additional level of diversity may have been introduced by the existence of an error-prone DNA polymerase. Previous studies have suggested that wild-type HSV strains have such an enzyme (Hall et al., 1985); analogous results have been interpreted as accounting for the extensive divergence of retroviruses, such as HIV. To account for the wide variations in G+C content (even among closely-related members), non-selective forces resulting from intrinsic mutational biases in the DNA replication machinery have been postulated (lioness, 1984), aided by the help of recombination (McGeoch, 1987- suppl). Further changes may have occurred as a consequence of different viral enzymes involved in nucleotide metabolism (lioness, 1984). 35 ii. Convergent vs. divergent evolution. At present, HSV-1 (McGeoch et al., 1988), VZV (Davison and Scott, 1986), HCMV (Chee et al., 1990b), and EBV (Baer et al., 1984) have been completely sequenced. Compared to other viruses, their genomes are complex, ranging between 124,884- (VZV) and 229,354-bp (HCMV) in length and containing 70- (VZV) to 200- (HCMV) genes. Together they represent at least one member of each of the three herpesvirus subfamilies. These four herpesviruses represent at least one member for each of the three herpesvirus subfamilies. Because of their clinical significance, more is known about the nature of human herpesvirus genes and their gene products in comparison to those of animal systems. In conjunction with this information, these sequence databases provide an especially important foundation for phylogenetic- and molecular studies involving less characterized non-human systems. Homology comparisons between these genomes indicate that members of all three lineages share a common ‘core' of about 30 related genes located in the unique long regions of their respective genomes (Chee, 1990b). The presence of these common genes suggests that members of the three herpesvirus subfamilies have all evolved from a single progenitor by a process referred to as divergent evolution. Positional localization of these core genes in the alpha-, beta-, and gammaherpesvirus lineages suggests that such an evolutionary process would have required large-scale UL region rearrangements (Davison and Taylor, 1987; Chee et al., 1990b; Kouzarides et al., 1987). Figure 3 depicts the rearrangement and organization of homologous gene blocks in the genomes of HCMV, EBV, VZV, and HSV-l . 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F. . r: _..2 r. 9.. 7:6: 924 .>N> 5mm 520mm ZmEmm MOZMDOmm .mO 8:0qu figmmzoo 38 An alternative, non-mutually-exclusive possibility for herpesvirus evolution is convergent evolution This form of evolution would predict that the same mechanism from which herpesviruses originally evolved would have independently repeated itself. The recent sequence analysis of CCV (see lIl.D.3.) highlight genetic properties which are entirely distinct from the alpha-, beta-, and gammaherpesvims lineages. Such findings provide support for the view that herpesvimses have evolved both divergently and convergently. HI. Difierenoel charactuidng the three phylogenetic W- Aside from the ‘core’ of genes conserved by all herpesviruses (except CCV), each lineage contains a unique set of genes which are specific to its phylogenetically-related members. Some of these appear to have been transduced from cellular genes (Sugden, 1991). For example, the EBV BCRP-l gene encodes a homolog of interleukin-10. Gammaherpesviruses, appear to have evolved their own characteristic set of unique genes important for maintaining latent infections, as well as specifying functions responsible for immortalization and/or transformation (Kiefi and Liebowitz, 1990). Despite their structural difierences, EBV and HVS encode serologically-related proteins and share a common organization of coding sequences which difiers from that of the alpha- and betaherpesviruses (Davison and Taylor, 1987; Efstathiou et al., 1990; Gompels et al., 1988; Nicholas et al., 1992b). However, recent studies indicate that these two gammaherpesvimses significantly diverge in at least two locations (Nicholas et al., 1992b). This includes the absence of two blocks of genes in HVS which correspond to EBV genes (encoding EBNAs) and cis-acting signals (oriP) associated with latent growth, as well as those which disrupt latency and promote lytic growth (e.g. BZLPI). Although the nature of genes responsible for latency in HVS are not yet characterized, HVS has incorporated its own unique 39 repertoire of genes, including some which bear a close resemblance to members of the D-typo cyclin- and G-protein-coupled receptor (GCR) family of proteins (Nicholas et al., 1992a). With a 229,354 bp genome, the betaherpesvims, HCMV is much larger than the genomes of alpha- and gammaherpesviruses. As such, it has incorporated a more diverse set of genes, a number of which bear cellular counterparts. This includes an MHC class-I-related gene (Beck and Barrell. 1988) involved in preventing immune surveillance (Browne et al., 1990) and a family of 3 GCR-related genes (UL33, U827, and U828; Chee et al., 1990a) related to recently described GCR homologs in HEN-6 (Neipel et al., 1991) and HVS (Nicholas et al., 1992a). The U5 localization of U827 and -28 may appear to suggest host-derived acquisition independent from those of HHV—6 and HVS. On the other hand, a common UL localization for the HHV-B GCR homolog (ORFS) and HCMV UL33 suggests a common phylogenetic origin, possibly distinct from U827 and -28. The latter are tandemly arranged, presumably evolved by duplication and divergence. The HCMV OCR-related gene family represents one of at least nine different sets of homologous gene families, the majority of which are located in the Us region of HCMV (Weston and Barrell, 1986; Chee et al., 1990b). These account for at least 21 genes, each distinguished by a unique a motif pattern characteristic for each family (Chee et al., 1990b). Members of the U86 glycoprotein family (086, -7, -8, -9, ~10, and -l l) exemplify recent findings indicating that HCMV’s Us region specifies an important locus for genes that are nonessential for growth in cell culture (Jones et al., 1991; Jones and Musithras. 1992; Kolbert-Ions et al., 1991). HCMV and HSV-l both contain invertible 8 components with genes that are unique to alpha- and betaherpesviruses; however, aside from their basic 40 structural similarity, these two regions bear no sequence homology with one another. Nevertheless, these two regions exhibit remarkable parallels in their nature. Each encodes a set of genes unique to members of their subfamily. Like HCMV, alphaherpesw’ruses contain a similar cluster of Us region genes that are nonessential for growth in cell culture (de Wind at al., 1990; Longnecluer and Roizman, 1987; Longnecker et al., 1987; Weber et al., 1987). These appear to encode supplementary essential functions that have presumably evolved to facilitate the efficient dissemination of virus in their host tissues (Roizman, 1990a; further discussed in V.B.). Furthermore. like HCMV, alphaherpesviruses, such as HSV-l, contain a set of tandernly arranged glycoprotein genes which appear to have similarly evolved by a process of gene duplication and divergence (McGeoch, 1990). It will be interesting to see whether other betaherpesvirus 8 regions exhibit a similar genetic divergence which characterizes alphaherpesviruses (Brunovskis and Velicer, 1992; Davison and Willtie, 1983; Davison and Scott, 1986; further discussed in V. A.). Although the acquisition of a progenitor 8 region appears to have been an event coinciding with establishment of the alphaherpesvinis lineage (Davison and McGeoch, 1986; Davison and Scott, 1986; Davison and Taylor, 1987; McGeoch, 1990), this may not be the case with betaherpesviruses. Despite a closer phylogenetic origin between HCMV and HHV-6 (Lawrence et al., 1990; Neipel et al., 1991), the latter contains a smaller (appr. 162-168 kb) group A genome structure (Figure 2) lacking an 8 component (Lindquester and Pellett, 1991; Martin et al., 1991). HCMV may have acquired an 8 component relatively recently in its evolution from a progenitor betaherpesvirus. Alternatively, HHV-6 may have lost its 8 component, perhaps reflecting a move towards a more lymphotropic existence. 41 2. Lahncy. 1. Introduction: EBV and HSV. In recent years, numerous laboratories have sought to identify viral products which maintain the latent state. In the EBV system, such studies have identified a complex transcriptional pattern responsible for the expression of a small subset of EBV-encoded gene products in latently infected B-cells. Interestingly, the particular pattern of expression can vary between different B-cell subtypes EBV-transformed epithelial cells (Klein, 1989). While some of the functions expressed relate to immortalisation and/or transformation, one unifying feature characterizing persistence in these difierent infections is the consistent identification of EBNA—l, a trans-acting factor necessary for replication and maintenance of episomal EBV genomes. Recent data suggest that such results may be flawed by the artifactual nature of the in vitro culture systems employed raising important concerns about the relevance of EBNAs to normal latent infections. A recent PCB-based study (On and Rowe, 1992) using uncultured peripheral blood lymphocytes from healthy, latently- infected individuals has found, contrary to expectations, an apparent dispensability for EBNA expression, instead implicating a central role for the T? membrane protein, previously identified in ‘latently’ infected tissue culture cells (Kiefi and Liebowitz, 1990). HSV-l studies have thus far failed to identify a single viral gene actively involved in neuronal latency. In spite of the initial enthusiasm concerning identification of non-polyadenylated transcripts expressed in latently-infected cells (e.g. latency-associated transcripts, LATs), LAT mutants are nevertheless able to facilitate the establishment and reactivation of latent infections (Steiner et al., 1989). These findings suggest that, in some cases, latency may be more a function of the host limiting the expression of viral genes important in productive 42 infection either by repressing their expression (Lillycrop et al., 1991) or failing to express critical transcription factors necessary for their activation (Garcia- Blanco and Cullen, 1991). ii. CpG content In actively dividing cells, CpG dinucleotides of vertebrate genomes are susceptible to methylation. This leads to the production of S-rrtethylcytosine, which is deaminated at high frequency to form thymine. As a consequence of this propensity for methylation, all vertebrate genomes thus far analyzed have disproportionately low CpG frequencies and disproportionately high frequencies of TpG dinucleotides compared to those predicted on the basis of mononucleotide compositions (Bird, 1980). Herpesviruses lack methylation system of their own, yet recent studies have illustrated a striking pattern of CpG frequencies largely consistent with the current biologically-based classification system (Honess et al., 1989). Ganunaherpesviruses, such as EBV and HVS are characterized by significant CpG deficits associated with corresponding excesses of TpG. In contrast, alphaherpesviruses, such as HSV, VZV, and PRV fail to exhibit discrepancies in their dinucleotide frequencies, while the betaherpesvirus, HCMV, has properties intermediate between those of the other two families. Local CpG deficits and TpG excesses were resticted to the immediate early genes of HCMV. The latter result, while dificult to interpret, reflects the biological ambiguities associated with this unusual group of herpesviruses. The CpG deficiencies common to all gammaherpesviruses thus far analyzed (EBV, HVS, and MIN-68; Efstathiou et al., 1990; Honess et al., 1989), have been interpreted as a consequence of their long-term maintenance in actively-dividing lymphocytes subject to methylation, in contrast to alphaherpesviruses, which are latently-maintained in a non- dividing methylation-free environment. 43 Consistent with their close phylogenetic relationship with alphaherpesviruses, MDV has not been found to exhibit CpG deficiencies (Honess et al., 1989; Ross et al., 1991; Brunovskis and Velicer, 1992a). 0n the surface, this would appear to conflict with the fact that MDV can go latent in lymphocytes. The lack of CpG deficiencies for MDV have been interpreted to suggest that the epidemiologically significant form of MDV transmitted in nature 'I unlikely to be one whose precursor derives from latently-infected T- lymphocytes (Honess et al., 1989). Although MDV has been shown to be methylated in lymphoid tumor cells (Kanamori et al., 1987), these findings are not irreconcilable with such predictions. It is worth questioning whether MDV persistence and shedding are actually derived from ‘latently'-infected lymphocytes. There is surprisingly little known about the long-term maintenance of MDV. Only one study has been conducted that would support the notion of long-term MDV latency in lymphocytes (Witter et al., 1971). Unlike classic co—cultivation assays for latency, virus could only be rescued from a small proportion of convalescent birds 76 weeks postinfection. This raises a number of questions. Does MDV employ an active, genetically-determined strategy for maintaining long-term latency in lymphocytes (like EBV)? Could the difficult-to—detect viremia possibly reflect persistence in PPS cells from which periodic bouts of productive replication serve to non-productively re-infect lymphocytes in a chronic fashion? It should be noted that, despite their ability to protect against T-cell-induced lymphomas, vaccinated birds still manage to shed superinfected MDV (Purchase and Okasaki, 1971). Does this reflect persistence in immunologically-protected FFE cells? As described in II.D., MDV is extremely eficient in horizontal transmission. Unlike other herpesviruses, which are usually transmitted during 44 episodes of acute infection, MDV is released in a form that can remain infectious for long periods of time (>200 days) at ambient room temperatures (Carozza et al., 1973). Residual MDV associated with keratinized material near the feathers has been previously suggested to contribute to the long-term shedding of infectious virus (Johnson et al., 1975). MDV’s ability to initiate a fully productive infection is intimately associated with the state of PFE cell diflerentiation. Nonproductive infections involving the basal layers of HE cells can eventually proceed to a productive infection upon differentiation Oohnson et al., 1976). It may be worth considering whether MDV can maintain itself in a non-productive state in basal layers of the FFE from which it ultimately proceed to a productive infection (and shedding) upon diflerentiation. The latter could also account for chronic, non-productive lymphocyte reinfections, as well as reinfections of new basal layers in order to maintain MDV's persistence for subsequent periodic sheddings. A similar idea has been proposed to emphasize a more prominent role for oropharyngeal cells in EBV persistence (Allday and Crawford, 1988; Rickinson et al., 1985). However, consistent with predictions based on CpG deficiencies, recent findings appear to rule out such a hypothesis for EBV (Cratama et al., 1988; Niedobitek et al., 1991). Finally, with regard to MDV, it may be worth adding that a chicken succumbing to a tumor following a ‘latent’ lymphocyte infection would be unable to subsequently transmit virus if it were dead! iii. Codon usage. Despite new questions concerning the nature of genes expressed in cells latently infected with EBV, Karlin and colleagues (1990) have recently identified a potentially important contrast in codon usage between genes thought to be specific for productive- or latent infections. In particular, they found a statistically significant decrease (20%) in the percentage 45 of C or C in codon site 3 (83 percentage) for genes expressed in latent infections. This and other disparate features of codon usage were interpreted as reflecting an adaptation to minimize the deleterious consequences to the host during latent infections. 3. Sequence analysis as a marker for put event. If we assume that the evolution of herpesviruses is synonomous with the evolution of their hosts, then it follows that sequence companions between viral genes and their cellular counterpart ofler a potential strategy which can lend insight into the past history of a given vims or its host. Phylogenetic trees have often been used to estimate the divergence time between host organisms and virus which infect them. Amino acid sequence comparisons between the thyrnidine kinase genes of HSV and marmoset herpesvirus (MI-1V) suggest that HSV-l and -2 diverged fi'om one another 8-10 million years ago (Gentry et al., 1988). This time span was consistent with other phylogenetic trees similarly constructed using herpesvirus sequences from three independent sets of related proteins. HSV-l and -2 are primarily transmitted by oral and genital routes of infection, respectively. Considering the fact that the closely related B virus is transmitted by both routes, in addition to the above divergence time and a number of other factors, a provocative suggestion has emerged. By this, the 8-10 million year divergence time was predicted to result from changes in human sexual behavior influenced by (i) adoption of a generally upright position resulting from a change towards bipedalism; (ii) adoption of close face-to-face ventral-ventral mating, and (iii) an increased female sexual appetite, not limited by menstrual cycles (Gentry et al., 1988). These changes were considered necessary and appropriate for the microbiological isolation required to bring about the above divergence. 46 V. Chanaerlsetlon and significance of the alphaherpesvirus 8 region. As previously noted (III.D.), alphaherpesvirus 8 regions (or 8 component) are covalently linked to an L region and consist of a unique short (Us) segment bounded by a pair of inverted repeat (IRs/TRs, or simply Rs). The 8 regions of alpha- and betaherpesvimses bear no phylogenetic relationship to one another. The remainder of this review will summarize the significance and current state of knowledge concerning the various genes and product encoded by alphaherpesvirus 8 regions of human and animal systems. Figure 4 identifies alphaherpesvirus homologs which have been thus far identified. Names common to each system are listed below each box. Inasmuch as most 8 region homologs have HSV-l counterpart, for which much of our current knowledge is derived. their identification will be simplified by the use of nomenclature emphasizing their homologies to HSV-l . Thus PRV gp50, a homolog of HSV gD (Figure 4) will be referred to as PRV 9D or PRV U86. The term ‘l-ISV' will be used in situations equally applicable to both HSV-l and -2 (as before). A. 8 region divergence. Except for two HSV-l UL region genes (UL46, UL56), the remaining 64 all possess an equivalent in VZV (McGeoch et al., 1988). Unlike the L region, which shows a great deal of conservation, alphaherpesvirus 8 regions appear to be much more diverse. This was first observed in DNA-DNA hybridization studies comparing homologies between HSV-l , or HSV-2 and with EHV-l, PRV, and or VZV (Davison and Wilkie, 1983). Despite obvious homologies between their UL regions in a colinear fashion, no homology could be detected between the Us regions of HSV-1 and -2 and their alphaherpesvirus counterpart. Only after adjusting the formamide concentration to 30% and the hybridization temperature to 37 C, could a hybridization signal be observed (between HSV-l and VZV; Davison and Wilkie, 1983). The overall divergence of 47 .39 45mm :05 H058 .33 N59.— 05 30:00:28 3050 5023 0020805 2 0&8 84805054 .885 .40 “0 0:055:89 :05055 gab 05 no :ou00o5 005080 05 958008 3508005. «0058 :058 my? 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Tune? xx 5. “$.«hiéfixlm ' '8 .szo V. . . nNU “tUsI V.>Im v . . . . . .. : .3... t. .N.H.m.....\..w..v. <. 05:119.”? in mm. 00. . . on. ....5? H .1... .( \qyzfl . “mam 33m 33m 8:” “ma” Fmam ./ 1.x. 3‘ 3 . ,, 01.0...- . 28 x: a 4 ”MU nNU Ru" n L . .w : . u 0 m 2. on 50.80: a. u. _ A N F9 :0: _. or “”20: am: 09.00: 3.5: 3.8: mm: 00.3: {.83 «m: «8.5: rrnm Wu H A i. W A J F 0 J 8 ”UH H H Pc>mI .. o— m w a o m e n u No.83 3.5m: 090m: vumOm am: «m: atEOm arm: 5: «atom H A HFH .JJBF NJ. V.FV JH BB m. h m n N OFF >N> >65. mmmeu ZOHUNM m mbgmmmmgmé D72 >02 .mO ZOmEEZOU 49 Us regions is further emphasized by the lack of hybridization between the Us regions of closely-related viruses. Despite widespread hybridization between the genomes of EHV-l and -3 (Baumann et al., 1986) and MDV and HVT (Igarashi et al., 1987), their corresponding Us regions failed to exhibit detectable homology. Sequence analysis has confirmed the diversity characterizing alphaherpesvirus 8 regions as their most divergent coding region (Bnmovskis and Velicer, 1992a; Davison and McCeoch, 1986). Figure 4 illustrates the nature of this divergence. For example, the Us region of HSV-1 is 13 kbp, compared to VZV's which is just over 5 kbp. VZV lacks six HSV-l-related homologs (U82, -4, -6, -6, -11, and -12; Davison and McCeoch, 1986); MDV lacks five such homologs (US4, -6, -9, -11, and -12), yet it contains at least four ORFs not conunon to alphaherpesviruses (Brunovskis and Velicer, 1992a). EI-IV-l contains three such ORFs. The relative position of repeat junctions and homologs can vary greatly (Figure 4). Single-copy Us homologs may be similarly- (e.g. U83, - 4, -6, -7, and 8) or differently arranged (U82, -9) relative to one another. Some may be present either as single-copy Us region genes or as two-copy repeat region genes (U 81, -10). 8 region gene homologies are generally apparent only at the as level. Although conserved amino acid regions are readily detectable, they are often restricted to particular locations. Such homologs often exhibit significant difierences in length, transcriptional characteristics, genomic localization, and functional activity (Figure 4). B. Presence and significance of supplementary essential genes. To understand the nature of viral functions responsible for diflerences in pathologic- and biologic- properties it is necessary to identify and characterize the so-called ‘nonessential’, ‘dispensable’, or supplementary essential genes (for discussion, see Roizman, 1990a). In contrast to minimally 50 . essential genes, which are necessary for replication. packaging and infection of cells in culture, the supplementary essential genes confer functions allowing for the eficient dissemination, growth. and maintenance in various tissues in the face of an immune system poised for its elimination. Achieving such goals has necessitated the development and evolution of elaborate, poorly understood mechanisms to cause irnmunosuppression, avoid immunosurveillance, and allow for the establishment and maintenance of a latent growth state and it subsequent reactivation to facilitate further dissemination to new host. Many of the distinct virus type-specific biological properties are likely to be determined by the supplementary essential genes, whose true function is probably only apparent in viva. While these functions are likely to benefit the firus in furthering it existence, their presence is often associated with marked pathologic consequences to the host. In recent years clusters of supplementary essential genes have been found in the 8 regions of HSV-1 and HCMV Oones et al., 1991; Iones and Muzithras, 1992; Kolbert-jons et al., 1991; Longnecker and Roizman, 1987; Longnecker et al., 1987; Weber et al., 1987). Such clustering can extend to other regions as well (Barker and Roizman, 1990; Baines and Roizman, 1991). Of the 12 HSV-l Us region genes, ll have have been found to be dispensable for growth in cell culture (Longnecker et al., 1987). A viable PRV mutant has recently been created which lacks all four of the known nonessential PRV glycoprotein genes, including three Us region genes (Mettenleiter et al., 1990b). Glycoprotein D (U86) is considered the exception to nonessential Us genes. However, unlike HSV, PRV gD mutant can still grow in a cell-cell manner. (Posters et al., 1992; Rauh and Mettenleiter, 1991). VZV lacks such a homolog altogether. Some ‘dispensable' genes can become ‘essential’ when the function of another nonessential gene is lost. PRV 91 (homologous to HSV gE) was 51 recently shown to be essential for growth in a g!!! (homolog of HSV gC)-minus background (Zsak et al., 1992). Unlike their wild-type parents, HSV Us region gene mutant grow poorly in animal host models and are consistently associated with reduced levels of virulence and/or the capacity to induce latency (Meignier, 1988). Similarly, most (but not all) PRV Us mutant are markedly attenuated and are less able to spread in both pigs and rat (Card at al., 1992; Kimman et al., 1992; Pol et al., 1991). The importance of ‘nonessential’ genes is highlighted by a failure to identify naturally occurring deletion mutant. MD vaccine studies have indicated that protection against tumor induction is largely efiected by neutralizing the initial spread of primary infections. An understanding of mechanisms for dissemination, spread and characterization of tissue-specific factors that efiect these processes are essential if we are to comprehend the pathologic- and biologic properties associated with herpesvims infections. Despite sharing similar as sequences, herpesviruses exhibit strikingly difierent biologic- and pathologic expressions. This is not only true of closely related vimses such MDV and HVT, but is equally applicable comparing HSV-l with -2 (Craig and Nahmias, 1973; Nahmias and Roizman, 1973). If we consider the proposal that the lymphotropic properties of MDV and HVT are unlikely to be determined by molecules homologous to those of EBV (Lawrence et al., 1990), questions arise concerning the nature of genes conferring this ability (as well as it inability to grow in neurons). It has been suggested that ‘the delineation and evolutionary relatedness of genes responsible for biological properties may be a more significant criterion for both evolutionary relatedness and classification than the arrangement and evolution of genes conserved throughout the family Herpesviridae, although they are not yet known’ (Roizman, 1990b). Although such a proposal is clearly debatable, the fact that Us- and 52 other 8 region genes originate from an alphaherpesvirus-specific area exhibiting significant genetic diversity, pathogenic potential, and putative supplementary essential gene functions, together suggest the likelihood of such genes fitting the ‘although they are not yet known’ category above. C. Regulation of herpesvirus gene expression: immediate-early genes. All herpesviruses thus far analyzed undergo a temporal program of coordinated gene expression characterized by the initial expression of immediate-early (IE) (or alpha) gene product critical in triggering the subsequent expression of latter classes of viral product, whose synthesis may lack a dependence on viral DNA replication (early, E or beta) or require it (late, L or gamma) (Honess and Roizman, 1974). Activation of HSV-1 IE genes during productive infections is largely attributed to the activity of the UL-encoded (UL48) gamma product, VP16 (also called Vmw65, a-TIF) which comes already packaged in the virion (Roizman and Sears, 1990). Because of their importance to the coordinate, sequential regulation of herpesvirus gene expression and the control of latency, product and homologs of ICPO, -4, -22, -27, -47 and VP16 have been the focus of numerous studies in recent years. The five IE HSV-l infected cell polypeptides (ICP) and their transcript are listed in Figure 6 and Table 1. Nucleotide sequencing nomenclatures have alternatively referred to «:27, a0, a4, a22, and a47 as UL64, IEllO, IE176, U81, and U812, respectively (McGeoch et al., 1985; McCeoch et al., 1986; McCeoch et al., 1988; Perry and McCeoch, 1988). Similarly, their VZV counterpart (except «247 which is lacking) are represented by genes 4, 61, 62, and 63/70, respectively (Davison, 1986b). The UL-encoded ICP27 homologs bear a distant relation with the EBV BMLFl gene (Davison and Taylor, 1987), in contrast to the other HSV IE genes and their homologs, which appear unique to alphaherpesviruses. 53 FIGURE 8. Map of HSV imrnediate-early mRNAs. A simplified map of the HSV genome and the positions and orientations of the immediate-early mRNAs. From Whitton and Clement, 1984b. TABLE 1. 'l'hetwoperallelnornenclatmeeoftheHSVIBgene Shown are the sizes of the proteins as estimated by polyacrylamide gel electrophoresis in the presence of SDS, the sizes of the primary unmodified amino acid sequence deduced from the DNA sequence and the size (in nucleotides) of the corresponding mRNAs. From Everett, 1987. 54 MAP OF HSV IMMEDIATE-EARLY mRNAs 6 011 ’£077 038 019 To ' 5 u PH _ M . Us EL 4% sun. I n. m fl 4 — l 2 V4 3 I Table l. 11:: [Exam of HSV-1 and rlm'rproduce. fem W 0.5“” ”mm Size on Sis: from Size of Gene Product Gene Procter SOS-PAGE sequels: mRNA tE-t Vmwilo 00 lCPO 110:0 78452 2684 lE-Z Vmw63 021 ICP27 63kt! $5376 1690 15-3 Vmwl‘ls c4 ICP4 l‘lSkd 132833 4259 [24 Vmw68 022 ICP22 68“ 46521 1666 [8-5 Vim-12 047 1C?” 12kt! 9792 1172 55 Figure 5 shows the relative polarity and location of HSV-1 E genes. It is important to emphasize that, except for ICP27, all of the E product have promoters and/or product located in the repeat regions. Considering the enhanced mutability of the repeat regions, this observation underscores the potential for regulatory divergence among the various alphaherpesviruses (see below). Unlike ICP4 and ICP27, the other three E product are produced from spliced transcript. The E-4 and E-6 transcript employ identical promoters located in the IRs and TRs regions (Figure 5). These spliced transcript share a common non-coding exon located in the repeat which is spliced to unique ICP22- or ICP47-specific sequences located at either end of the Us region (Waton et al., 1981; Rixon and Clement, 1982). In contrast to the unspliced ICP4 message of HSV-1, ICP4 transcript of EHV-l and BHV-l are characterized by a complex pattern of splicing and regulation (Harty and O’Callaghan, 1991; Wirth et al., 1991; Wirth et al., 1992). With the exception of ICP47 (Marsden et al., 1982), all of the HSV-l E product are phosphorylated and predominantly localized to the nucleus (for review of properties, see Everett, 1987). Most attention has been focused on ICPO, ICP4, ICP27, and their homologs, as they are known to be involved in the regulation of E, E, and L gene expression. Despite impaired growth under certain circumstances (Post and Roizman, 1981; Longnecker and Roizman, 1986; Sacks and Schaffer, 1987) ICPO, ICP22, and ICP47 have been found to be dispensable for growth in cell culture, in contrast to the ‘essential’ genes which code for ICP4 'and ICP27 (Roizman and Sears, 1990). Since ICPO can trans- activate E promoters (like VP16), it is thought to play an important role during the initial stage of reactivation which occurs in the absence of virion proteins such a VP16 (Cai and Schaffer, 1992). In this way ICPO could be functionally analogous to the EBV ZEBRA product implicated as the key regulator 56 responsible for the shift between latency and lytic growth (Cai and Schafier, 1992; Harris et al., 1989; Miller, 1990). D. Charm (1 S regkm genes. 1. E176 (ICP4). ICP4 is the only ORF located in the IRs/TRs regions of HSV (McGeoch et al., 1986) and is the major regulatory protein involved in activation of beta- and gamma genes, as well as repression of alpha genes, including itelf (for review and references, see Papavassiliou et al., 1991). The HSV-l N- terminal half apparently contains most of the functional domains responsible for nuclear localization, phosphorylation, DNA binding, transactivation, autoregulation (DeLuca and Schaffer, 1988); the role of the C-terminal half is more obscure. The nuclear-localized ICP4 product is found as a homodimeric complex (Metzler and Wilcox, 1986), with dimerization properties that map to it DNA-binding domain (Everett et al., 1991). Extensive posttranslational modifications, including phosphorylation, ADP-ribosylation, adenylation, and guanylation account for numerous bands on two-dimensional gels difl‘ering in apparent molecular weight and charge (Ackermann et al., 1984). Three phosphorylated forms (ICP4a, 4b, and 4c) have been recognized by one- dimensional SDS-PACE (Vthcox et al., 1980; Ackerrnann et al., 1984). Phosphates have been reported to cycle on and 08 ICP4 (Wilcox et al., 1980) and to afiect its interaction with infected- and uninfected cell factors in the activation of beta and gamma genes (Papavassiliou et al., 1991). Other features appear consistent with properties of GTP-binding proteins that function in transcriptional activation (Blaho and Roizman, 1991). HSV-l and VZV ICP4 have reportedly been associated with the tegument of purified virions (Yao and Courtney, 1989; Kinchington et al., 1992) and/or plasma membranes (Yao and Courtney, 1991), suggesting a possible adjuvant to 57 VP16—mediated trans-induction. However, recent evidence has challenged this view, suggesting that ICP4 is instead associated with a novel class of non- infectious enveloped tegument structures lacking capsids or DNA (L particles) common to alphaherpesvimses, including HSV-l (McLauchlan and Rixon, 1992; Sailagyi and Cunningham, 1991). Without exception, ICP4 genes have been found as two copies relegated to the W or Rs regions; these genes and their corresponding polypeptides, ranging in size between 170- and 200-kDa, have been identified for EHV-l (Caughman et al., 1988; Grundy et al., 1989; Roberton et al., 1988; Telford et al., 1992), PRV (Cheung, 1989; Ihara et al., 1983), VZV (Davison and Scott. 1986; Forghani et al., 1990; Shiraki and Hyman, 1987), BHV-l (Wirth et al., 1991), and MDV (R. Morgan, pers. comm). Analysis of the predicted amino acid ICP4 sequences of HSV-1 and VZV have identified five highly-related sequence blocks (McGeoch et al., 1986). These domains are conserved to varying extent in PRV and VZV. EHV-l was found to contain a similar pattern of homology, with a number of clear differences nonetheless (Cnmdy et al., 1989); on the other hand, regions 1, 3, and 6 were found to lack homology with PRV (Cheung, 1989). All of the ICP4 genes are expressed as immediate-early transcript, however, in addition to the single, spliced E 6.0 kb transcript (Harty et al., 1989), EHV-l contains a 3’-coterminal 4.4 kb E transcript synthesized during the E and L stages which encodes a 130—kDa polypeptide in-frame with it larger 200 kDa product (Harty and O’Callaghan, 1991). Interestingly, the BHV-l transcript shares a promoter and a short exon with it ICPO-homologous transcript. However, it coding region is contained within the intron of the latter (Wirth et al., 1991). 58 Although PRV ICP4 is clearly known to trans-activate cellular- and heterologous viral genes, it efiects on PRV gene expression are poorly understood. Transient expression assays have indicated that the ICP4s of EIN- l and VZV can negatively autoregulate their own promoters (Disney et al., 1990; Smith et al., 1992), in addition to activating E-L promoters (Inchauspe et al., 1989; Smith et al., 1992). These are properties shared by HSV’s ICP4. However, unlike HSV, VZV’s ICP4 promoter appears to be negatively regulated by it own ICPO homolog (ORF61; Nagpal and Ostrove, 1991); this lends further support to the view that mechanisms for controlling latent VZV infections differ from those of HSV (Croen et al., 1988). 2. U81 (ICP22). In contrast to ICP4, the roles of ICP22 and ICP47 are more obscure. ICP22 homologs exhibit significant difl'erences in length, a conservation, and transcriptional kinetics. Compared with the ICP22 homologs of MDV (Brunovskis and Velicer, 1992a; Ross et al., 1991b), VZV (Davison and Scott, 1986), PRV (Zhang and Leader, 1990), and EHV-l (Holden et al., 1992a; Telford et al., 1992), HSV-l ICPZZ has approximately 100 additional as at it amino-terminal end. Length divergences are particularly pronounced in comparing the 179 aa ICP22 ORF of MDV with the 481 aa ORF of HSV-1. Apart from the others, PRV ICP22 contains a remarkable C-terminal acidic region in which nearly three-quarters of the 138 residues are either aspartic- or glutamic acid (Zhang and Leader, 1990). In contrast to their E HSV-1 counterpart, ICPZZ homologs of MDV, EHV-l , PRV, and BHV-l exhibit strikingly difierent patterns in their expression kinetics. MDV U81 encodes an abundantly expressed 27-kDa late class cytoplasmic phosphoprotein, pp27 (Brunovskis and Velicer, 1992b). PRV has only one E gene which codes for it ICP4 homolog (Cheung, 1989); failing to find immediate-early regulation of PRV U81 was therefore not a 59 surprise (Zhang and Leader, 1990b). The bovine- and equine ICPZZ homologs are apparently regulated at two difierent levels. BHV-l ICP22 is an abundantly expressed 66-kDa phosphoprotein, regulated at both E and late times (M. Schwyzer, pers. comm); EHV-l ICP22 is expressed from both early and late promotors, resulting in independent transcript which could code for proteins of 293- or 469 aa, respectively (Holden et al., 1992a). These recent observations appear consistent with a much earlier prediction (lioness, 1984) invoicing the recombination/isomerization process to account for the possible dislocation of normal regulatory element from genes (or their homologs) with transcript crossing the boundaries of the repeat-unique junctions (e.g. a22, a4?) thereby altering their normal gene regulation. While MDV’s U81 is located entirely in it Us region, PRV, EI-IV-l, BHV-l, and VZV U81 genes are found as two copies in the adjoining repeat (Figure 4). The above discrepancies in gene regulation kinetics are reminiscent of similar findings for other genes of EHV-l (Harty and O’Callaghen, 1991), PRV (Cheung, 1991), BHV-l (Wirth et al., 1992), and HCMV (Stenberg et al., 1989). Although the function of U81 family genes is not yet clear, a number of interesting findings have emerged. Some of these suggest ICP22 encodes a determinant for tissue tropism. This is based on two observations. First, while ICPZZ is dispensable for growth in some cell lines, ICP22 mutant grow very poorly in others. This led to the suggestion that certain cell lines (e.g. Vero) contain a host ICP22-like function which can allow for the growth of the mutant (Sears et al., 1986). These observations appear consistent with the fact that ICP22-less mutant are less vinilent or capable of establishing latency in mice (Meignier et al., 1988; Roizman et al., 1982). It should be pointed out that although HSV ICP22 is considered ‘nonessential’ for growth in cell culture, such a conclusion is based on the use 60 ofa mutant which maintains the ability to express the NHz-terminal 200 as of HSV-1’s ICP22 (Ackermann et al., 1986; Post and Roizman, 1981; McCeoch et al., 1986). This result in the expression of a 33.7-kDa phosphoprotein in I'Ep-2 cells and at least three distinct 33.7-39-kDa phosphoproteins in BHK cells (Ackermann et al., 1986). Like the full-length ICPZZ, the truncated polypeptides were similarly phosphorylated and localized to the nucleus. These truncated derivatives may be relevant to cell culture growth in view of the report that an oligo(nucleoside methylphosphonate) dodecamer derivative complementary to the splice junction of the ICP22 mRNA 4 eflected a 98% decrease in HSV-l titers with little, if any, deletarious effect on host-cell macromolecular metabolism (Kulka et al., 1989). The functional nature of ICP22 homologs may relate to gene regulation. HSV-l ICPZZ is a predominantly nuclear protein (Fenvvick et al., 1978; Fenvvick et al., 1980) which is known to associate with chromatin (Hay and Hay, 1980). An HSV-l ICPZZ mutant has been characterized as defective for late gene expression (Sears et al., 1986). In another study, use of a temperature sensitive mutant in rat XC cells at the non-permissive temperature resulted in a severe reduction in L protein synthesis; this defect was correlated with a lack of ICP22 protein expression (Epstein and Iacquemont, 1983; Jacquemont et al., 1984). Although it is still too early to say whether ICP228 have any particular preference for activation or repression, BHV-l ICP22 has been recently found to express a 66-kDa phosphoprotein inhibiting target promoters of all kinetic classes tested (M. Schwyzer, pers. com.). The VZV ICP22 protein has been reported to repress the E promoter of it ICP4 homolog, but stimulate the E promoter of it TK homolog (Jackets et al., 1992). The consequences of this repression in VZV are as yet unclear, however, recent evidence suggest a potentially important role in the maintenance of a 61 latent growth state. By in situ hybridization, VZV gene 63 transcript have been found in latently-infected neurons (Vafai et al., 1988a) or in neurons infected with VZV in vito (Merville-Louis et al., 1992). ICPZZ-homologous sequences in HIV- 1 defective interfering particles (DIPs) may potentially contribute to the oncogenic transformation and persistence associated with hamster embryo fibth infections. Interestingly, a unique recombination event has been reported to juxtapose a majority of EHV-l’s ICP22 ORF in-frame with a potential zinc-finger motif derived from the C-terminal portion of it ICPZ7 homolog (Holden et al., 1992a; Yalamanchili et al., 1990). 3. U82. The role of this gene is completely obscure. U82- homologous genes are known to be encoded by HSV-1 (McGeoch et al., 1986), HSV-2 (McGeoch et al., 1987), VZV (Davison and Scott, 1986b), PRV (van Zijl et al., 1990), EHV-l (Breeden et al., 1992; Colle et al., 1992; Telford et al., 1992), and MDV (Cantello et al., 1991, Ross et al., 1991, Bnmovskis and Velicer, 1992a). These are the only group of 8 region homologs with a highly conserved N- terminus; all of which begin with the sequence M-C—V contained within a notably hydrophobic N-terminal region, possibly a signal peptide for membrane- bound translation (McGeoch et al., 1986). The highly conserved glycine residue may be a target for myristylation (Towler et al., 1987). A myristilated hydrophobic region might be expected to play a role in mediating transient membrane interactions (Schultz and Oroszlan, 1984). Specific immunological reagent have been created and used to identify the 28- and 29-kDa U82 polypeptides of PRV (van Zijl et al., 1990) and MDV (Bnmovskis and Velicer, 1992c). However, further information regarding their functional or structural nature is lacking. 62 Specific U82 mutant, have been generated for HSV-l (Weber et al., 1987), PRV (de Wind at al., 1990), and MDV U82 (Cantello et al., 1991); in all three cases U82 was found to be dispensable for growth in cell culture. Unlike the other Us mutant, U82 mutant have thus far been found to maintain their virulence in animal host. HSV-1’s U82 mutant failed to exhibit a significant loss in the ability to replicate in the central nervous system of mice (Weber et al., 1987); PRV’s U82 mutant was found to maintain a comparatively normal level of virulence in its natural pig host (Kimman et al., 1992). 4. U83 (PK). Protein kinases (PK) constitute a large and diverse family of enzymes difiering in their substrate specificities, regulatory control, and the amino acids that they phosphorylate. Nevertheless, all eukaryotic PKs thus far studied show similarities in a clearly identifiable domain of about 250 as, which contains a number of conserved subdomains important for catalytic functions (Hanks et al., 1988). McGeoch and Davison (1986) were the first to note that HSV-l (US3. McGeoch et al., 1986) and VZV (gene 66, Davison and Scott, 1986b) shared a common gene that was related to members of the protein kinase family. Additional U83 PK—homologous genes have since been identified in HSV-2 (McGeoch et al., 1987), PRV (van Zijl et al., 1990; Zhang et al., 1990), EHV-l (Colle et al., 1992; Telford et al., 1992), and MDV (Bnmovskis and Velicer, 1992a; Ross et a1, 1991). While all of these share sequences corresponding to the predicted catalytic domain, each contain an unrelated 100- 200 aa NHz-terminal region. Because these homologs are an integral and evolutionarily conserved part of their respective genetic repertoires (in contrast to oncogenic retrovims PK genes), Leader and Purves (1988) characterized the U83 homologs as the first authentic eukaryotic viral PK genes described to date. 63 Further inspection has revealed that U83 PK-homologous members contain sequence motifs characteristic of serine-threonine PKs (Leader and Purves, 1988). The U83 PK family appears to define a distinct subfamily within the serine-threonine protein kinase superfamily. It is thought that related cellular counterpart exist and await future characterization (Hanks et al., 1988). Interestingly, one of three MDV U83—directed antisera was recently found to irnmunoprecipitate a 68-kDa cellular protein (Brunovskis and Velicer, 1992c). The interaction appeared to be specific, insofar as the fusion protein immunogen used to generate this antisera was able to block this precipitation. Further work will be necessary to determine whether this cellular protein is in fact a cellular counterpart to the U83 family of PKs. The PRV and HSV-l PKs were previously discovered in cytoplasmic extract of infected hamster fibroblast and distinguished from known cellular protein lo'nases (Katan et al., 1986; Purves et al., 1986a). It enzyme was purified to near- or complete homogeneity (Purves et al, 1987a; Frame et al., 1987) and shown to have the following characteristics. (1) Constitutive activity, requiring no efiector; (2) Autophosphorylation capabilities (Purves et al., 1987a; Frame et al., 1987); (3) A high KCL concentration optimum (Katan et al., 1985; Purves et al., 1986a); and (4) Transfer of phosphate from ATP to seryl or threonyl residues present in basic (but not acidic) artificial substrates (such as protamine) and in synthetic peptide substrates containing several arginyl residues on the NHz-terminal side of serine or threonine (Katan et al., 1986; Purves et al., 1986b; Leader et al., 1991). Evidence that the HSV-l U83 gene codes for the HSV-l PK enzyme above was based on studies showing that; (1) an HSV-1 mutant with a U83 deletion did not exhibit the PK activity above (Purves et al., 1987b) and (2) an anti-peptide antisemm directed against a C terminal octapeptide of the U83 ORF was found to react with highly purified PK 64 preparations from HSV-l-infected cells (Frame et al., 1987). Using an antiserum raised against a PRV PK fusion protein expressed in E. coli, Zhang et al. (1990) confirmed the cytoplasmic localization of PRV and HSV PK, and further demonstrated it presence in purified PRV and HSV-l virions of which the former was shown to phosphorylate the major 112-kDa PRV virion phosphoprotein in vitro. The antisera directed against HSV-l PK was also found to precipitate a 68/69 kDa doublet, a result analogous to the smaller PK doublet identified for MDV (Brunovskis and Velicer, 19920). Purves et al. (1991) have recently identified an apparently essential virion- containing phosphoprotein encoded by the UL34 gene (McGeoch et al., 1988; Purves et al., 1991) which is posttranslationally modified by HSV-l PK. Evidence was based on the fact that site-specific mutagenesis of serine or threonine residues present in a potential U83 PK consensus target site (Leader et al., 1991; Purves et al., 1986b) led to the replacement of the 30-kDa wild-type UL34 phosphoprotein with a slower-migrating 33-kDa polypeptide of similar mobility to a novel product identified in HSV-1 PK' infected cells. Mutant altered in the PK consensus site were further characterized by greatly impaired growth properties. ‘ Although HSV-l and PRV PK mutant are known to grow in cell culture, altered or impaired growth properties have been cited in both cases (de W'md et al., 1990; Purves et al., 1991). PRV and HSV-l PK mutant also show a significantly decreased vimlence in pigs (Kimman et al., 1991) and mice (Meignier et al., 1988), respectively. Although the PRV mutant did not appear to be adversely affected in it tissue tropism, it was found to display an altered morphogenesis, possibly contributing to its lower replication in vitro (de Wind et al., 1990) and in vivo (Kirnman et al., 1992). 65 6. U84 (g6). Reflecting their close evolutionary relationship, HSV-l and -2 Us homologs share as sequence identifies of 70-80% (McGeoch et al., 1987). Their corresponding gG polypeptides exhibit pronounced serotype-specific difierences. These difierences provide the basis for serological approaches which facilitate discrimination between infections of these two serotypes (Sanchez-Martinex et al., 1991). Two groups (Marsden et al., 1984; Roizman et al., 1984) originally identified and mapped (to the Us region) HSV serotype-2- specific glycoproteins with apparent molecular masses of 92,000 and 124,000, respectively. These polypeptides were subsequently found to represent identical glycoproteins (Balachandran and Hutt-Fletcher, 1986); the above size discrepancies were attributed to difierences in the cross-linking agent used in the two gel systems. Soon thereafter, a smaller glycoprotein was mapped to a similar location in the HSV-l genome; this suggested that the new glycoprotein was the HSV-l equivalent of gC-2 (Ackermann et al., 1986; Richman et al., 1986). Sequence analysis of the HSV-2 U84 gene, in conjunction with the generation and use of a serotype-common anti-peptide antiserum confirmed this proposal and showed that gC-l had approximately 400-600 a deleted relative to gG-2 (McGeoch et al., 1987). Sequence conservation is mainly limited to the C- terminal portions of their respective proteins. Additional gC homologs have been sequenced and mapped to the Us regions of PRV (Rea et al., 1986) and HIV-1 (Colle et al., 1992; Telford et al., 1992). In both cases, the conserved regions contain sequences homologous to the 96-2 specific portion. MDV (Bnmovskis and Velicer, 1992) and VZV (Davison and Scott, 1986b) appear to lack gG-l or gG-2 counterpart. PRV (Rea et al., 1986) and HSV-2 (Su et al., 1987) ng are known to undergo proteolytic processing event which result in the secretion of partial 66 (HSV-2)- or nearly full-length (PRV) product. However, nothing is known about these or other alphaherpesvirus gG proteins. HSV-l (Weber et al., 1987) and PRV (de Wind at al., 1990; Mettenleiter et al., 1990b) gG mutant are not impairsdintheirabilitytogrowincellcflture. WhileanI-ISV-l gGmutantdid show a reduced ability to replicate in the central nervous system of rat (Weber et al., 1987), PRV gC mutant appeared to exhibit normal virulence in both mice (Thomsen et al., 1987) and pigs (Kimman et al., 1992). From an immunological perspective, anti-PRV gC antibodies failed to protect animals against lethal challenge (Thomsen et al., 1987), while vaccinia-gC-l recombinant failed to induce a detectable neutralizing antibody response (Blacklaws et al., 1990). 6. U88 (g1). The USS gene encodes a 92-aa ORF, apparently specific to HSV-l and -2 (McGeoch et al., 1986; McCeoch et al., 1987). The predicted a sequence has features characteristic of membrane-bound glycoproteins; it putative polypetide has recently been designated as g], although details of it characterization have not yet been published. Tnseinserted HSV-l U86 mutant have been reported to be unaffected for replication in cell culture or in mouse central nervous system (Weber et al., 1987). 7. U86 (gD). Glycoprotein D (U86) is considered the only Us region polypeptide essential for replication in cell culture. This reflect it obligate role in vian-cell penetration, initially demonstrated by work showing that; (l) anti-gD neutralizing antibodies monoclonal antibodies (MAbs) permit adsorption but inhibit penetration (Fuller and Spear, 1987; Highlander et al., 1987); and (2) an HSV mutant in which gD sequences are replaced by B-galactosidase sequences binds to, but is unable to penetrate into cells (Ligas and Iohnson 1988). A similar role in penetration has since been demonstrated for the gD 67 homologs of EHV-l (Whittaker et al., 1992), BHV-l (Fehler et al., 1992), and PRV (Rauh and Mettenleiter, 1991; Peeters et al., 1992). In spite of its importance to vims-cell penetration, PRV’s gD is dispensable for cell-cell spread (Peeters et al., 1992; Rauh and Mettenleiter, 1991). VZV lacks such a homolog altogether. All other alphaherpesviruses thus far analyzed encode gD homologs, including HSV-l (McGeoch et al., 1985), HSV-2 (McGeoch et al., 1987), PRV (Petrovskis et al., 1986a), EHV-l (Audonnet et al., 1990; Flowers et al., 1991; Telford et al., 1992), BHV-l (Tikoo et al., 1990), and MDV (Brunovskis and Velicer, 1992a, Ross et al., 1991b). Interestingly, MDV has an intact gD ORF that does not appear to be expressed in cell culture (Bnmovskis and Velicer, 19920). On the other hand, FFE cells, which support fully-productive MDV infections do appear to express this polypeptide (R. Witter, pers. comm). These observations suggest that the cell-associated behaviour of MDV and VZV may be attributed to a lack of gD expression. Further support for this hypothesis is based on findings which show that HSV-I gD contains a domain which restrict fusion of the envelope with cytoplasmic membranes (Campadelli-I-‘iume et al., 1990). Alteration of this domain promotes cytoplasmic deenvelopment of vinis upon egress, resulting in the accumulation of unenveloped capsids alone or juxtaposed to cytoplasmic membranes (Campadelli-Fiume et al., 1991). Fibroblast cell cultures infected with MDV also accumulate unenveloped capsids (Nazerian et al., 1968), possibly reflecting an inability to prevent deenvelopment. An interesting property of cell surface-expressed gD is it ability to render permissive cells resistant to further infection by homologous- or heterologous alphaherpesviruses (Chase at al., 1990; johnson and Spear, 1989; Petrovskis et al., 1988). Such an interference phenomenon appears to represent an economical strategy for eficient viral dissemination. Iohnson et al. (1990) 68 suggested that the interference is due to the sequestration of host receptors necessary for mediating subsequent entry. This view has been disputed by others who argue that the restriction is conferred by a specific domain within gD itelf (Campadelli-Fiume et al., 1990). Glycoprotein D is a major structural envelope component for HSV-1, -2. PRV, BHV-l, and EHV-l. This may account for the fact that it represent a primary immunogen and target for neutralizing antibodies in these different systems (Eloit et al., 1988; Para et al., 1986; Whittaker et al., 1992). In one study (Para et al., 1985), all 33 of the MAbs found to bind to purified virions of HSV were shown to immunoprecipitate one of five glycoproteins; all six MAbs exhibiting potent neutralizing activity were gD-specific. Two other anti-gD antibodies and the 26 others specific for either gB, gD, gE or gC had much less potent, if any, neutralizing activity. Anti-gD MAbs have been found to block both virus penetration (Highlander et al., 1987) and virion-induced cell fusion (Noble et al., 1983). Cattle or pigs with purified BHV-l gD (Babiuk et al., 1987), PRV gD (Marchioli et al., 1987) or PRV gD-directed monoclonal antibodies (Marchioli et al., 1988) were found to be protected from disease. These result account for a great deal of interest in the use .of gD as a potential subunit vaccine. gDs are also important for inducing cellular immune responses. BHV-l gD appears to be the most important glycoprotein antigen responsible for such responses (Hutchings et al., 1990). Virus vaccine vectors have increasingly gained favor in recent years because of their added ability to induce CD4+- and CD8+-mediated T-cell responses. Compared with other glycoprotein- expressing vectors, gD-expressing vaccinia virus recombinant were reported to induce the best neutralizing antibody titers, the best clearance of HSV from infected ears, protection from the establishment of latency in the sensory 69 ganglia following lethal doses of HSV (Blacklaws et al., 1990). Similar studies employing vaccinia \n'rus-PRV recombinant showed that mice could be protected against lethal challenge following expression of PRV gD, gB or gC homologs. However, protection in their natural host required co-expression of PRV glycoproteins; PRV gD and gB were particularly effective (Riviera et al., 1992). The latter study raises concerns about overinterpreting the significance of result obtained using non-natural host models. 8. U87 (gl) and U88 (g3). These two glycoproteins have been grouped together to reflect their close association with one another. At present, sequences have been obtained for HSV-l (McGeoch et al., 1985), HSV-2 (McGeoch et al., 1987), VZV (Davison and Scott, 1986), PRV (Petrovskis et al., 1986b), EHV-l (Audonnet et al., 1990; Elton et al., 1991; Telford et al., 1992), EHV-4 (Cullinane et al., 1988), and MDV (Bnmovskis and Velicer, 1992a; Ross et al., 1991b). Both glycoproteins have been found to coprecipitate as a complex in HSV (Johnson and Feenstra, 1987; Johnson et al., 1988), PRV (Zuckermann et al., 1988), VZV (Vafai et al., 1988b; Vafai et al., 1989), and MDV (Chen et al., 1992) systems; in the case of VZV, these two glycoproteins are reported to share a common epitope (Vafai et al., 1988b; Vafai et al., 1989), consistent with the recent isolation of CD4-positive T-cell clones that can lyse target cells expressing either one of these glycoproteins (Huang et al., 1992). VZV and HSV gl/gE complexes have been shown to act as Fc receptors (Johnson et al., 1988; Litwin et al., 1992). The HSV-l Fc receptor (FcR) can exist in two forms. The gI/gE complex has a particular specificity for monomeric IgG; gE alone, for lgC complexes (Dubin et al., 1990). The HSV-1 FcR has been shown to utilize an antibody bipolar bridging mechanism (Frank and Friedman, 1989) to protect HSV-infected cells from antibody-dependent cellular cytotoxicity 70 (Dubin et al., 1991). MDV and PRV studies have thus far failed to associate FcR activity with gI/gE (Chen et al., 1992; Zuckermann et al., 1988). In contast to the FcR activities of HSV and VZV, the PRV gI/gE complex ha been shown to possess a function deleterious for growth in some cell types, but not others; serial passage of PRV in CEFs invariably select for gI and/or gE mutant which have a growth advantage not found in rabbit kidney cells (Mettenleiter, 1988b; Zuckermann et al., 1988). In conjunction with unknown cellular functions, these mutant exhibit an enhanced ability for virus release (Zsak et al., 1989). Interestingly, nearly all of the attenuated vaccine stains used against PRV are derived from genetically engineered stains lacking g1 or cell culture- attenuated stains (such as Bartha or Norden) which have undergone spontaneous deletions involving 91 (Wittmann and Rziha, 1989). Extended cell culture passages of MDV and EHV-I have also been associated with deletions and/or rearrangement involving the gI/gE region (Colle et al., 1992; unpublished observations; H.-J. Kung, pers. com). The latter is exemplified by the EHV-l Kentucky A cell culture stain which contains a 3.9 kb deletion of Us sequences coding for gl, gE, and an additional 130 aa ORF (Figure 4). To understand the nature of attenuation as it relates to gl/gE, it is necessary to consider their flmction apart from a possible role in immunoevasion. PRV’s gI/gE region has been implicated as a marker for neurovinilence prior to any knowledge of it genetic sequence properties (Lomniczi et al., 1984; Berns et al., 1986). These observations have been recently confirmed by work showing that a specific deletion of PRV gE significantly reduced the spread of infection in both rat (Card et al., 1992) and pig (Kimman et al., 1992) cental nervous systems. It has been suggested that the neuroinvasive attibutes of PRV gE result from cell-specific differences in 71 virus recognition mediated by distinct gE-cell receptor interactions (Card at al., 1992). Another study has correlated the presence of gE with the ability to spread in the nasal mucosa and to promote nuclear- rather than cytoplasmic envelopment (Pol et al., 1991). Inactivation of gl or gE is known to stongly reduce virulence in pigs (Kimman et al., 1992). In contast to pigs, PRV gl-, gE-, or gC-homolog mutant were found to essentially retain wild-type vinllence levels in l-day old chickens. on the other hand, gI/gC or gE/gC double mutant were found to have an ' avirulent phenotype in chickens (Mettenleiter et al., 1988a). To understand the nature of these result, it is necessary to consider recent findings which have provided important clues about the particular roles that supplementary essential genes play in the context of the host. Like gI/gE, gC is considered dispensable for growth in cell culture (Roizman and Sears, 1990). It chief role is associated with virion-cell attachment (Herold et al., 1991; Mettenleiter et al., 1990a; Okasaki et al., 1991). Although the role of 91 is not yet clear, recent evidence has implicated gE homologs in cell-cell spread (Chatterjee et al., 1989; Zsak et al., 1992). In the latter study PRV gE’ mutant were found to primarily spread by the release of vims and it subsequent gC-mediated adsorption; in contast, gC' mutant were found to spread by cell-cell tansfer. Based on these observations it was argued that the avinllence associated with the gC/gl double mutant was attibuted to their inability to spread by either of these two mechanisms. Further support was provided with the observation that mice passively immunized with mouse anti-PRV fared better against challenge with gE‘ rather than gC" mutant; this is consistent with the fact that cell-cell tansfer is generally less sensitive to the effect of neutalization (Ahmed and Stevens, 1990). Since MDV is mainly disseminated by cell-cell spread, such findings 72 raise concerns about overinterpreting the significance of virus neutalization result which involve cell-free virus. In animal systems, g1 and gE (and other Us genes) have been targeted as insertion sites in the creation of attenuated live vaccines (Quint et al., 1987). Pigs were found to be completely- (gE) or partially (gI) protected from lethal challenge following vaccination with these two mutant (IGmman et al., 1992). Human systems have examined gI/gE as potential subunit vaccines. Mice were recently shown to be protected from lethal challenge following vaccination with baculovirus-expressed HSV-1 gI (Chiasi et al., 1992a) or gE (Chiasi et al., 1992b). . gE is the most prominent antigen of VZV and is known to stimulate hurnoral and cell-mediated immunity and provide protection against lethal VZV challenge and establishment of latency in animals (Arvin et al., 1987). Much attention has been focused on cell-mediated immunity (CMI), partly because protection against VZV infections fail to correlate with the presence or level of specific glycoprotein-directed antibodies (Bnmell et al., 1987). CMI is likely to be of particular importance for protection against cell-associated herpesvinlses, such as MDV and VZV. This is emphasized by the finding that bursectomy of 1- day old chicks was not found to influence the immunity conferred by an attenuated MDV stain (Else, 1974). g1 and gE may be important for CM! against MDV and VZV. Both of these glycoproteins have been reported to stimulate CD4+ and CD8+ T-cell clones reactive against VZV-infected cells (Arvin et al., 1991; Huang et al., 1992; Yasukawa and Zarling, 1986). HSV and VZV gE homologs are known to undergo extensive posttranslational processing. Both contain N- and O-linked glycans and are phosphorylated, palmitylated, myristylated and sulfated (Crose, 1990; Harper and Kangro, 1990; Spear, 1984). Little is known about the ftmctional significance 73 of these processing event. Recent evidence suggest that the phosphorylation of VZV gE may mediate an interaction between VZV and mannose 6-phosphate host cell receptors that is responsible for withdrawal of newly synthesized virions tom the secretory pathway and their diversion to prelysosomal structures where the viral envelope is disrupted (Cabal, 1989). This was ofiered as a possible rationale for VZV’s cell-associated nature; a similar possibility might have important implications for MDV’s strict cell-associatedness. 9. U89 (10D. HSV-1 U89 is predicted to encode a 10-kDa product (McGeoch et al., 1986). Anti-peptide antisera directed against HSV-l U89 has been found to precipitate at least 12 related electrophoretically-distinct polypeptides ranging between 12-20-kDa in size (Frame et al., 1986). The various forms of 10K difiered in abundance and phosphorylation; the lower MW forms were precipitated from virions, probably as tegument proteins associated with nucleocapsids during, or shortly following, their tanslocation to the nuclei of infected cells (Frame et al., 1986). The consequences of this association may be related to envelopment at the nuclear membrane. P01 et al., (1991) observed a correlation between envelopment at the nuclear membranes and the presence of PRV’s U89 and gE product. Although the significance of herpesvirus envelopment at nuclear membranes has recently been questioned (Rixon et al., 1992; Whealy et al., 1991), it is interesting to note that MDV appears to lack a U89 homolog and has previously been reported to undergo cytoplasmic envelopment in the feather follicle epithelium (Johnson et al., 1976). Other hornologs have been identified in PRV (Petovskis, 1987), EI-IV-l (Elton et al., 1991; Telford et al., 1992), and VZV (Davison and McCeoch, 1986; Davison and Scott, 1986) 74 10. U810. HSV-1 U810 codes for a 33-kDa polypeptide identified by hybrid-selection and cell-free tanslation (Lee et al., 1982; Rixon and McCeoch, 1984). It corresponding mRNA is initiated within the coding region of U811 and is translated in a leftward direction, such that it contains an unusual 110 codon out-of-frame overlap with the 3’ end of U811 (Rixon and McCeoch, 1984). It has been cited as a virion protein (McGeoch et al., 1988), although no evidence of this has yet been published. A number of HSV-1 and -2 mutant with a deleted U810 (in addition to others) have been isolated and shown to promote viral growth . in vito (Brown and Harland, 1987; Longnecker and Roizman, 1986; Umene, 1986). Homologs are known to be encoded by EHV-l (Holden et al., 1992b; Telford et al., 1992), EHV-4 (Cullinane et al., 1988) and MDV (Brunovskis and Velicer, 1992a). In examining PRV’s 8 region gene organization (Figure 4), it appears unlikely that a U810 homolog will be found. Based on their analyis of a region highly conserved among alphaherpesvimses U810 homologs, Holden et al., (1992b) have identified a potential zinc-finger domain, possibly implicating a role in gene regulation. MDV U810 has been found to express a 26-kDa phosphoprotein that appears to be translated from an unusual bifunctional message apparently responsible for the expression of the 27-kDa MDV U81 homolog. as well (Bnmovskis and Velicer, 19920). HVT appears to lack a U810 homolog (Brunovskis and Velicer, 19920; M. Wild, pers. com.). 12. U811. This gene codes for an extemely basic 21-23-kDa polypeptide, originally identified by hybrid-selection (Lee et al., 1982; Rixon and McCeoch, 1984). As noted above, it C-terminal portion overlaps out-of-frame with the N-terminal region of the 33-kDa U810 polypeptide and has been further noted to contain 24 tandem repeat of the tipeptide X-P-R (Rixon and 75 McCeoch, 1984). Except for HSV-l and -2, homologs have yet to be identified elsewhere. This gene has been considered a tue late gene (gamma-2), whose expression is stringently dependent on viral DNA replication (Johnson et al., 1986). Although it function appears dispensable for growth in cell culture (Brown and Harland, 1987; Longnecker and Roizman, 1986; Umene, 1986), recent studies have uncovered some interesting observations regarding it functional nature. The U811 product was originally considered a nucleolar DNA-binding protein (MacLean et al., 1987) thought to bind to ‘a’ sequences (Dalziel and Marsden, 1984). Recent evidence indicates that the U811 product is in fact a sequence-specific RNA-binding protein (Roller and Roizman, 1990) that negatively regulates the accumulation of a tuncated tanscript coding for an essential protein encoded by UL34 (Roller and Roizman, 1991). Additional evidence has identified U811 as a \n'rion product with a potential regulatory role that stems from a specific association with 608 ribosomal subunit (Roller and Roizman, 1992). 13. U812. Little is known about the role of the immediate-early ICP47 product. Except for HSV-2, homologs have not been identified in other systems. It product appears to be dispensable for growth in cell culture (Brown and Harland, 1987; Longnecker and Roizman, 1986; Mavromara-Nazos et al., 1986; Umene, 1986). The polypeptide is is not phosphorylated and is localized to the cytoplasm (Marsden et al., 1982). Transient expression assays have thus far failed to identify a role in gene regulation. 76 14. Non-HSV-rslated 8 region genes. At least four non-HSV-related genes have been identified in MDV (SORFI, -2, -3, and -4; Figure 4; Brunovskis and Velicer, 1992a). Close inspection of their predicted amino acid sequences fail to have provided any obviolt stuctural clues regarding their nature or flmction. SORF3 was found to exhibit significant homology to an uncharacterized fowlpox virus ORF (ORF4; Tomley et al., 1988); the other three ORFs failed to exhibit significant homologies to any protein sequences in the current databases (Bnmovskis and Velicer, 1992a). Gene product for these ORFs are yet to be characterized. Non-HSV-related genes have been identified in EHV-l as well. These correspond to genes 67/77, 71, and 76 (Figure 4; Telford et al., 1992). Gene 71 encodes a particularly interesting ORF. Located at a position corresponding to HSV-l g], the 797-aa ORF 71 contains features characteristic of glycoproteins (signal peptide, tansmembrane domains, glycosylation sites etc.), yet it lacks any detectable relatedness to the smaller, 92 as g] sequence. It sequence is identical to the 383-aa EUS4 ORF of the Kentucky A cell culture stain (Colle et al., 1992) at both N- and C-terrnini; however, compared to EUS4, the corresponding ORF of the Ab4p field isolate stain contains a unique 1242-bp in- frame insertion following position 74. It would appear that these sequences (like g1 and gE) have been deleted from the Kentucky A stain following extensive serial passages. The additional 414-aa are particularly rich in serine, threonine, and alanine residues. Some of the threonine-rich sequences derive from a 226-bp region containing two different 15-bp element repeated 6-7 times each. Mainly due to their threonine/serine/alanine-rich nature, homology searches yield several high FastA scores (over 60 scores between 100-380) that primarily reflect homologies between the additional sequences and a diverse set 77 of cellular proteins; such diversity complicates any obvious or meaningful functional predictions. Gene 67/77 is present as two copies in the repeat regions and has been alternatively referred to as IRB (Breeden et al., 1992; Holden et al., 1992b; Figure 4). Gene 76 encodes a 130-aa ORF identified by two labs (Eton et al., 1991; Telford et al., 1992) which has been deleted from the Kentucky A stain (Figure 4). ' ORF 76 contains residues highly conserved in EHV-4, however, the loss of one nucleotide in E-IV-4 (relative to EHV-l) would lead to premature tuncation (relative to EIV-l ORF 76) resulting in a smaller 77-aa ORF. Leaving out a nucleotide in their original EHV-4 sequence (Cullinane et al., 1988) would theoretically lead to a 123-aa sequence that is 61% identical to the 130-aa ORF 76 sequence. Neither of these latter ORFs (67/77 or -76) contain any obvious stuctural features that would predict a possible ftmction. E. In condition. The broad nature of this literature review provides a useful framework for thinking about MDV as an alphaherpesvirus. From this we can see that more genetic- and biological parallels appear to exist between the oncogenic MDV and other alphaherpesvinls members than perhaps previously thought. Alphaherpesvinls 8 region genes code for product potentially responsible for specifying the varied biological properties which distinguish alphaherpesviruses from one another and from those of other phylogenetic lineages. Moreover, they offer a particularly useful model system for studying virus-cell interactions which naturally occur in nature. 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P., T. Mettenleiter, C. Schreurs, N. Sugg. and T. Ben-Pant. 1988. Complex between glycoproteins g1 and gp63 of pseudorabies virus: it efiect on vinis replication. J. Viral. I:4622-4626. surHaiisen.H.I‘I.Schulte-l-Iolthaiisen. G.Klein.W.Hen1e.P.Cliflord.andL. Saritessari. 1970. EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature 228:1056. Cluptrll StuchnalclnractdsafianaffinMamk’sdteasevhuMmiiquashat region: mdalplulnrpesvhinJiamologmn,bwlpaavhit-hamologommdm- spacificgenes Submitted for publication. 101 ABSTRACT Despite it classification as a garnmaherpesvirus, primarily due to it lymphotropism. Marek's disease vinis (MDV), an oncogenic avian herpesvirus, is phylogenetically more related to the "neurotopic" alphaherpesviruses, characterized by it prototype. herpes simplex virus (HSV) (Buckmaster et al., 1988. J. Gen Virol. 69:2033-2042). This raises interesting questions regarding the seeming incongruence between MDV’s genetic- and biologic properties. Alphaherpesvirus Us- and other S region genes originate from an area specific to members of this group, arguably their most divergent coding region. Moreover, this area contains a cluster of glycoprotein genes potentially important in protective immunity and possesses supplementary essential functions presumably evolved for adaptation to unique and diverse cell environment. In this report we present the nucleotide sequence of an 11,286 base pair DNA segment containing MDV's entire 11.160 bp long Us region C'vinilent" GA strain; vMDV). Eleven open reading frames (ORFs) likely to code for proteins were identified; of these, 7 represent homologs exclusive to alphaherpesvinis 6 component genes. These include MDV counterpart of HSV U61 (ICP22), U62, U63 (a serine-threonine protein kinase), U66, U67 and U68 (HSV glycoproteins gD, g1 and gE, respectively) and U610. Three additional ORFs were identified with no apparent relation to any sequences currently present in the SwissProt or GenBank/EMBL databases, while a fourth was found to exhibit significant homology to an uncharacterized fowlpox vinis (FPV) ORF. Having precisely identified the IRs-Us and Us-TRs junctions, we have corrected and clarified their previously reported locations. By characterizing genes encoding three new alphaherpesvirus-related homologs (U 61, U68 and U610), completing the sequence for a fourth (U 67) and identifying two new MDV- 102 103 specific ORFs (SORFl and -3) and a fowlpox homolog (SORF2), our sequence analysis extends upon that of a 5,266-bp segment located in the Us region of the Very virulent" RBlB stain of MDV (vaDV) (Ross et al.. 1991, J. Gen. Virol. 72:939-947; 949-964). The vMDV and vaDV sequences above were found to be 9996 identical at both nucleotide- and predicted amino acid levels. Combined with the fact that MDV Us sequences failed to show statistically significant CpG deficiencies, our analysis is consistent with MDV bearing a closer phylogenetic relation to alphaherpesviruses. Inasmuch as the alphaherpesvirus-specific Us region is known to specify a cluster of supplementary essential fimctions thought to be important in defining biological properties, our sequence provides a foundation for further MDV studies aimed at resolving the apparent discrepancy between MDV’s genetic- and biologic properties. INTRODUCTION Marek’s disease virus (MDV) is a highly pathogenic herpesvinis of chickens. which can cause; (i) T cell lymphomas as early as 3 weeks post- infectian; (ii) peripheral neural lesions, characterized by lymphoproliferative infiltation and demyelination, occasionally leading to paralysis and/or blindness; (iii) various phenomena of acquired immnodeficiency and (iv) atherosclerosis in normacholesterolemic chickens, bearing a remarkable resemblance to the human disease, both in character and distibution of arterial lesions. Marek’s disease (MD) is the clinical outcome of these various pathogenic manifestations (reviewed in Calnek 8: Witter, 1991) and was the first naturally occurring lymphomatous disorder to be effectively contolled by vaccination (Churchill et al., 1969). Because of similar biological properties, especially it lymphotopism, MDV and it antigenically related, apathogenic vaccine virus, herpesvirus of turkeys (HVT) have been provisionally classified as gammaherpesvinises (Roizman et al., 1981). In contast to gammaherpesviruses, MDV and HVT have genome stuctures more closely resembling those of alphaherpesviruses (Cebrian et al., 1982; Fukuchi et al., 1985; Igarashi et al., 1987). Consistent with their stuctural relatedness to alphaherpesviruses. recent data indicate that MDV and HVT are phylogenetically more related to alphaherpesviruses than gammaherpesviruses (Buckmaster et al., 1988). This raises interesting questiort regarding the seeming incongruence between MDV’s genetic- and biologic properties. To understand the nature of these differences, and to search for new glycoproteins potentially important in vinis-host cell interactions, as well as the mechanism of protective immunity against MD, we have become particularly interested in the MDV Us region. This stems from the observation that 104 105 alphaherpesvirus Us regions are known to contain a cluster of glycoprotein genes and appear to specify determinant for pathogenesis and viral dissemination, rather than those essential for vinis production (Roizman, 1990a). These determinant are encoded by a cluster of "non-essential"- or supplementary essential genes which are likely to account for many of the unique in vivo properties characteristic for a given alphaherpesvirus. The natural host MDV system affords a unique opportunity to examine the in viva role and function of this putative class of supplementary essential genes. The alphaherpesvirus Us region is flanked by a pair of inverted repeat sequences (inverted- and terminal repeat short, [Rs and TRs, respectively, or simply, repeat short. Rs). Together, these component make up the 6 region. Alphaherpesvirus 6 regions are distinguished by marked differences in content, genetic organization and evolutionary divergence (e.g. HSV-1 Us = 13.0 kbp, 12 genes (McGeoch et al., 1985); varicella-zoster virus (VZV) Us = 6.2 kbp, 4 genes (Davison 8: Scott, 1986). Secondly, they specify a cluster of glycoprotein genes potentially important for protective immunity. A similar cluster in MDV would be particularly significant given the paucity of specific details regarding protective immunity against naturally occurring Marek’s disease (MD) tumors. The DNA sequence of a 5,265-bp segment from Us region of the "very virulent" RBlB stain of MDV (vaDV) was recently reported (Ross et al., 1991). This region was found to contain open reading frames (ORFs) homologous to proteins encoded by HSV U62, U63 (protein kinase), U66 (glycoprotein D), part of U67 (glycoprotein I), and an additional MDV-specific ORF. In this report, we extend upon these result and present a sequence analysis of the entire 11.2 kbp MDV Us region ("virulent" GA stain, vMDV). Compared with the 6.3 kbp RBlB segment of MDV, the corresponding GA sequence was 99% identical at both nucleotide- and predicted amino acid levels. In addition to the 106 alphaherpesvirus Us homologs shared in common with the RBlB stain, we have completed the U67 (g1) sequence and have identified three additional ORFs homologous to HSV U61 (ICP22), -US8 (gE), and -U610; a fowlpox virus (FPV) homolog; and at least two additional MDV-specific ORFs. Recomblrunt pl-tnih. M13 subalaning and DNA sequeming. Pathogenic MDV GA stain subclones included EcoRI-O, -I and -V cloned into pBR328 (Gibbs et al.. 1984) (pE328-O, pE328-I, and pE328-V, Figure 1B); BamHI- A and BamHI-Pl, cloned into pACYC184 and pBR322. respectively (Fukuchi et al., 1986) (pBACYC-A and pB322-P1 (Figure 18), kindly provided by Dr. Meihan Nonoyama of the Tampa Bay Research Institute, St. Petersburg, FL); and GA-02. a phage clone containing a partially digested MDV 6au3A insert cloned into the Sall site of EMBL3. ltindly provided by Dr. Paul J. A. Sonderrneijer, Intervet International, Boxrneer, The Netherlands. The latter clone contains most of BamHI-A. all of BamHl-Pl and additional 3’-flanking sequences, including some of those present in pE328-V. This phage clone was used to generate the pUCl8 subclone, p6Pl8-A (Figure 1B). This clone contains a 2.5 kb SalI insert with approximately 20 bp of EMBL-3’s multiple cloning site at its 3’ end. Together, the above clones (Figure 1B) were used to generate Ml3mp18 and -19 subclones for use as templates for nucleotide sequencing. DNA sequencing of both stands was performed by the dideoxy-chain termination method (Sanger et al., 1977) using single-standed M13 templates. Reaction product were synthesized and labeled using a 17-mer M13 primer, a modified T7 DNA polymerase (Sequenase). [35$] thio-dATP (NEN) and appropriate deoxy- and dideoxynucleotides according to instuctions by the manufacturer (Sequenase sequencing kit; United States Biochemical Corp., Cleveland. Ohio) and electophoresed through 796 polYBCIfiMdB/BO“ urea/Tris-Borate-EDTA gels. Remaining sequence gaps were determined by substituting M13 primers with synthetic 17-mer oligonucleotides (under similar 107 108 reaction conditions, 0.5 pmoles/reaction) generated based on previously determined sequences. Analyst d sequent” data. Sequences were assembled and analyzed with an IBM Personal System 2/Model 60 microcomputer utilizing Genepro (Version 4.10; Riverside Scientific Enterprises, Seattle, WA) sequence analysis software packages or programs obtained from the University of Wisconsin Genetics Computer Group (UWGCG, Versions 6.2 and 7.0; Devereaux et al., 1964) and run through a VAX 8660 minicomputer. Homology searches of the SwissProt (Release 18.0, 6/91), GenBank (Release 71.0, 3/92) and EMBL (Release 30.0, 3/92) databases were performed using the UWGCG programs FASTA and TFASTA (Pearson 8: Lipman, 1988). FASTA was utilized against both protein (SwissProt) and nucleic acid (GenBank/EMBL) databases, while TFASTA was used to compare protein sequences against GenBank/EMBL. Briefly, these programs employ an algorithm to locate regions of similarity, a PAM260-based scoring system to provide a qualitative and quantitative homology assessment and an alignment procedure to join together, when possible, the highest-scoring, non-overlapping regions in order to derive an alignment and it resulting, optimized score. Dot matix homology plot were generated using the UWGCG program DOTPLOT with the output file from UWGCG’s COMPARE. To create multiple alignment, successive GAP comparisons were conducted between MDV and it homologous sequences (in descending order of homology), generating gapped output files to be used as input sequences for subsequent mm of GAP until the alignment of these gapped sequences could no longer be expanded by the addition of new gaps. Following alignment, the gapped output files were displayed and a consensus sequence calculated using the UWGCG program, Pretty. For optimal result, manual editing, based on visual inpection, was employed (using UWGCG’s LINEUP). When using FASTA. TFASTA, 109 COMPARE and GAP, "similar' amino acids were defined as amino acid comparisons equal to, or exceeding +0.5, as derived from Dayhofi's mutational matixtablebyrescalingthevalues bydividingeachbythesumofitrowand column, and normalizing to a mean of 0 and standard deviation of 1.0. where perfect matches are set to +1.5; these and other values ranging between -1.1 and +1.6 are described in the UWGCG User's Guide appendix or accessible by typing the command FETCH. followed by ComparPep.Cmp or NW6GapPep.Cmp. Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number XNNNNN. RESULTS DefinhigtlnMDVUsregianandthelocafianaftluimlque-repeatreglon jinictlal. Figure l containsamapoftheareathatwassequenced. This segment is bounded by a pair of Pqu sites and spans the 3’ half of BamHI-A. extending an additional 1.5 kbp to the right of the 3’ end of BamHI-Pl (Figs. 18 and 2). This 11,286-bp segment spans the entire Us region, which is 11,160 bp inlengthandflankedattheS’ and3’ ends bya63bpstetch ofIRsandTRs DNA. respectively, each inversely complementary to the other (Figs. 1B and 2). Based on Southern blot analysis, the IRs-Us junction was previously localized to a 1.4 kb Bgl I fragment (Fukuchi et al., 1986) located in the second of five EcoRI subfragments of BamHI-A (for BamHI-A/EcoRl map see Wen et al., 1988). Our sequence analysis provides conclusive proof that this junction is actually located in the middle of the third EcoRI subfragment, approximately 2-3 kbp downsteam from the reported position. This is further supported by our own Southern blot analysis (data not shown). The Us/‘I'Rs junction is located 263 nucleotides downsteam of the U68 termination codon (following position 11.223, Figure 2). Identifying the MDV Us/TRs junction necessitated the characterization of previously unreported clones mapping 3’ of BamHI-Pl , extending into the TRs region. Assuming that the MDV IRs is identical to it TRs, the availability of these new clones should now allow for the complete characterization of MDV’s 6 region. Nucleotlh sequence and ldentificafion of open reading frames. The overall guanine plus cytosine ratio of the region sequenced was found to be 41%, somewhat below the reported genomic value of 4696 (Calnek 8: Witter, 1991). Observed frequencies of CpG dinucleotides in the whole sequence, or in the coding regions only, did not differ significantly from those expected from 110 111 HGUREI. MaplacationafareaeequencedandarganisatlananDVUsORfi. A) MDV genome stucture and BamHI restriction map outlining area 3) sequenced. Boxes define plasmid clones with BamHI, EcoRl or Sell- bound insert that were used to generate Ml3mp18 and -19 templates for sequencing. Organization of MDV Us ORFs. Boxes represent location of MDV ORFs. Arrows define direction of tanscription/tanslation. Names of ORFs are displayed above boxes. Basis for nomenclature is outlined in RESULTS. A 0 20 4O 60 80 100 120 140 160 180 . L L A L A A A A A A A L L A I Kb TRL IRL IRS TRS ULI 9," UL: us Z'lsieilta I : i . : : ‘9” 5 Ship ‘9” (02) 1 ' Lgiz o 0.6 c F K2K3l3 E e21. JOgR s\\\\|ax.n lger A P,: hetero Barn HI H 'H' ' _.-- I I l I B. _________ —~\ ------------- ‘\\‘ ---------- \\‘ ........ ‘\ UI‘T‘ pBACYC-A l r 98322-1" | “e " : I psaze-o T pesza-i I I pens-vi 7n I L pSPlB-A j i”— l I . I I | I i : P E S E P P B B S E B E 8 BP Kb h i L l l i 1 1 J 1 1 i l #4 ' I I Y r T I l I fir ] I I 1' j T I T I I I f 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 lfisd—HUS usH-erns SORF1 SORF2 US1 U810 SORF3 U82 U53(PK) SORF4 U56(gD) U87(g|) US8(9E) mmmmfiszQJfi)4C§jfi) )fiL 7&1 113 their mononucleotide compositions (data not shown). This result agrees with those obtained from alphaherpesviruses, while sharply contesting with those obtained from all gammaherpesviruses thus far studied (Efstathiou et al., 1990; Honess et al., 1989), including the A+T rich herpesvirus saimiri (HVS) and the G+C rich Epstein-Barr vinis (EBV), which are both deficient in CpG dinucleotides. The region sequenced contains at least 11 ORFs likely to code for proteins (Figure 1B; basis for names is defined below). This prediction was primarily based on homology and positional organization comparisons to other alphaherpesvinis genes. This identification was further guided by the observation that alphaherpesviruses such as HSV and VZV tend to contain relatively tightly packed, unspliced coding regions (Davison 8: Scott, 1986; McGeoch et al., 1985, 1987, 1988). Methods for detecting protein coding regions based on the use of MDV-derived codon frequency tables (using these and previously published MDV sequences; Binns and Ross, 1989; Ross et al., 1989; Scott et al., 1989) or analysis of compositional bias (using the UWGCG programs CODONPREFERENCE and TESTCODE) were inconclusive. However, as pointed out previously (Ross et al., 1991), MDV does appear to contain a detectable bias for A-T residues in the wobble position. Furthermore, using the UWGCG program FRAMES, together with the MDV-derived codon frequency table above, the 11 identified ORFs clearly show a significantly low pattern of rare codon usage not observed following computer-based tanslation of the remaining reading frames (data not shown). The predicted amino acid sequences of the putative ORFs (beginning from the first ATG codon) are shown relative to the nucleotide sequence in Figure 2. Due to the A-T rich nature of MDV, there are numerous TATA-like sequences for tanscriptional initiation, more than are likely to have functional 114 FIGURE 2. Nucleotlh and predicted amino acid sequences. The nucleotide sequence is given as the rightward 6’ to 3’ stand only (numbered 1 to 11,286). Rs and TRs sequences are located at the 5’ and 3’ ends, respectively. and are depicted using lower case symbols; Us sequences are in upper case. Rightward- and leftward-directed predicted amino acid sequences are shown above and below the corresponding nucleotide sequences, respectively, in single-letter code. The name of each ORF is given to the left of the first line of it respective sequence. Amino acid sequences are numbered from the N terminus. beginning with the first in-frame methionine codon and ending with the amino acid at the C-terminus. which precedes the termination codon. Dotted lines identify potential polyadenylation signals. Putative signal peptide and tansmembrane domain regions of MDV U66 (gD), - U67 (g1), and -U68 (gE) are overlined at the amino- and carboxy ends, respectively. Signal peptide overlining continues through to the last amino acid to the left of the predicted cleavage site (von Heijne, 1985). Potential N- glycasylation sites (N -X-6/T) are indicated by dashed lines. SCIF1 SOIFZ 061 0610 101 76 201 44 301 11 401 1 501 26 601 61 701 16 2101 50 2201 63 2301 116 2401 150 2501 163 2601 2701 2601 2901 321 3001 3101 255 115 F1 2 < lIs cegct tattttcccc t etctcatacc ccattttt tegeggrerertiiEQEEAGCCAAAIcaiAitccrcaaastrreACAAnaccrer ‘c' 9 9° 9‘ '3' as * s r a i e P L r v r t t CAAACTCACACGCTCCAAGCGAAACTCGAAAAAAAAAGGGCCGGGGGGAGAATATTCTGTAGGACCCGCAGAACTTCTCAAGCCAGAGGAAAGATACACA L S V I 0 L I F E F F F P P P P S Y E T P 6 A S S I L A 6 6 L Y V TTATTTTTTCTTAGATTTACGCAAGTTTTGCAGAACCTGCAGGGAATGTATACACCATCAAATCTACTCGACTTATTGCTTGACTCCAATTTAACAGAAA I I K T L I L C T K C F I C P I Y V 6 0 F I 6 S K I 6 S 0 L K V S ITAtAAIAT¢TTgAT3TT:CC:CA:ATaCALCCTCGCATATCGCGCTCCCACACACGACCATTATATCCCCAGACATGAACCTCAAACTGCCATTTTGAT CCCATCATTCGAGAGACAAATTCCCATACATCCTACTTATCCCACACATTGGATGTCCGTCTTTATTCAGGCCATATCAGCTTTCACGGGCGCAAATTCC I 0 I 0 T G I I E 0 K K I T 6 L E S 0 G T E I A F 6 0 TATTCATAGATCCGTCATCGATGCAGCGCCAAACCGCACATATCCAAGACAAAAAGAGAACCCGTTTGGAATCGCACGGGACCGACAATCCTTTTTCAGA 6 I 0 6 K 0 C L L I E G l I E P l L l P S T l A 0 L E G l I E L V TGGCAGACATGGCAAAGATCGATTGTTACATCAAGGAATTAATGACCCCATTTTCATTCCCTCTACCATCGCAGATCTCCAGGGCATTCCTGAATTCCTC I K F I C I L L P F E K C P D F C L I l 6 G L E A 6 F I K 6 0 E E L CCAAAATTCCCTGCTCGTCTACTGCCCTTTGAAAAGTCTCCCGATTTTTGTCTGAGAATTGGCGCTTTGGACGCCAGCTTTCATAAACGGCACGACCACC L E Y C E A L Y L P 0 P V K I E l V G l V D 0 V P C L A T 6 I 0 L TCTTAGAGTATTCTGAAGCACTTTATTTACCACAACCTCTTAAGATGGAAATAGTAGGCATTCTAGACGATGTGCCATCTCTGGCAACGGCGATGCAATT L 1 L V A E G C E V Y A Y E E 0 T L I K L A T S F 6 E F L E l C V ACTCATTCTTCTTCCCGAGGGCGCACACGTATATGCCTATGAAGAAGATACTCTGCATAACTTAGCCACGAGTTTTTCCGAATTCCTTGAAATTGGACTC K 6 L C I E V Y I C B E Y 1 E 0 V V I ' 179 AAATCTTTACCGACCGACGTTTACCATTCTGCAGAATATATAGACCAAGTAGTACATTAGGCGCTGCGTTAAACACCAACTAATTTTTCACCGGATATCA 4 CGTGATGTAAATTCTAGCAATTATTCTTCCTAGCAGAAGATAAAAGCTCSTAGCTATATAATACAGGCCAAACTCTCCAAATTACACTTGACCAGAAAAC I S I 0 I 0 I A I P 0 T I L S 6 6 0 I E S 0 0 E 0 CTGCTTTCGGCTCCATCGGAGCCAACATGAGTCGTGATCGAGATCCAGCCAGACCCGATACACGATTATCATCGTCAGATAATGACAGCCACGACGAAGA Y 0 L P I S I P E Y 6 S 0 S 6 0 0 0 F E L I I V C K F C P L P I K TTATCAACTCCCACATTCACATCCGGAATATCGCAGTGACTCGTCCCATCAAGACTTTGAACTTAATAATCTCCCCAAATTTTCTCCTCTACCATGCAAA P 0 V A I L C A 0 T I K L F I C F I I C I L I 6 6 P F I 0 A L I I A CCCGATGTCGCTCCCTTATGTGCCGATACAAACAAACTATTTCGATGTTTTATTCGATGTCGACTAAATAGCGGTCCGTTCCACGATGCTCTTCGGAGAC L F 0 l I I I C I I G Y I L K 0 A E I E T I I I L T P I 0 S L I L CACTATTCCATATTCATATGATTGCTCGAATGGCATATCGACTAAAACAACCCGAATCGGAAACTATCATGAATTTGACCCCACGCCAAAGTCTACATCT I I T L I 0 A 0 6 I 6 A I P l S D 1 Y A 6 0 6 1 F I P l A A 6 6 C GCGCACGACTCTCACCGATGCTGATAGTCCAACCGCCCATCCTATATCCGATATATATGCCTCCCATAGCATTTTTCACCCAATCGCTGCCTCCTCCGGA V K G I I 0 L S V 0 6 K L I ‘ T l S S 0 C 0 ‘ACTATTTCTTCAGACTGCGATGTAAAAGGAATGAACGATTTGTCCCTAGACACTAAATTCCATTAACTATCCACACTTGAACAGAAACCTCTTATTATAT AATTTTAATTGTT AGACATACAGCCCACATTCTTTGATCTATCTAATGAGATAAAATAATAGATTTTGGATTTATTTCTCATGATCTCTT CTGACCCCCCCCATCCATGAAGGGGCGTGTCAAATAACCTGTTGCCTTTTTGTTGTATATGAAGATATTTAATGTCCCGTTGAGCCTAATGAGAGCAGAA I A I I 6 L I I K S 6 I 6 V 0 CGTCTTTGAATACTCGAGACGAGCGCCGTCTAAGATTAAAACATATTGGAGACCTATGGCCATGTCCTCTCTACGGCGCAAATCTAGCAGCAGTGTGCAA L I V 0 6 P K E 0 S Y 0 l L S A 6 6 E I V A L L P K 6 V I 6 L A I T CTCCGGCTAGATTCTCCAAAAGAACAGAGTTATGATATACTTTCTGCCGCCGGGGAACATGTTGCGCTATTCCCTAAATCTGTACGCACTCTACCCACGA l L T A A T l S 0 A A I K A C K P P 6 S I L I C E I F 0 I I T V T CCATATTAACCCCCGCTACGATCTCCCAGGCTGCTATGAAAGCTGGAAAACCACCATCGTCTCCTTTGTGCGCTCAGATATTCGACACAATCACTCTCAC L I E Y 0 l S A 6 P F I P T 0 P T I K I V G I A L I C 1 E I A P L GCTTAACCAATATGATATTTCTGCTTCGCCATTCCACCCCACAGACCCGACGAGAAAAATTGTAGCCCGGCCTTTACGCTGTATTGAACCTGCTCCTCTT T I E E I 0 T I F T l I I Y I C C L C I A G Y C T V 6 I L Y E K I V ACACACGAAGAAATCGACACTCCCTTTACTATCATGATGTATTGGTGTTCTCTTGGACATCCTGCATACTCTACTCTTTCCCGCTTATATGACAACAATB I L I 0 1 V C 6 A T C C C l 6 P L P E I E 6 Y I K P L C I A V A T TCCCTCTTATCGACATAGTACGTTCCGCAACGGGCTGTGCAATAAGTCCACTCCCCGAAATAGAGTCTTATTGGAAACCTTTATCTCCTCCCCTCGCTAC K C I A A 1 6 0 0 A E L A I Y L T I L I E S P T C 0 C E S Y L F 213 TAAGGGGAATGCAGCAATCCCTGATGATGCTGAATTCGCACATTATCTGACAAATCTTCGCGAATCGCCAACAGGAGACGCGGAATCCTACTTATAACTA ATCCCACAATTATTAATAGGATTTTAGGAAAAACTGCTACTAACGTTGTTTAAATAATAAAATTT1A1TTTCAATAAGGCATTACAGTGTTGTCATGATT GTATGTATIATATGGGGTATGCATGAGGATTACTTCGATTGAAACTTTGTCTAAATGTCTGTAGGATTTTACTATTCATTAGTCTGGATCGAGGCGGACG 351 - i P v A n P u s a I r s o a r r o L l K s I a L I s r P r t TAAATGGAGATTGCGCCAAATCTAGCCCTGCTCCTACATAAGACCTCCAACATCCATTCGACTCATCGGCCTGCGTCCAAATGGATATGTTGATGTACCT L I L I C I Y P I 0 Y I L 6 6 V 0 I I S I P I I 6 F P Y T 6 T G TCTAAACTTATGACATTAGAAGATCCATGGTCAATAGTGCGATCTATATCCATGCTATTCTCAATATTGCATGATATGCAATGTTCCCCGTTAGGTTTGA 0 L T l V I 6 6 I I I l T P 0 l 0 I 6 I E l I C S l C I E I I P K TAAGATCATGTATGGTTCTATAATACAACTCCTCTTCAGAAGAATCA111A11TTATGTCCACTGTCCTTGGATATTCCAGTTTCTGTCAATCGATTCGC i L o u l t n v r L E a s s s o u i r u a s o r s i a T E T L t u A 92 1500 125 1600 156 1700 SOIF3 3201 221 3301 166 3401 155 3501 121 3601 3701 55 3601 21 3901 4001 4101 266 4201 234 4301 201 4401 166 4501 134 4601 101 4701 66 4601 34 4901 5001 30 5101 64 5201 97 5301 130 5401 164 5501 197 5601 230 5701 264 5601 297 5901 6001 364 6101 397 6201 116 TTGCATTTGCCTGCACCATCTCTTGATGGCATTTCCTATGCTATCATCCGGCACGCCTAAGCCTGTTCTATACTCCCACACACCTAGACCAAGAACCACC C I 0 T C C T K 1 A I 6 l 6 0 0 P L G L P T I Y E C V P L A L V V CCATATCGACCTACCTCTATTGCCCCGCTAAGCACATTTCTTGCACACTGTATTGTCATGAACATATTTCCTGTATTCTCTCGATCATAACCCTTGTTGA A Y I A V E l A 6 6 L V I I A 6 0 l T I F I I I T I I I 0 Y 6 K I TTCCTATCGAAAGCATTGTGETCCAGTTTTCCAGATGAAATCAAAACAATGCGGGCAAAAATGGTCCCACCTCTTTCATCTTCAATCCATCTCTCACATC l 6 l 6 L I T T I I E L I F 6 F L A P L F P C V 0 K I K L A 0 I V 0 CCAABTTCTATACAATATTCTCCACTGACCACTTTCCGTAACATCAGTTTCTGTAAAATTTCTGATACTTTCAATCCAAAACATTTTCTCCATCATGCCA I T I Y F l I I 0 6 T E T L D T E T F I T I T E l 6 F I K 0 I I A AAAAATCTATAGGCAGACCAGATAACCATTTGACACCACATATCCTTGTGTATATCAAACGATCTAATACATCCCTCGTTACTAGATATGGTACATAAAA F F I Y A 6 I l V I 0 C I I 0 K I l 0 F 6 T l 6 6 E I T 6 l T C L GCCCTAATCTCTCTCCCCCTTCCATACATTGAACGATTCCTTCTCTGAATTCATCAACAACCACATCCCAAAAATTTACATTACTAATCTTTCTCCCTEE L G L I E I A E I C 0 V l C E T F E 0 V V V I I F I V I T I K I P P CTTACCAAATCCTCCTCTTGCTATATCCATATCATCCAACATTGTACCATTGACTCTGCTCATCCTTCTCTTTCAAATCCCCTCCATTCTTGAATCTCTC K 6 F I 6 I P l 0 I 0 0 F I T A I V I S I CTGATCTTAGAACTATATGCAACATACCCTGCATACATAABTGATCTAGAAGCCTTTCTTATTGCACTAATATACAAATTATACCTGACACTATACCGAC CSTTCTACCGATGCACCTAATCCTAATGTCTATACGCCCCATCATGTAATTATATCTAATTGCTACCAAGTAGCTCTGTCGAATAA:98CTAAT:AC;AC CGGCTCTACATTTTTTCTGTATTCGTGACTTTCCTCTCCCACTCTAACGAACCGGAATTGCAATCGCATCTCTATCTTCTTTCTTGCAACATTTTCCACA G A I C K K 0 l I 6 K 6 T A T Y I V P l A I A 0 I 0 E K K C C K C C ACACAATAATCTCCCCCCTGTACTACTCATTTGACCTCCTTCCATTTCCCGAGCTTTTAGACGATTGGCTCCCCACCCCACGATTTTCTATACACATACC C F L I 6 P T 6 6 I 0 P P E l E P P K L P I P P 6 6 L l K Y V C V ATATCACTGTCCCAAAAATGCCCTCTATCTTCTCCGCTGTCGAACTTCCGTTCCCATGTAGATCTCAAGAGAGTTTGAATATTGTCCCGAATGCCCCACC I 0 6 0 C F I A I 0 E P T 0 F K P E T 6 T L T l I 0 l A I GCATACCGGACCACCTCCCAGACACTTTGATTGCAACTAACCTTTTTGGCAAAGGAATACATTCGAGCGCAATCCCACATATATCTCCCCCCCCAACTAT P I 6 6 I T G 6 V K 1 A L L I K P L P 1 C E L A l A C l 0 A 6 I CCACAACCTATCTGGAGCATTACCACAAACTTCACATTCCAACATCAAATATCCAGATACAACATCCTGCCATTCTGTGCAACATCCTGCAACATCTTCA I L 6 I P A I 6 6 V E 6 E L I L Y C 6 L V 0 0 I E T 6 C 6 A V 0 E AATACCCCCACTATAAACGAATCCCTACTTCCGCCCAATCCGCTACCACCAACTCCACTTCCATCTCGTGGCTTTGTCCTTACTATCCCTCCATCTTGCC F L I V I F 6 0 I T 0 A L 6 T C I V 6 T C 0 P P K T I V l P I I 0 GACGAAGAATTAACAT666TTTCGCAAAACGCAATAGCTCTGCAGCTCTCCCGATTATGCCCACACCCACATCATCCTGTATTTCTTCCATACATTGCTT I P L I L I P K A F I F L 0 A A I I l P V 6 0 0 l 0 E C K TATAACCAATATCCATAAACTACATCCACCATCTCTAGATCTTCCTCCCAATCGATCGCATTCATCTAGAAGTGTGACTATACTTATCATGCACACACCC I L F l I L T S A 0 I 6 I 6 P L I 0 C E 0 L L T T I 6 6 A:CTTCACCTCCACCAATAATCTTTTTTATTGTTAATAACTCGCCCGGTCTGATCTCCAAATCTTATACTCTCCTAGAATATGAAACACGGTTAAAACTA I S 6 T P E A E T I E C G l 6 S 6 K V I 0 S K T I T T Y C CCTAATAGACTGGATGTCTTCGACTCCGCAGCCAGAAACGATGGAATGTGGCATTTCTTCCTCGAAAGTACACGACTCTAAAACTAATACTACCTACCGA 1 l 6 l I C T 0 T L F 0 T F P D 6 T 0 I A E V T C 0 V 0 D V K ATTATACATAACAGCATCAATCCTACGGATACGACCTTGTTTGATACTTTTCCCGACACTACCGATAACCCCGAACT TGACGGCGGATCTGGACGATCTCA T E 6 6 P E 6 0 6 E 0 L 6 P F 6 I D 6 I E 6 P E T V T 0 l 0 A V 6 AGACTCACAGCTCTCCCGAGTCCCAATCTGAAGATTTCTCACCTTTTGGCAACGATGGAAATGAATCCCCCGAAACGGTGACCGACATTGATGCACTTTC A V I I 0 Y I l V 6 6 L P P 6 6 E 6 Y 1 Y V C T K I 6 0 I T K I K ACCTGTGCCAATCCACTATAACATTCTTTCATCGTTACCGCCCGGATCTGAACCGTATATCTATGTTTCTACAAACCCTGGGGATAATACCAAGACAAAA V 1 V K A V T 6 C K T L 6 6 E l 0 l L K K I S I I 6 l l I L V I A Y 6TCATTGTGAAACCTGTCACTCCTGCCAAAACCCTTGCGACTGAAATTGATATATTAAAAAAAATGTCTCACCCCTCCATAATTAGATTABTTCATGCTT I I K 6 T V C I V I P K Y K C 0 L F T Y 1 0 I I 6 P L P L I 0 I l ATAGATGGAAATCGACACTTTGTATCCTAATGCCTAAATACAAATGCGACTTCTTTACGTACATAGATATCATGCGACCATTGCCACTAAATCAAATAAT T i E I 6 L L 6 A L A Y I I E K 6 l l I I 0 V K T E I 1 F L 0 K P TACCATACAACGCGCTTTGCTTGGACCATTGGCATATATCCACGAAAAGGGTATAATACATCGTGATGTAAAAACTGAAAATATATTTTTGGATAAACCT E L 6 0 C A A C K L 0 E T 0 K P K C Y C I 6 C T E T I 6 GAAAATCTAGTATTGGCCGACTTTCCCCCAGCATCTAAATTAGATGAACATACAGATAAACCCAAATCTTATGOATGGACTGGAACTCTGGAAACCAATT E L L 0 P Y C T K T 0 1 I 6 A C L V L F E I 6 V K I l T F F CGCCTGAACTGCTTGCACTTCATCCATACTCTACAAAAACTGATATATGGAGTGCAGGATTAGTTCTGTTTGAGATGTCAGTAAAAAATATAACCTTTTT C K 0 V I 6 6 6 6 0 L I 6 l l I C L 0 V I P L E F P 0 I I 6 T I L TCGCAAACAAGTAAACGGCTCAGCTTCTCAGCTGAGATCCATAATTAGATGCCTCCAACTCCATCCCTTGGAATTTCCACAGAACAATTCTACAAACTTA C K I F K 0 Y A l 0 L I I P Y A l P 0 l l I K 6 C I T I 0 L E Y A I TGCAAACACTTCAAGCACTACGCGATTCAGTTACGACATCCATATGCAATCCCTCACATTATACGAAACAGTGGTATGACCATGGATCTTGAATATGCTA A K I L T F 0 0 E F I P 6 A O D 1 L I L P L F T K E P A 0 A L Y T TTCCAAAAATCCTCACATTCCATCACCAGTTTAGACCATCTGCCCAACATATTTTAATGTTGCCTCTTTTTACTAAAGAACCCGCTGACCCATTATACAC l T A A I I * 40 GATAACTCCCCCTCATATGTAAACACCCGTCAAAAATAACTTCAATGATTCATTTTATAATATATACTACGCCTTACCTGCAATAATGACAACATTCCAA i -3 e3 :3 s§ t3 §§ a? at $5 st at §§ 5000 29 5100 63 5200 129 5400 163 5500 196 229 5700 263 5600 296 5900 363 6100 396 6200 “F4 066 (ID) 067 (0‘) 6301 24 6401 56 6501 91 6601 124 6701 1 6901 21 7N1 54 7101 67 7201 121 7301 154 7401 167 7501 221 7601 254 7701 267 7601 321 7901 354 6001 367 6101 1 6201 15 6301 6401 61 6501 115 6601 146 6701 161 6601 215 6901 246 9001 261 9101 315 9201 117 IAPSGPTPYSIIPCIKIYCTFSD 6TCTTT6AA6ATTCCCACACCTTTTTT666AAT66CACCTTC666ACCTACGCCATATTCCCACACACCGCAAATAAACCATTATCEAACATTTTCCMT CIIYTLIDESKVDDICSDIIISLACSIVTSSISV TCCATCACATATACTCTAAAC6AT6ACA6TAA66TA6AT6ATA6AT6TTCAGACATACATAACTCCTTACCACAATCCAATCTTACTTCAAGCATGTCTC 0 A E 0 P K S V F Y K I K P 0 I 6 IIDSEECPLIICPSI TAAT6AAC6ATTC66AA6AAT6TCCATTAATAAAT66ACCTTC6AT TAAAA616TTTTTTATAAA6TTC6TAAGCCT6ACCGAA6 IOF6IOILI6I6I66LIIEKYlISSKIIIKIPE TC6T6ATTTTTCAT666AAAATCT6AACTCCCAT66CAATA6T66TCTAC6TC6T6AAAAATATATAC6TTCCTCTAA6A66C6AT66AA6AATCCC6AC lFKVSLKCESI6A6I61K16F6FF'147 ATATTTAAGCTATCTTT6AAAT6T6AATCAATT6666CT66TAAC66AATAAAAATTTCATTCTCATTTTTCTAACATTATAATATATCAMTC6TTTCT murmurmuttercasein!«truestAnetmniimmmcrccnmrcwuimsmfiiiflunnmm WWI—3W 6TCTCCTATAACTCTTATATTGCCACCTTTTAGAGCTTC66TAT6AATA6ATACAGATAT6AAA6TATTTTTTTTACATATATCTCATCCACGAGAATGA 0 6 I6LKKOIS llPTLIPK6I TTCTTATAATCTGTTTACTTTT666AACT6666ACAT6TCC66AAT666ACTTAA6AAA6ACAATTCTCC6ATCATTCCCACATTACATCCIMAA66TAA E I L I L I E Y K 1 P L F 0 T L 0 I 6 E T K I V I Y 0 T6AAAACCTCC6666TACTCTCAAT6AATACAAAATCCC6TCTCCACT6TTT6ATACACTT6ACAATTCATATGAGACAAAACACCTAATATATAC66AT I .C- S L P F 6 0 P K Y T L L L L 6 Y 0 L A I AATTCTACTTTTCCTCTTTTGAAT CCATTT66C6ATC66AAATATAC6CTTCTCACTTTACT6TT6AT666AC6AC6CAAATAT6AT6CTCTA6TA66AT FVL6IAC6IP1YLIEYAI-C-STIEPFCTCKLKSL 66TTT6TCTT6666AMGCAT6T666A6ACCAATTTATTTAC6T6AATATGCCAACT6CTCTACTAAT6AACCATTT66AACTT6TAAATTAAA6TCCCT 6IIOIIYAITSY10IOELKL1lAAPSIELSGLY A66AT66T666ATA6AA6ATATGCAAT6ACGABT TATATCGATCCACAT6AATT6AAATT6ATTATTCCA6CACCCA6TC6T6A66TAA6T66ATTATAT TILllII6EP16601LLTVK6TC6FS I61KOIK AC6C6TTTAATAATTATTAAT66A6AACCCATTTC6A6T6ACATATTACTGACT6TTAAA66AACAT6TA6TTTTTCG:6AC66666ATAAA66A AAA66ATAACA LCKPFSFFVI‘G'TTILLDIVITCTPIAIEEIVKC AACT AT 6CAAACC6T TCACTTTTT TT6TCAAT66TACAACAC66CT6TTAGACAT66T6CCAACAGGAACCCCGA6AGCCCAT6AA6AAAAT6T 6AA66A ILEIIGGKILPIVVETSIOOVSILPISFIDSYL 6T66CTT6AAC6AAAT66T66TAAACATCTACCAATCCTCGTCCAAACATCTATGCAACAAGTCTCAAATTTCCCGAGAAGTTTTAGAGATTCATATTTA K6 0 KYIO K 6 T I-l-TTS 0 YT6LTII AAATCACCT6AC6AC6ATAAATATAAT6AC6TCAAAAT6ACATC66CCACTACTAATAACATTACCACCTC66T66AT66TTACACT66ACTCACTAATC PEDFEKAPYITKIPIISVEEASSCSPKISTEKK 66CCC6A66ACTTT6AGAAAGCACCATACATAACTAAA66ACC6ATAATCTCTCTCGACCAGGCATCCA6TCAATCACCTAAAATATCAA SITCTTISLVVLCVI IKIIK ATCCCCAACCCAAATAATAATTTCACTA6TT6TTCTAT666TCAT6TTTT6TTTCATT6TAATC666TCT66TATAT66ATCCTT TVIYDIIIPSIIAYSIL‘403 AC66T6AT6TAT6ATA6A66TC6TCCATCAAGACCGCCATATTCCCGCCTATAACACGTCTTTGGTAT666C6T6TC6CTATA6TGCATAA6AA6TT6AC I'VEILLFITIFFI TACATTGATCAATCACATTATATA6CTTCTTT66TCACATA6AC6666T6T6T6ATTCCGATCTATGTACTACAATTATTATTTT66ATCC6CCTCTTTC 61ISlVYT6T6VTLST006ALVAFI6LOKIVIV 6AG6CATCT66TCTATA6TTTATACT66AACATCT6TTAC6TTATCAAC66ACCAATCTGCTCTT6TT666TTCC6666AT TAGATAAAAT66T6AAT6T I60LLFL600TIT66YT6TTEILKIDEEYKCYS AC6666CCAACTTTTATTCCT666C6ACCACACT666ACCAGTTCTTATACACCAACGACCCAAATCTT6AMT666AT6AA6AATATAAAT6CTATTCC VLIATSYIDCPAlDATVFIGCIDAVVYAOPI6IV 6TTCTACATGC6ACATCATATAT66ATT6TCCT66TATA6AC6CCAC66TATTCA6AGCCT6TA6A6AC6CT6T66TATAT6CTCAACCTCAT66TA6A6 OPF E LII E I 60T66Y1IV6L 6II TACAACCT TTTCCCGAAAA666AACATT6TT6ACAATT6TCGAACCCAGA6TATCACATACACGCA6CTATTACATAC6T6TAT CT CT CGCT 66AA6AAA TSDIFIIVVIlISSKSIACI--SA66FOAIKCII TAT6A6C6ATATATTTA6AAT66TT6TTATTATAA66A6TA66AAATCTT6666CT6TAATCACTCT6CTA6TTCATTTCA66CCCATAAAT6TATT66C YVDIIA EIYL16IV6ILL0606ELIA1YI-l-TPO TAT6TC6ACC6TAT6666TTT6AAAATTATCT6ATT66ACAT6TA66CAATTTGCT 66ACAGT 6ACTC66AATT6CAT6CAATTTATAATATTACTCCCC SISTDlIlVTTPFYDISGTIYSPTVFILFII-I-S AATCCATTTCCACACATATTAATATTGTAACGACTCCATTTTACGATAATTCG66AACAATTTATTCACCTAC66TTTTTAATTTGTTTAATAACAATTC IVOAII‘S’TCI LKYTLPILIYFST lVLCl CCAT6TCCATGCAATGAATTCGACT66TAT6T66AATACC6TTTTAAAATATACCCTTCCAAGCCTTATTTACTTTTCTACCATGATTCTACTATGTATA IALAlYLVCEICISPIIIIYI6EPI60EAPL1T6 ATACCATTCCCAATTTATTT66TCT6T6AAA66T6C66CTCTCCCCATCGTA66ATATACAT666T6AACCAA6ATCT6AT6AGGCCCCACTCATCACT T AVIESFOYOYIVKETPSDVIEKELIEKLKKKVE CT GCA6TTAAC6AATCATTTCAATAT6ATTATAATGTAAAGCAAACTCCTTCACAT 6TTATTGAAAA66A6TT6AT66AAAAACT6AAGAA6AAA6TC6A 7100 120 153 non 166 7500 220 76% 253 7700 320 79M 353 6000 366 6100 14 47 64W 114 147 6700 160 6600 214 247 260 9100 314 9200 347 066 (IE) 346 9301 1 9401 13 9501 46 9601 79 9701 113 9601 146 9901 179 10001 213 10101 246 10201 279 10301 313 10401 346 10501 379 10601 413 10701 446 10601 479 10901 00 1 add add 3: ‘d‘ llfi L L E I E E C V 355 ATT6TT66AAA6A6AA6AAT6T6TATA66TTT6A6AAACTATTATA66TA66166TACCT6TTA6CTTA61ATAA6666A66A6CC6TTTCTT6TTTTAA I’Pc v* r*1r41* i: 1’ 1’ V' r r AGACACGAACACAAGGCCGTAAGTTTTATAIGTGAAYTTTGTGCATGTCTGCGAGTCAGCGTCATAATGTGTGTTTTCCAAATCCTGATAATAGTGACGA T7 K’ V7 A 6' T A I l I I 1 0 V P A 6 I 6 A T T T 1 P I Y P P V V 0 6 C6ATCAAA6TAGCT66AAC66CCAACATAAATCATATACACCTTCCTGCAGGACATTCTGCTACAACGACGATCCCCCGATATCCACCA6TT6TC6AT66 T L Y T E T I T I l P I I C I -E- T A T 6ACCCTT 66AAC6AAAC66CAA 6 Y V C L E 6 A I C F T 0 L TACACC6A6AC6T66ACAT66ATTCCCAATCACT CA66CTAT6TAT6TCT66AAA6TCCTCACTCTTTTACCCATTTC lLGVSCIIYAOEIVLITDKFIVDAGSIKOIESLS AtAtiAeuetAtcmscAmAwAmcseAieAAAtcatcrtAceAAcreAtAAAtrtAnstceAteceseAiccAnAMtAAAtAeAArcacuA LIGVPIIFL! it surLEiLl 6L Attire srcrmtmrtcceAAiAtAttcctAicrAcaAAAeuAetAACAAsitwinimtmrmrmrmrncrmnmu I I e 1' A It L e L s s i ncrmrmcsgeum GCAAAGCTGGATGTTGTTGTGGTTGGCGTTT16661CAAGCAAGGGATCGCCTACCCCAAATGTCCAGTCCTATGATC 6 6 I A 0 1 K L 6 L K I F K A L V Y I V 6 0 T 1 I _V- 3 T A V 1 L 6 P TCATCCCAC6CC6ATATCAA6TTGTCATTAAAAAACTTTAAAGCATTACTATATCACCT666A6ATACTATCAAT6TCTC6ACC6CC6TTATACTA66AC 6 P E 1 F T L E F I L F L I Y I -P-’T' C K F V T 1 Y E P C 1 F I P CTTCTCC66A6ATATTCACATT66AATTTA666T6TT6TTCCTCC6TTATAATCCAACCT6CAA6TTCGTCACCATTTAT6AACCTT6TATATTTCACCC K E P E C 1 T T A E 0 6 V C I F A 6 I 1 0 1 L 6 1 A A A I S E I ‘6- CAAA6AACCA6A6T6TATTACTACTGCAGAACAATC66TAT6TCATTTCGCATCCAACATTGACATTCT6CA6ATA6C66CC6CAC6TTCTGAAAATT6T S T 6 Y I I C 1 Y 0 T A 1 0 E 6 V 0 A I L T F 1 E l P 6 F K I K A66ACA666TATC6TA6AT6TATTTAT6ACACC6CTATC6AT6AATCT6T66A66CCA6ATTAACATTCATA6AACCA66AATTCCTTCCTTTAAAATCA 0 V 0 0 Y V A L Y I 6 I P S A 1 Y L 6 T E T AA6AT6TCCA66TA6AC6AT6CT66ATT6TA16T66TT61666TTTATACAATGGACCTCCAACTGCAT66ACTTACATTTATTTCTCAAC66T66AAAC Y L I V Y E I Y I K P 6 F 6 Y K 6 F L 0 I "6 6 1 V 0 E I E A 6 0 I ATATCTTAAT6TATAT6AAAACTACCACAA666666ATTT666TATAAATCATTTCTACAGAACA6TA6TAT66TC6AC6AAAAT6A66CTA6C6ATT66 6 6 6 6 1 I '6' T l l 0 V TCCACCTCCTCCATT AAACCGAGAAATAATGCTACTATCATTTATCATATTTTACTCACATCGCTATCAAT1666666ATTATTATCCTCATACTA6666 V C ’1’ A 1 L 1 I I I I I I I T I 6 L F 0 E Y P K Y I T L P 6 I 0 L 6T61TT6TATTGCCATATTAATTA66C61A66A6AC6ACGTCGCAC6A66666TTATTCGAT6AATATCCCAAATATAT6AC6CTACCA66AAAC6ATCT 6 6 I I V P Y 0 I T C 6 6 I 0 V E Y Y 6 E K S A K I K I I 6 6 6 Y 666666CAT6AAT6TACCCTAT6ATAATACAT66TCT66TAACCAA6TT6AATATTATCAAGAAAAGTC666TAAAAT6AAAA6AAT666TTC666TTAT T A I L K I 0 K 1 I K I L 0 L I ‘ 497 ACC6CTT666TAAAAAAT6ATAT6CC6AAAATTA66AAACGCTTAGATTTATACCACT6ATAT6TACATATTTAAACTTAAT666ATATA6TATAT66AC BTCTATATGACGAGAGTAAATAAACTGACAATGCAAATGAAGCTGATCTATAT1GTGCTTTAYATTGGGACAAACCAC CTCGCACAAGCTU "CAACACA TCCACTCTTGGACAGCTTCATGT1AAAA1AAACTGTAAATCATTCAATGATAATGGGAGAAGAATGTGAGCAAGGATCCATGGTGTCTGCTTTTYATAGA MACCGCAATGCTACATATAA::teeeaatatacctctecccaeaaatgggcggtatgagatgcacggggsaaetacgcegcto 11266 a----> 34 10500 10600 412 10700 119 relevance. Therefore, these sites have not been highlighted in Figure 2. Proposed ORF and predicted polyadenylation signal locations, identification of the -3, +4 translational context nucleotides (Kozak. 1989), as well as the lengths, predicted molecular masses and predicted isoelectric points of the predicted translational products are shown in Table 1. Predicted polyadenylation site locations are based on the pattern of HSVl, in which Us genes have been found to share these signals, utilizing the nearest available site located within or near intergenic regions, resulting in the transcription of 3’ coterminal mRNA families (McGeoch et al., 1985; Rixon 8: McGeoch, 1985). Because these ORFs have not yet been characterized and to simplify identification. seven have been named (Figure 18, Table 1) based on homology (see below) to HSV-l-encoded Us ORFs (McGeoch et al., 1985). When appropriate, the letters MDV will preface the homolog’s name to indicate the ORF's origin. The four non- alphaherpesvirus related ORFs have been arbitrarily named SORF3 l, -2, —3 and 4 (unique short region open reading frame). According to the scanning model for translation, the 408 ribosomal subunit binds initially at the 6’-end of mRNA and then migrates, stopping at the first AUG (ATG) codon in a favorable context for initiating translation (reviewed in Kozak. 1989). As long as there is a purine in position -3, deviations from the rest of the consensus only marginally impair initiation (Kozak. 1989). In the absence of such a purine, however, a guanine at position +4 is essential for eficient translation. In the absence of SI nuclease and/or primer extension analysis, definitive start sites for translation cannot be predicted with any certainty. Except for SORFZ and 4, all of the ORFs contain a potential initiation codon with a purine residue in the -3 position. 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Z... . .,.-8.10.2... .>....v...8s!..2.f:.to&§.t .giflgr. .5... .0232....ix.§0.£.e.o=§.&2:2..o...._...9o.§>§.&§0- . firing-30.0.... mm ...- 0c~ —->:w «NN >¢s son >N> —- —->mz ~n~ >0: \ awn. mm: W.. ...- m —->zw on >u0 —m >~> mm —->m: >0: n~0v km: . M.” ...- pop p->=n np— —->:w ~o >¢a opp —->m: opp >02 “on. 00: «\c «\n 0000 no. >¢a sup >~> ns~ p->m: cop >0: 3200 mm: nxm nxu 0:00 — >¢¢ — —->ns — >0: ~03 «\0 «\n 0000 on v->=w we >N> «up p->nz he >0: 0.»: WWW ...- e >2w mm >80 0n >N> our p->uz Na >0: pa: (1 udauum U83 U52 U810 US1 """L'-)\"s'H' ....... . l t MDV U88 U87 U86 400 E 200 MDV ------------------- MDV 129 amino acids to target for functional analysis employing site—directed mutagenesis. Overall homologies were alternatively viewed using dot matrix homology plots (Figure 4B), in which conserved regions are depicted as diagonals. These conserved regions generally include, and in some cases extend upon, the regions depicted in Figure 4A. Talnen together, these plots illustrate that the conservation of predicted amino acid sequences corresponding to Us genes is often limited to particular regions, emphasizing their evolutionary divergence (Figure 43)- ArulylildMDVglycoproteirmguglandgE. IncomparingthegB homologs of seven different herpesvimses included in the alpha-, beta- and gammaherpesvims subfamilies, there is complete conservation of 10 cysteine residues (Ross et al., 1989). Alphaherpesvims S component glycoproteins have also been found to contain similar patterns of conserved cysteine residues (McGeoch. 1990). HSV-l U86 (gD) contains 7 cysteine residues, 6 appear critical for correct folding, antigenic structure and extent of oligosaccharide processing (Wilcox et al., 1988). Not only are these same 6 cysteines conserved among gD homologs of HSV-2 (McGeoch et al., 1987), PRV (Petrovslds et al., 1986a), EHV-l (Audonnet et al., 1990; Flowers at al., 1991) and BHV-l (Tikoo et al., 1990), but they are conserved by the MDV gD homolog as well (full alignment not shown). Similar cysteine conservation patterns apply to US? (g1) and 088 (gE) homologs (McGeoch, 1990) Figure 4A depicts partial cysteine conservation patterns observed among the gD, g1 and g8 homologs, in which 4, 3 and 6 conserved cysteines are shown, respectively. Careful inspection of the N-terminal regions of the MDV gD, g1 and gE homologs has revealed that all contain the three basic building blocks of signal peptide sequences: a basic, positively charged N-terminal region (n-region), a 130 central hydrophobic region (h-region) and a more polar terminal region (c- region) that seems to define the cleavage site (von Heijne, 1965). Figure 2 shows the likely position of these sites using a recently improved method for predicting signal sequence cleavage sites (von Heijne, 1986). Also included are the location of other characteristic features of membrane glycoproteins, namely, the presence of potential N-glycosylation sites (i.e. N-X-SI'I') and putative hydrophobic transmembrane and charged cytoplasmic domains near the C- terminal end. Like other gI homologs, MDV's counterpart contains a relatively long cytoplasmic domain. However, in contrast to the other 9D homologs, MDV gD’s signal peptide contains a longer n-region (18 residues), that is unusually highly charged (+4; Figure 2) considering an overall mean value of +1.7 among eukaryobs, which generally does not vary with length (von Heijne, 1986). Although a methionine codon exists directly before the hydrophobic h-region at position 6997 in Figure 2 (as PRV’s gD homolog, Petrovskis et al., 1986a), the scanning model for translation (Kozak, 1989) favors usage of the more 6’- proxirnal initiation codon (at position 6943, Figure 2). Despite this prediction, a possible mRNA cap site location between these two ATG sites would preclude the more upstream initiation at position 6943. Comparison of MDV sequences to those previously published. Comparison of sequences of the 'yinilent" GA strain of MDV (Figure 2) with those derived from a 6.6 kbp region of the "very vimlent" RBlB strain of MDV (Ross et al., 1991) has revealed over 99% identity at both the nucleic acid and predicted amino acid levels. One difierence results in an extension of five additional amino acids at the 6’ end of the GA US6/gD (M-N-R-Y-R) relative to its RBlB sequence counterpart (URI-‘6; Ross et al., 1991). In addition to the 6 aa extension, the next four positions in the GA strain (Y -E-S-I) would difier from the corresponding RBlB positions (M-K-V-F). It would be interesting if these signal 131 sequences were found to differentially afiect gD processing in these two strains. The GA 083 sequence is nearly identical to the R313 U83 sequence, however the translational context criteria in this case would predict initiation most likely to begin with the 2nd in-frame methionine codon (position 10 of of the R313 sequence). Our preliminary findings indicate that MDV U83 is expressed as two polypeptides (47/49 kDa), possibly consistent with initiation from both rnethionines (P. Brunovslds, unpublished observation). The predicted amino acid sequence of the GA U82 (Figure 2) is identical to that published in a recent report (Cantello et al., 1991), except for the presence of an alanine in place of an arginine at position 143. This minor difference is due to the inversion of a guanine and a cytosine relative to each other in the two GA sequences. The RBlB counterpart of SORF4 (ORF4 in their report) was recently proposed to be a probable homolog of HSV-1 gG (Ross 8: Binns, 1991). It is tempting to propose such a homology, given their similar locations relative to other Us region genes. We have further tested this proposed homology by similarly aligning these two sequences with CAP, following repeated shufiles of either of the two sequences while maintaining length and composition, (using the IRANdomizations command line option for 100 randomizations). This analysh was performed twice (each time with one of the two sequences shufiled). In doing so, we failed to find a significant difierence in homology score ratios between the actual- versus randomized alignments (1.12 +/- 0.07). In some cases, the homologies of the randomized alignments actually exceeded the proposed MDV ORF4/HSV-l gG alignment. Therefore we don’t consider the proposed homology to be statistically significant. In fact, when using the type of stringency as in the above example, more significant homologies are observed following almost any database search involving a given ORF (data not shown). While we can not absolutely rule out that the two sequences are evolutionarily 132 related. any functional homology would appear absent, since the supposed MDV gG homolog lacks hydrophobic domains representing signal peptide- and transmembrane domain regions. Thus, it would appear that, at the very least, selection pressure for the maintainance of a common glycoprotein function appears to have been lost in this case. DISCUSSION In this report we have identified 3 new ORFs homologous to HSV USl (ICP22), U88 (gE), and U510; 2 new MDV-specific Us ORFs (SORFl and -3), a fowlpox virus homolog (SORF2), and a complete HSV US7-homologous sequence. This extends upon the sequence analysis of a 6,256-bp segment located in the Us region of the R818 stain (Ross et al., 1991; Ross 8: Binns, 1991) and indicates only minor sequence differences in this region between two oncogenic serotype 1 stains, the “Very vinilent" RBlB- and the "virulent" GA stain. With completion of the entire 11,160-bp Us sequence (GA stain), we have precisely determined the IRs-Us and Us-TRs junctions; these were somewhat of a surprise, since previous results using the same MDV stain as ours (GA) mapped the IRs-TRs junction to a difierent fragment located 2—3 kb upsteam of this new location (Fukuchi et al., 1985). Completing the Us sequence necessitated the characterization of entirely new MDV clones; these should now allow for the complete characterization of MDV’s S region, or alternatively serve as probes for finding new homologs among avirulent serotype-2- (naturally avirulent MDV) or serotype-3 (HVT) stains. Alphaherpesvirus 8 regions are characterized by a set of homologs which are specific to members of this taxonomic subfamily (Davison 8: Taylor, 1967; McGeoch, 1990). The identification of 7 alphaherpesvirus S region homologs in this study is consistent with MDV bearing a closer relation to alphaherpesvimses than gammaherpesviruses (Buclcmaster et al., 1988). Furthermore, like other alphaherpesviruses, MDV’s Us region failed to show statistically significant CpG deficiencies that are common to all gammaherpesviruses thus far analyzed (Efstathiou et al., 1990; Honess et al., 1989). 133 134 Molecular dlfierencss between MDV and alphaherpesvirus 8 component assstrategyfordefiningbiologlcaldlvergencesndpsthogenesis. SinceMDV has been taditionally regarded as a gammaherpesvims, much of the previous work interpreting MDV’s properties has proceeded by analogy with the association between EBV and B cells (Wen et al., 1988, for example). Because of the closer genetic relationship between MDV and other alphaherpesviruses, we agree with others (Lawrence et al., 1990) that the lymphotopic properties of MDV and HVT are unlikely to be determined by molecules homologous to those of EBV. In fact, it can be argued that MDV has little in common with gammaherpesvimses, other than a capacity to latently infect and tansform lymphocytes. Moreover, lyrnphotopism (and epitheliotopism) is probably common to all herpesviruses and is largely responsible for the widespread disseminat'on of HSV and VZV in cases involving neonatal and immunocompromised patients, often resulting in death (Nahmias 8: Roizman, 1973; Grose, 1982). These infections are characterized by a biphasic viremia similar to that observed in MDV-infected chickens; in the absence of maternal antibodies, young chicltens can often die from an early mortality syndrome lacking any tumor involvement (Jakowski, et al., 1970; Witter, et al., 1980). Equine herpesvirus-l, an alphaherpesvirus, can also establish latent infections in T-lymphocytes (Welch et al., 1992). This lends support to an earlier proposal characterizing EHV—l as a T-lymphotopic herpesvinis (Scott, et al., 1983). In addition to latent T-lymphocyte infections, MDV also appears to establish latent infections in both Schwann- and satellite cells (Pepose, et al., 1981) like VZV (Croen, et al., 1988). Such complexities suggest that a biologically-based classification system is overly simplistic, potentially misleading, and guided by biases dictated by the limited availability of systems to manipulate and study these vinises in vito. Upon further exarninination, more parallels exist between 135 the "lymphotopic" MDV and the “neurotopic" alphaherpesviruses than previously appreciated. To account for the difierent biological expressions that exist, a renewed focus on molecular difierences between MDV and other alphaherpesviruses may be in order. To account for such difierences, the MDV Us region (and adjoining repeat) may be particularly important. Fifty-three of the fifty-five unique long (UL) region genes of HSV-1 possess an equivalent in VZV (McGeoch et al., 1988); a considerable number of these are related to beta- and gammaherpesvims genes as well (29 of 67 EBV genes are counterpart to VZV UL genes; Davison at Taylor, 1987). In contast, alphaherpesvirus 8 component are specific for members of this taxonomic subfamily and appear to represent their most divergent coding region (Davison 8: Wilkie, 1983; Davison 8: McGeoch, 1986). In comparing MDV with other alphaherpesviruses, significant divergence also extends to the UL -t1anking repeat regions (Buckmaster et al., 1988) which are known to be expressed in tumor cells (Schat et al., 1989; Sugaya et al., 1991). A comparison of the genetic organization of alphaherpesvirus S segment genes is presented in Figure 6. Despite obvious similarities, there are marked difierences in (i) gene content, organization, and localization; (ii) sequence conservation; and (iii) positioning of IRs/Us and Us/TRs junctions. Nevertheless, these overall gene layout are consistent with a model to account for the divergence of alphaherpesviruses from a common ancestor by a number of homologous and semi-homologous recombination event which result in expansion or contaction of the inverted repeat regions and a concomitant loss or gain of Us gene(s) (Davison 8: McGeoch, 1986). In the case of VZV, homologs of six HSV-l Us region genes are missing (U82, U84, U86, U86, U811, U812). Unlike all other alphaherpesvimses thus far analyzed (Figure 6), MDV appears to lack a U89 homolog. 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RUB >05 m h m m N or _. 138 known to encode a differentially phosphorylated 12-20 kDa tegument protein which becomes associated with nucleocapsids at or, soon after, their formation in the nuclei of infected cells (Frame et al., 1986). A recent study has suggested that PRV’s U89 homolog has a function associated with envelopment at the nuclear membrane (Pol et al., 1991). Lacking such a homolog might explain MDV’s characteristic inability to become stably enveloped in tissues other than the feather follicle epithelium. Presence of MDV-specific and fowlpox virus-homologous genes. Unlike other alphaherpesviruses, MDV contains at least 3 MDV-specific ORFs in it Us region SORFs 1, -3 and -4 (Figure 6). Virus-specific 8 component ORFs have also been identified in HSV-1 and -2 (USS, USll and U812; Davison & McGeoch. 1986; McGeoch et al., 1986) and in EHV-l (ORF67, 71, and 76, Telford et al., 1992; EUS4, Colle et al., 1992). Further sequence analysis of other alphaherpesvirus 8 regions will be necessary to confirm whether such genes are truly unique to given herpesviruses. SORF3, located in the EcoRI-O subfragment (Figure 18), specifies a 351 aa MDV-specific ORF. Considering it location, preliminary tanscriptional mapping of the other genes mapping in EcoRl-O (e.g. MDV U81 and -10) (P. Brunovskis, unpublished observations) and previously reported data (Schat et al., 1989), it appears possible that SORF3 may code for the 1.1 kb A, tanscript, one of four immediate-early tanscript consistently identified in all MDV tumor cell lines tested (Schat et al., 1989). A major surprise from this work was finding a FPV-related ORF. We are not aware of any other examples of such conservation across vinis family lines, except a few cases that include cellular counterpart as well. MDV‘s FFV homolog, SORF2, was found to be 67 96 similar and 42% identical (over 100 a) to FPV ORF4 (Tomley et al., 1988). With a Fast A score of 237 and the alignment 139 in Figure 3, the level of conservation is in many respect more stiking than that which characterizes alphaherpesvirus 8 region homologies (Figure 4). Interestingly, compared with FPV ORF4, SORFZ contains an amino-terminal extension of 82 aa; conversely, ORF4 carries a carboxy-terminal extension of 41 aa. The block of conserved sequences may encode one or more functional domains that have independently evolved following host cell-acquired gene tansfer. On the other hand, it is intriguing to consider the possibility of virus- virus gene tansfer. Individual cells have been found to be simultaneously cohabited by MDV and FPV (Tripathy et al., 1976). Given the difierent modes of replication for MDV and FPV (e.g. nuclear vs. cytoplasmic) such a possibility could point to a possibly novel form of gene tansfer. WVUsreglongemsupotentlaldeterminantforpathogenssband tissue to”. Recent studies have shown that 11 0f 12 open reading frames contained in the HSV-1 Us region are dispensable for growth in vitro (Longnecker et al., 1987; Roizman 8: Sears, 1990). These, and other “dispensable“ genes appear to specify functions for optimal survival, maintenance and dissemination among the host (and it population at-large), rather than the presence of functions necessary for replication (Longnecker et al., 1987; Roizman 8: Sears, 1990). The significant divergence of alphaherpesvirus 8 component may reflect this region’s capacity for determining distinct tissue- and host cell growth potentialities. Previous result have suggested that the product of HSV U81 (ICP22) encodes a determinant for ttsue topism, since it function appears to be dispensable for growth in some cell lines, but not others (Sears et al., 1985). Considering the extensive genetic divergence among a cluster of different glycoprotein homologs, each potentially subject to glycosylation, phosphorylation, palrnitylation, myristylation and/or sulfation (Crose, 1990), a potentially large window exist for the creation of 140 multiply distinct virus-cell interactions which can afiect host range, tissue tropism, invasiveness and cell-cell spread. Previous result have demonstated that “nonessential” alphaherpesvirus glycoproteins encode functions associated with virulence (Lominiczi et al., 1984; Meignier et al., 1988; Mettenleiter et al., 1968; Roizman 8: Sears, 1990). This may reflect their ability to promote the infection and spread of vinis in vivo (Lominiczi et al., 1984; Longnecker et al., 1987; Mettenleiter et al., 1988; Pol et al., 1991; Card at al., 1992). Consistent with this proposal is the observation that a specific deletion of PRV g1 (homolog of HSV gE) was found to reduce the spread of infection in both rat (Card, at al., 1992) and pig (Kimman, et al., 1992) cental nervous systems. This defect could reflect the inability of PRV gI mutant to promote cell-cell spread (Zsak et al., 1992). Recent findings (M. Wild, personal communication) indicate that the HVT Us region is no more than 7.6 kb and contains homologs of alphaherpesvinis Us region genes; the latter result conflict with an earlier hybridization-based report indicating a lack of homology between MDV and HVT Us regions (Igarashi et al., 1987). A difierence of at least 3.6 kb suggest that avinilence may reflect an absence of Us homologs or other ORFs that are common to pathogenic MDV stains. If MDV Us region genes specify virulence determinant, these could indirectly affect oncogenic potential by afiecting any number of critical event which precede tumor induction. Previous studies have shown that oncogenic potential appears to be directly correlated with cell-associated viremia levels and the capacity to cause irnmunosuppression (Calnek 8: Witter, 1991). The sequence of event leading to tansforrnation include (i) an initial lytic growth phase in B cells, which is thought to cause activation and expansion of T-cells; (ii) latent growth phase involving infected T-cells; (iii) a second wave of lytic 141 infection, coincident with permanent immunosuppression; and (iv) oncogenic tansformation (Calnek, 1986). Attenuated MDV strains (derived from oncogenic serotype 1 stains), as well as nononcogenic MDV and HVT stains (serotypes 2 and 3, respectively), are deficient in inducing the early cytolytic infection of B cells in chickens, suggesting that their cell topisms difier from oncogenic stains (Schat et al., 1986; Shek et al., 1982). This is reflected in additional findings which show that attenuation of MDV leads to a marked reduction in infectivity and/or replication in lymphocytes (Schat et al., 1986). In conchlsiat. The current herpesvirus classification system has been described as ‘simple, fortuitously appropriate. and defective’ (Roizman, 1990b). It has been further suggested that ‘the delineation and evolutionary relatedness of genes responsible for biological properties may be a more significant criterion for both evolutionary relatedness and classification than the arrangement and evolution of genes conserved throughout the family Herpesviridae, although they are not yet known’ (Roizman, 1990b). While such a proposal is subject to debate, inasmuch as the alphaherpesvirus-specific Us region is known to specify a cluster of supplementary essential functions thought to be important in defining biological properties, our sequence provides a foundation for further MDV studies aimed at identifying some of the genes responsible for the apparent discrepancy between MDV’s genetic- and biologic properties. 142 ACKNOWLEDGEMENTS We thank O. McCallum, R. Stinger, K. Nickels, S. Peacock and C. Mulks of Michigan State University for their excellent technical assistance; H.-J. Kung and D. jones for sharing unpublished sequence data; M. Nonoyama and P. Sonderrneijer for kindly providing clones; P. Coussens, B. Silva, X. Chen, R. Patterson and S. Spatz for many helpful discussions and suggestions; and T. Smith for help with some of the gE subclones. 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Latent equid herpesvinises l and 4: detection and distinction using the polymerase chain reaction and cocultivation from lymphoid cells. I. Gen. Virol. 73:261-268. MW. C., D. Long. D.L.Sodora.R.I.E-iisenberg.andG.H.Cohen. 1988. The contibution of cysteine residues to antigenicity and extent of 147 194111ng47°f herpes simplex vinis type 1 glycoprotein D. I. Virol. Wilts. R. L.. I. M. Sharma. and A. M. Fadly. 1980. Pathogenicity of variant Marek's disease virus isolant in vaccinated and unvaccinated chickens. Avian Dis. 24:210-232. Dung. G. and D. P. Leader. 1990. The stucture of the pseudorabies virus genome at the end of the inverted repeat sequences proximal to the 'unction with the short unique region. I. Gen. Virol. 71:2433-2441. Isak. F. Zuckermann. N. Sugg. and '1'. Ben-Porat. 1992. Glycoprotein gI of pseudorabies virus promotes cell fusion and vinis spread via direct cell- to-cell tansmission. I. Virol. 66:2316-2325. mm Marek'sdiseassvhuexmessesanmunualfl-klhlatclasscytoplasnfic phosphou'othihomologomtomsBB-kDaherpesshnplethrmdiats-early mislearphosphoprotetrLICPZB 148 ABS'I'RAL‘II Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus thatcancauseTcelllymphomasasquicldyasthreeweekspostinfection (fora recent review, see Calnek and Witter, 1991). The recent knowledge that Marek's disease virus is phylogenetically more related to alphaherpesviruses than gammaherpesviruses raises interesting questions regarding the seeming incongruence between herpesvirus genetic- and biologic properties. To examineflienanteoftiesedifierences,wehavebegimtofocttourattenfion on the MDV 8 region, inasmuch as alphaherpesvinis 8 regions are known to contain clusters of genes specifying functions associated with pathogenesis and viral dissemination instead of those essential for vinis production. In this study we have characterized the MDV homolog of HSV-1 ICP22, an immediate-early polypeptide thought to specify a determinant for tissue topism. Northern blot analysis of RNA from MDV-infected cells identified a 1.9 kb PAA-sensitive USl tanscript. Sl-nuclease protection analysis mapped it 5’ and 3’ ends to sites located near TATA- and poly (A) + consensus sequences; the length of the 81- protected region was consistent with that of the full-length tanscript. Protein studies were carried out by irnmunoprecipitation analysis of PMSF-teated lysates from MDV-infected cells using monospecific, polyclonal antibodies generated from rabbit immunized with bacterially-synthesized tpE—USI fusion proteins. In the absence of multiple protease inhibitors, the MDV USl polypeptide was characterized as a 27-kDa MDV U81-encoded phosphoprotein, designated pp27; in their presence, a less abundant 24-kDa phosphoprotein was coprecipitated. In contast to the 68-kDa nuclear HSV ICP22, MDV pp27 synthesis was almost completely dependent on viral DNA replication and its localization was resticted to the cytoplasm of infected DEF cell cultures. Pulse-chase experiment identified at least three distinct polypeptides of 149 150 approximately 27 -kDa that were characterized by reduced mobility with longer chase periods. A larger. difiuse band approximately double in size (53-kDa) was observed to undergo a similar processing pattern. The 63-kDa polypeptides appeared to bear a direct relation to pp27 .24 since it polypeptides could be precipitated from lysates boiled in the presence of 2- mercaptoethanol and SDS under conditions that led to the loss of all non- specifically precipitated polypeptides. Identification of the larger phosphoprotein(s) was enhanced following electophoresis under non-reducing conditions; together these findings suggest that pp27,24 forms disulfide-linked dimers under native conditions. INTRODUCTION Marek's disease virus (MDV) is an acutely tansforrning herpesvirus which causes T-cell lymphomas in chickens. Marek's disease (MD) isolates have also been shown to cause lymphoproliferative neural lesions, peripheral nerve demyelination, blindness. and paralysis (for review. see Calnek and Witter. 1991). Based on it lymphotopic properties. Marek’s disease virus (MDV) has been classified as a gammaherpesvirus (Roizman et al., 1981). However, recent evidence (Brunovskis and Velicer, 1992a; Buckmaster et al., 1988; Ross et al., 1989; Ross and Binns, 1991; Ross et al., 1991; Scott et al., 1989) indicates that MDV bears a much closer phylogenetic relationship to alphaherpesviruses, characterized by it prototype, herpes simplex virus (HSV). MDV is known to contain at least 7 alphaherpesvinis-related genes in it unique short (Us) region (Bnmovskis and Velicer, 1992a). Alphaherpesvirus Us (and other 8 region) genes are unique to members of this taxonomic subfamily (Davison and McGeoch. 1986; Davison and Taylor, 1987; McGeoch. 1990). They are particularly noted for encoding a cluster of genes that are nonessential for growth in cell culture (de Wind at al., 1990; Longnecker and Roizman, 1987; Longnecksr et al., 1987; Sears et al., 1985; Weber et al.. 1987). Unlike their wild-type parents, HSV Us region gene mutant grow poorly in animal host models and are consistently associated with reduced levels of virulence and/or the capacity to induce latency (Meignier et al., 1988). The strikingly different biologic- and pathologic expressions that characterize herpesviruses are largely thought to be determined by the nonessential or supplementary essential genes which confer functions enabling eficient dissemination and growth in various tissues in the face of an immune system poised for it elimination (for discussion, see Roizman. 1990). 151 152 Unlike the L region, which appears to exhibit significantly greater conservation (McGeoch et al., 1988) and specifies genes shared by representatives of all three herpesvirus subfamilies (Davison and Taylor, 1987; Kouzarides et al., 1987). alphaherpesvinis 8 regions are substantially more diverse (Brunovskis and Velicer, l992a; Davison and Taylor, 1987). This is apparent from both genetic organization- and sequence homology levels. HSV-1 ICPZZ and it related alphaherpesvirus counterpart exhibit significant differences in length, amino acid (a) conservation, and tanscriptional kinetics (Holden et al., 1992; Zhang and Leader, 1990; M. Schwyzer, pers. comm). Unlike other HSV immediate-early (IE) proteins, such as ICP4. the role of ICPZZ is comparatively obscure. Evidence suggest that the ICP22-encoding gene of HSV-1 (i.e. U81) specifies a tissue topism determinant (Sears et al., 1985). Although the 68-kDa ICP22 polypeptide is dispensable for growth in some cell lines. ICPZZ mutant grow very poorly in others. This has led to the suggestion that certain cell lines (e.g. Vero) contain a host ICPZZ-like function that allows for the mutant to grow (Sears et al., 1985). Such a function may be related to gene regulation. Defect in ICP22 expression are associated with a reduction in late (L) gene expression (Epstein and Iacquemont, 1983; Iacquemont et al., 1984; Sears et al., _1985). VZV’s ICP22 homolog was recently reported to repress the IE promoter of its ICP4 homolog, but stimulate the early (E) promoter of it TK homolog (Iackers et al., 1992). Specific immunological reagent for ICP22-related proteins have only been created for HSV-l; however, while this synthetic peptide-derived antiserum was able to react with full-length and tuncated ICP22 derivatives by Western blot analysis. the antiserum was unable to irnmunoprecipitate these polypeptides (Ackermann et al., 1985). In this study we have characterized MDV U81 expression at the tanscriptional level by Northern blot- and 81 nuclease 153 protection analyses and at the tanslational level by immunoprecipitation studies using polyclonal antisera specific for the polypeptide encoded by MDV USI . Compared with it HSV-l ICP22 counterpart, the MDV homolog exhibited a number of stildng contast of potential significance to MDV's unique biological proportion. MATERIALS AND METHODS Cells and viruses. The preparation of primary duck embryo fibroblast (DEF) cells was by established methods (Solomon, 1975). Cells were seeded in lOO-mm diameter plastic tissue culture plates or large roller bottles (for RNA isolations) with 1x107 or 4x108 cells. respectively and a Medium 199/nutrient mixture F-10 combination containing 4% calf serum (C8). Pathogenic MDV GA stain-infected cells (passage level 6) and HVT vaccine virus stain FC-126- infected cells (passage level 13) frozen in liquid nitogen were used to infect 85- 9096 confluent DEF cell monolayers at a 1:6-1 :8 dilution in growth medium containing 4% CS. The following day and thereafter, virus-infected cells were maintained in growth medium containing 0.2-1.096 CS. In some experiment. cells were teated with cycloheximide (Cl-DI; l00 pg/ml) or phosphonoacetic acid (PAA; 200 pg/ml) beginning 2 hr before- or 24 hr after infection, respectively- Aritbsra Immune chicken sera (ICS) was pooled together from convalescent chickens naturally exposed to MDV. 2BN90.1. a nuclear antigen- specific monoclonal antibody, was kindly provided by L. Lee (USDA Avian Disease and Oncology Laboratory, E. Lansing, MI). m bolafien and Northern blot analysis. Total cellular- and poly (10+- purified RNA (via oligo (dT) -cellulose chromatography) was isolated from mock- and vinis-infected DEF cells by the guanidinium isothiocyanate procedure as previously described (Sithole et al., 1988). fractionated on formaldehyde-1.2% agarose gels and blotted onto nitocellulose or nylon-based membranes by standard methods (Maniatis et al., 1982). More recent RNA isolations were carried out by the acid-guanidinium thiocyanate method (Chomczynski and Sacchi, 1987). Probes were labeled with 32P-dCTP, initially by nick-tanslation 154 155 and more recently by the random priming method. according to instuctions supplied by the manufacturer. Standard hybridization and wash conditions were employed (Maniatis et al., 1982). SI-nuclsus protectltm analysis. Sl-nuclease protection analysis was performed as described elsewhere (Maniatis et al., 1982) using 1 pg of poly (A) + mRNA per reaction. Probes were prepared by digestion of pBSM-O with Aval followed by 5’- or 3’ (end)labeling with polynucleotide kinase or Klenow Wm. mpectively. The 6’- and 3'-labeled probes were gel-purified from 0.796 low melting point agarose gels following secondary digestions with EcoRI or ClaI, respectively. A 3’ end-labeled (Klenow) pBR322/Hinfl digest was used as a molecular weight standard. Expressionsndpurlficatlonoffirdonprotelninmmogens. The 1.8kb NcoI-Clal fragment of pBSM-O was blunt-ended at the NcoI site with Klenow fragment and subcloned into SmaI/ClaI-digested expression vector, pATHZ (kindly provided by R. C. Schwartz) using CaClz-competent E'. coli RRl cells. The resulting constuct allows for the NHz-terminal 323-aa of trpE fused (by way of an inserted multiple cloning site) with MDV USl (a 56-172; Brunovskis and Velicer. 1992a). Induced expression and purification of trpE-USI was according to a procedure recently described (Koerner et al., 1991). Inmmixatlons with trpE-USI fusion proteins. 1.0-1.6 mg of 1x sample bufier-solubilized tpE-USI was electophoresed through a 3-mm thick 7.596 SDS-PAGE gel (preparative) and stained with Coomasie Blue. The entire stip containing tipE-USI was excised, washed in distilled water for 1 hr and homogenized in 1-2 volumes of Freund’s complete- or incomplete adjuvant by serial passage through 18-. 20-, 22- and 25-gauge syringe needles. Primary injections (subcutaneous route) of female New Zealand white rabbit involved the use of 750 pg of fusion protein in Freund’s complete adjuvant; subsequent 156 injections employed 376 pg of fusion protein in Freund’s incomplete adjuvant. The rabbit were boosted once a month; serum was collected 10-14 days following each boost. Iadlolabsling d protiru. Mock- and virus-infected DEF cells were labeled with so pCi/ml of [3551.methionine (specific activity, 1,000 pCi/mmol, ICN) at 48-72 hrs. post-infection for 2-4 hrs. as previously described (lsfort et al., 1986) with the following modifications. In later experiment, lysis bufiers containing phenylmethylsulfonyl fluoride (PMSF) were supplemented with the following additional protease inhibitors; 50 lug/ml tosyl-lysyl-chloromethyl ketone (TLCK). 50 rig/ml tosylarnide-phenylmethyl-chloromethyl ketone (TPCK), and 2 pg/ml aprotinin. DEF cells were similarly labeled with szP-orthophosphate with the following modifications. Phosphate-free Dulbecco modified Eagle medium wasusedfor3hrbefore-andduringthe2hrlabelingperiodinwhich250 pCi/ml 32F; (carrier-free; Amersharn Corp.) was used per 60 mm plate. Pulse- chase labeling with [358] -methionine was done as previously described (Chen et al., 1992), in this case employing a 6 min pulse followed by chases of 0, 5, 15, 30, 60, 180 and 300 min. WW and SDS-PAGE analyses. Immunoprecipitation/SDS- PAGE analyses were carried out as previosly described (lsfort et al., 1986). The specificity of tpE-USl-derived antisera was tested by preincubating 970 pl PBS with 20 pl lx sample bufier-solubilized trpE-USI and 10 ill U81-specific antisera (or preimmune contol serum) for 30 min at 4 C. Labeled cell lysates were then added and processed as before. In some cases, lysates were adjusted to 1% SDS and 1% 2-mercaptoethanol (2-ME) and boiled for 5 min prior to the addition of antibodies (Sirnek and Rice, 1988). Immunoprecipitates were washed 2x with 1x phospholysis bufier (PLB) and electophoresed on 10% SDS-PAGE gels. 3H- 157 labeled protein markers (BRL) were included to allow for molecular weight estimations. Subcellulu fiecfltmatlui. Cells from 100-mm mock- and GA-infected plats were labeled with (“SI-methionine when extensive cytopathic streets were observed (72 hr p.i.), washed 2x with ice-cold PBS (pH 7.2). and scraped from plates with 1 ml ice-cold PBS (pH 7.2). Cells were precipitated in a microfuge (1000g. 10 min) and teated for 10 min on ice with 1 ml NP40 lysis bufier containing PMSF, 'I‘PCK. TLCK, and aprotinin. Nuclei were pelleted 10 min at 1,000g, and the cytoplamic supernatant fraction was retained. The nuclei were again washed with 1 ml NP40 lysis buffer, and pelleted as before. Nuclei were disrupted by the addition 1 ml of 1x PLB containing the above protease inhibitors. Whole cell fractions were obtained by substituting the NP40 lysis buffer addition step (above) with 1 ml 1x PLB as above. The three fractions were adjusted to equal volumes using lx PLB. 60 pl of lysate were used for each irnmune-precipitation. RESULTS Northamblot-andSl-rmcleaseprotectlonanalysedeVUSl. Priorto the identification of USI -related coding sequences by nucleotide sequencing, tartcriptional mapping studies employing a radiolabeled EcoRI-O probe (Figure 1B; Silva and Witter, 1985) identified a 1.9 kb rightwardly-directed tartcript (Figure 2A, lane 2; Figure 2B, lane 4). In DEF cells teated with PAA. an inhibitor of MDV DNA replication (Lee et al., 1976). the 1.9 kb tanscript was reduced to scarcely detectable levels (Figure 2C, lanes 6,7). Identical result were subsequently obtained using a probe limited to MDV USl coding sequences (data not shown). Expression of an MDV late contol gene, gp67-65 (Coussens and Velicer, 1988) was found to be similarly affected by the addition of PAA (Figure 2. lanes 9,10). The reduced RNA levels were specifically attibutable to PAA inhibition, rather than differences in the level of RNA loaded. inasmuch as similar levels of 188 and 288 rRNA were observed in the PAA+ and PAA- lanes following ethidium bromide staining of the gel prior to Northern tansfer (data not shown). With longer exposures, the 1.9 kb USl tanscript could be detected at a low level in PAA-teated cells as well. suggesting a gamma-l (leaky-late) mode of temporal regulation. However, in the absence of tue one-step growth conditions for growth of MDV in cell culture, it is not clear whether the "PAA-insensitive“ tanscript may have been tue late (gamma-2) tanscript present in the infected cell inoculum used to infect new cells prior to the addition of PAA. To map the precise location of the 1.9 kb tanscript, an 81 nuclease- protection experiment was performed. Following hybridization of the 5’-labeled 0.72 kb EcoRI-Aval probe (Probe I, Figure 3C) to 1 pg of MDV-infected poly (A)+ and subsequent digestion with 81 nuclease, two fragment of 510 and 622 158 159 FIGURE 1. Localixafioriofthe 1.9kbMINEcoRI-Otanscrlpt. A) MDV genome stucture and BamHI restiction map. B) Eco RI and Barn HI restiction maps identifying the area of focus. 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Basmamoaadossaaa was 80 teas 3208;02:3de as a; tea: 308005-8an as, 332 as; 0:8 000 8083-803 8.30 >02 .8 -60.: 200 0.82 sewage»? :0: do 83020020 eds 00008.0 Gas: 2 .35.: .szasebee :0: >02 2: as 8:582 32.803 0.3 debatsastaas assesses..— .s 850:: 168 0w 0' NF p.. 1" I 12' co_u0~__000_ ..0_s__0oesm .0 ezoa< £0“: .06.: .0020 mem .l.I\H AOIN )IOOW 3_0_:0000 0030.0m .m dun m00 E9500: 0~00>_ll>_co umsE m N _. '0.? lemon lauds . I00 I00 . 000. Iluz_I|I + .+ I u. .I.. m d w: 3 w "5:5 . n .0503 m .0000 3.230000 Fmadm 00000328 F0: .6 eozeozzcs... .< 00 .0000 .mocbmdiod F0: >05. 0.: ...o zoFN> A032 .000=0> 0:0 0226ng >94 to 002080: GD 05 com 000003000 300 0550 03300:“ .8035. $000 “00380826053038: 0 00ch 183 Pm one .00 000 mu man an mun 90 oh _. 00 .00. 009050 £0 59.0.. m: 28 25220 .o Equaamm 0:0 05.3.3.8 .0 00 300: ...0 :00. :.>0: $05: 0030 50. SE .725. 082. "00:00 O? H A: 32:02 oxo .m>< mm H Amv 3:0:53 oxo .m>< \ ’ 0 , \ . A , [ll-LI .330. 0020200 $0040.20: ANNQU_:mD ....O mozmwmmao DZ< >00.._OS_OI com 80 con 8 F 0 A00. _ . . . . . . . _ . . m m "1| “Hg T>mI w J“ m m m m n. _lnm\NN_ _INnxomu— >m i m m T>Im _+20_ L : >N> m mm m «.30 m H mm m m >05. 184 sequences are primarily limited to a 103-as region which fails to exhibit any obvious structure-function relationships (Figure 7). Pairwise. comparisons between ICP22 homologs in this region result in average similarities and identities of 65% and 4096, respectively. However, even in this conserved region, the level of homology can range from as low as 45% similarity/26% identity (MDV, HSV-l) to 75% similarity/68% identity CEHV-l, PRV). Perhaps this region allows for the creation of unique protein-protein interactions responsible for diflerential gene expression depending on the cell type. It is possible that ICP22-related homologs exhibit a basic level of functional conservation conferring interactions with itself and/or transcription factors. ICP4 has previously been shown to homodimerize (Metzler and Wilcox. 1985) and operationally substitute for the cellular transcriptional factor Spl in promoting the eficient expression of the viral thymidine kinase gene. Our preliminary results suggest that pp27,24 can also homodimerize. Further work will be necessary to confirm this possibility and to determine the significance of its cytosolic localization in relation to MDV growth. REFERENCE WM..MSarndsnto.sndB.Rolxmm 1985. Applicationofantibodyto synthetic peptides for characterization of the intact and truncated «:22 protein specified by herpes simplex virus 1 and the R325 022' deletion mutant. J. Virol. 86:207-215. MLP..R.C.Costs.LE.Holland.andE.l.Wagnsr.1980. Characterization of herpes simplex virus type 1 RNA present in the absence of de novo protein 8 thesis. J. Virol. 34:9-27. Bechr.).,U.Leser.M. AngfordW.Jilg.H.Gelderblom.P. Relchart. and H. Wolf. 1991. 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The structure of the pseudorabies virus genome at the end of the inverted repeat sequences proximal to the junction with the short unique region. I. Gen. Virol. 71:2433-2441. ChapterV AmlysbofMusk‘sdlseuavhumdqueshonreglonpolypepfldeswlthuubara mummywmm 190 ABSTRACT Our recently completed nucleotide sequence of the Marek’s disease virus (MDV) unique short region identified 7 alphaherpesvinis-related open reading frames (ORFs), 3 MDV-specific ORFs, and a novel fowlpox vinis homolog (Brunovskis and Velicer, l992a). To facilitate characterization of the polypeptides specified by these ORFs, monospecific, polyclonal antisera were generated from a panel of 16 difierent bacterially-expressed trpE fusion protein immunogens representing 9 of the 11 Us region ORFs. The resulting antibodies were found to imrnunoprecipitate 6 of the 7 alphaherpesvinis-related Us homologs from MDV-infected cell cultures. U82-specific antibodies precipitated a 30-kDa polypeptide; USlO-specific antibodies, a 24-kDa phosphoprotein. Three different antisera were all found to precipitate a 47,49-kDa doublet con'esponding to the MDV protein kinase-related product, U83; one of these antisera specifically reacted with a 68-kDa cellular protein as well. In in other alphaherpesvirus systems. antisera was found to coprecipitate 46- and 62-72-kDa glycoproteins homologous to HSV U57 (g1) and U88 (gE), respectively. Boiling of lysates in 1% SDS/Z-mercaptoethanol failed to prevent the coprecipitation of gl and gE. This suggests that, like , MDV g1 and gE share common epitopes in a similar fashion as their related varicella-zoster homologs. Expression of polypeptides encoded by U53, -7, -8, and 10 was inhibited by phosphonoacetic acid treatment; this suggests that they are late proteins. possibly associated with virion components. Antibodies reactive with the U86 (gD) epitopes of three dihrent bacterially-expressed fusion proteins failed to precipitate gD fi'om avian cell cultures. A consideration of the results obtained raises a number of interesting questions highlighting the potential importance of Us region genes in determining a number of MDV’s unusual biological properties. 191 INTRODUCTION Marek'sdiseasevirusfldDWisanavianherpesvimsthatcancauseT cell lymphomas as quickly as three weeks postinfection (for a recent review, see Calnek and Witter, 1991). Although numerous studies have addressed its complex pathogenesis, molecular biological studies have lagged behind those of other herpesvirus systems. The major dificulty of working with MDV is that it is strongly cell-associated and lacks a suitable cell culture system amenable to fully productive infections. Primary chick- or duck embryo fibroblasts are permissive for MDV replication. Such cell cultures are characterized by slowly progressing, semi-productive infections, which fail to result in the production of enveloped cell-free vinis. These factors necessitate passage of infected cells onto uninfected cell monolayers in order to obtain suficient quantities of material with which to work. This precludes straightforward plaque purification; consequently, isolation of mutants is much more dificult. Such condin'ons also preclude the establishment of one-step growth conditions for efiective temporal gene regulation studies. Fully productive infections are restricted to the feather follicle epithelium (FFE); PPR-derived cell-free vinis titers are generally limited to just 104 PFU/ml. Together, these dificulties help account for the characterization of only four genes and their gene products reported in the current literature. These correspond to the MDV homologs of the HSV glycoproteins gB (Chen and Velicer, 1992; Isfort et al., 1986b), gC (Binns and Ross, 1989; Isfort et al., 1986a; Coussens and Velicer, 1988), and two MDV- specific products, pp38 (Chen et al., 1992a; Cui et al., 1991) and meg (Iortes et al., 1992). Recent data demonstrating a closer phylogenetic relationship between MDV and alphaherpesviruses than between MDV and gammaherpesviruses 192 193 (Binns and Ross, 1989; Brunovskis and Velicer, l992a; Buckmaster et al., 1988; Ross et al.. 1989; Ross and Binns, 1991; Ross et al.. 1991: Scott et al.. 1989) has focused renewed attention on alphaherpesvirus systems as a source for clues to shed light on the many complexities and paradoxes associated with MDV. These related human and animal herpesvirus systems provide a wealth of useful information that can facilitate rapid progress irt the MDV field. Complete nucleotide sequences are now available for three alphaherpesvinis members, herpes simplex virus (HSV: McGeoch et al., 1988), varicella zoster (VZV; Davison and Scott. 1986), and equine herpesvirus (EHV; Telford et al.. 1992). Gene sequences are ohen of limited use in the absence of additional information relating to the characterization of their products. Many of the latter studies have been carried out with HSV; new information resulting from these studies provide a foundation for further studies of their related counterparts in other alphaherpesvinis systems. We have recently determined the complete nucleotide sequence for the MDV Us region (Bnmovskis and Velicer, 1992a). Our analysis identified at least 11 open reading frames (ORFs) likely to code for proteins; of these, 7 represent homolog exclusively related to alphaherpesvirus S region genes. These include MDV counterparts of HSV USl (ICP22), U82, U83 (protein kinase), U86, U87 and U88 (glycoproteins gD, g1 and gE. respectively) and USlO. Three additional ORFs were identified with no apparent relation to any sequences found among herpesvintses or present in the existing databases, while a fourth was found to be homologous to a fowlpox virus ORF. Since the Us region exhibits significant genetic diversity, pathogenic potential, and the presence of functions nonessential for growth in cell culture, further MDV studies involving Us region genes are likely to shed light on the difierent pathogenic expressions associated with members of the 194 alphaherpesvirus subfamily (Bnmovskis and Velicer, 1992a). Unlike HSV or VZV, the MDV system offers unique opportunities for studying the interaction of these genes in their natural host. The facilitation of such studies often requires suitable immunologic reagents. In this report we present a preliminary analysis of MDV Us region polypeptides utilizing such reagents. We have employed inducible expression vectors (pATH series; Koerner et al., 1991) to create a series of trpE-MDV fusion protein irnmunogens. Sixteen different fusion proteins corresponding to nine of the eleven MDV Us ORFs were expressed irt E. coli. Our preliminary analysis succeeded in characterizing a group of polyclonal antibody preparations capable of precipitating all seven MDV Us region alphaherpesvinis homologs. MATERIALS AND METHODS Cells and virtues. The preparation and cultivation of primary duck embryo fibroblast (DEF) cells was by established methods (Solomon, 1976). Pathogenic MDV GA strain-infected cells (passage level 6) and HVT vaccine virus strain FC-126-infected cells (passage level 13) frozen in liquid nitrogen were used to infect 85-90% confluent DEF cell monolayers at a 1:6-1 :8 dilution in growth medium initially containing 4% calf serum. In some experiments, cells were treated with phosphonoacetic acid (PAA; 200 pg/ml; Lee et al., 1976) at 12 hr postinfection. Antisera. Immune chicken sera (ICS) was pooled together from convalescent chickens naturally exposed to MDV. RaUSl sera has been described (Brunovskis and Velicer, l992b). Normal rabbit serum (NRS), normal chicken serum (N CS) from specific pathogen-free (SPF) chickens, and rabbit anti-chicken IgG were obtained from Sigma Chemical Co. Antisera generated from the study are described in the text. Cloning of plasmid constructs for bacterial fusion protein expression. Sequence analysis of the MDV Us region (Brunovskis and Velicer, 1992a) led to the identification of useful restriction sites (Figure l) for cloning of in-frame Imions with the amino terminal 323-aa of ttpE. MDV inserts were gel-purified (LMP agarose). blunt-ended. when necessary, and ligated into the multiple cloning site of the appropriate pATH expression vector (kindly provided by R. C. Schwartz); CaClz-competent E. coli RRl-transforrned cells were mini-prep- screened to facilitate isolation of the desired recombinants. hpreselonandpurlficatlenoffitslonprotelnirmmogens. Induced expression and purification of trpE-fusion proteins was carried out according to a recently published procedure (Koerner et al., 1991). 195 196 Immunisation with trpEfttslen retains. 1.0-1.5 mg of 1x sample bufier- solubilised tips fusion proteins were electophoresed through 3-mm thick 7.596 SDS-PAGE gels (preparative) and stained with Coomasie Blue. Fusion proteins were washed in distilled water for 1 hr and homogenized in 1-2 volumes of Freund’s complete- or incomplete adjuvant by serial passage through 18-, 20-. 22- and 26—gauge syringe needles. Primary injections (subcutaneous route) of female New Zealand white rabbits involved the use of 750 pg of fusion protein in Freund’s complete adjuvant; subsequent injections employed 376 pg of fusion protein in Freund’s incomplete adjuvant. The rabbits were boosted once a month; serum was collected 10-14 days following each boost. Radlolabeling of proteins. Mock- and virus-infected DEF cells were labeled with so ..Ci/ml of [3531-methionine (specific activity. 1,000 ..Ci/mmol, ICN) at 48-72 hrs. post-infection for 2-4 hrs. as previously described (Isfort et al., 1986b). A similar approach was employed for [32?] -orthophosphate labeling of DEF cells, with the following modifications. Phosphate-free Dulbecco modified Eagle medium was used for 3 hrs before- and during the 2 hr labeling period in which 250 pCi/ml 3213i (carrier-free; Amersham Corp.) was used per 60 mm plate. Labeled bacterial lysates containing Tip]?- and trpE-fusion proteins were grown similar to the large-scale harvests (Koerner et al., 1991) with the following modifications. Overnight cultures were inoculated from frozen stocks into media supplemented with each of the 20 as at a concentration of 50 pg/ml. The following day. 0.5 ml was inoculated with 4.5 ml of the above media lacking methionine and tryptophan and incubated by vigorous shaking in a 50 ml tube at 37 C for 2 hr. The cells were then treated with 10 liq/ml indoleacrylic acid (IAA), and incubated for an additional hr prior to the addition of 250 ..Ci [355]- methionine. Following a 15 min pulse at 37 C, cells were pelleted at 1,000g and 197 lysed with 1 ml of 1x phospholysis bufier (PLB; Witte and Wirth, 1979) supplemented with protease inhibitors as previously described (Brunovskis and Velicer, l992b). 50 pl of lysate was used for each immunoprecipitation. WW and SDS-PAGE analyses. Immunoprecipitatiort/SDS- PAGE analyses were carried out as previously described (Isfort et al.. 1986b). The specificity of trpE-MDV-directed antisera was tested by preincubating 970 pl PBS with 10 pl of apE fusion antisera and 20 pl 1x sample bufier-solubilized tips-fusion proteins (or (1125' alone) for 30 min-1 hr at 4 C. Labeled cell lysates were then added and processed as above. In some cases, lysates were adjusted to 1% SDS, 1% 2-mercaptoethanol (Z-ME) and boiled for 5 min prior to the addition of antibodies (Sirnek and Rice, 1988). When using chicken antibodies, an equal volume of rabbit anti-chicken IgG was added for at least 1 hr prior to the Staph A precipitation step. Irnmunoprecipates were washed 2x with 1x PLB and electophoresed on 10% SDS-PAGE gels, unless otherwise noted. High-stringency wash conditions involved sequential washes as follows; twice with RIPA bufier; once with high salt wash (2M NaCl, 10 mM Tris-HCl, pH 7.4, 1% NP40, 0.1% SDS); once with 1M MgClzz once with Tris-HG], pH 7.4. 3H- labeled protein markers (BRL) were included for estimation of molecular weights. RESULTS Nine of the eleven MDV Us ORFs were targeted for antibody production (Figure 1). Not included were the two most recently identified ORFs, SORFl and SORF2 (Brunovskis and Velicer, l992a). The antisera generated was immunoreactive with all 7 of the alphaherpesvirus-related Us region polypeptides. USI polypeptide expression is described elsewhere (Brunovslds and Velicer, l992a). MDV U810. HSV USIO encodes a putative virion protein (McGeoch et al., 1988) containing a potential zinc-finger domain (Holden et al., 1992) that appears to be nonessential for replication in cell culture. Two rabbits were independantly immunized with trpE-USIO. The antisera generated (RaUSlO) was capable of immunoprecipitating a 24-kDa MDV-specific polypeptide from infected cells. The protein was detected with either [35$]-methionine (Figure as. lanes 2 and 3) or [32PJ-orthophosphate (Figure 26, lane 10) labelings at late times postinfection (72 hr) when significant cytopathic effects were observed. The immunoprecipitation appeared to be specific; preincubation of RaUSlO antibodies with trpE-USIO (but not trpE alone) blocked the precipitation (Figure 2B, lanes 5, 6). Lack of reactivity with HVT-infected cell lysates (data not shown) suggests that HVT lacls a USI 0 homolog. Nucleotide sequencing of HVT’s S region should resolve this point. Expression of the 24-kDa polypeptide was sensitive to PAA treatment of infected cells (Figure 2D); PAA-sensitive and PAA-insensitive controls are outlined in Figure 40, below. This result indicates that MDV U310 encodes a late protein, consistent with the expected properties of its HSV-l counterpart, previously described as a virion protein (McGeoch et al., 1988). 198 199 05855 00.5500 500 o5E0 0595000050 505 80 00 0000350 05 W90 2 00050000 0550 >92 0550: 50 000: 0050 50050002 A082 00050.? 95 0205558 2 0505000 05 055 sea-.5509 2338385339: ._ 005E 200 | hmvéhn “um—o 5000 ..G. mafia: nUfiuu 02.5..“ On 5050.. 9.0083: I 05.8. 5 3.0.. I 0.30. I «0.6.. I 09.00. 0 . 5 00...: 5 ......o. ' 80.03 | .8.... | 02.00 5 5.3 03.3. | 0.2» ' 2.70... m J L E 1 fa WU H N U w ...8. :.z .358 mu ...0 >080 =3. .30 ....0 ....5\_rm/ .82 :2.. :3.. ...00 .82 .82 2:30 .2: .2.: >080 mm: hm: om: vmmOm am: am: ”know o rm: 5: >DDPm $.15. Z. we... 0... Gums“. $20.05“. >05. ".0 0(5— 201 FIGURE 2. lrmmmoprecipitaflorVSDS—PAGE analylil d the M «loaded by MDV USIO. 1) Identification of the MDV USlO polypeptide. Lysates from mock-inflected cells were analyzed by immunoprecipitation with pooled sera from rabbits 1 and 2 immunized with trpE-USIO (lane 1); MDV-infected cell lysates (lanes 2.3) were analyzed with antiseras from rabbits 1 and 2, respectively; subsequent experiments employed RaUSlO from rabbit 1. 3) Specificity of RaUSlO antiserum. Lysates from mock (lane l)- or MDV (lanes 2,3) -infected cells were analyzed by immunoprecipitation with RaUSlO (lane 1) or RaUSlO preincubated with either an (lane 2) or apE-USIO (lane 3). C) Phosphorylation of U810. Lysates from mock (lanes 1,3)- or MDV (lanes 2,4)-infected cells labeled with [32P1-orthophosphate were analyzed by immunoprecipitation with RaUSl (Brunovslds and Velicer, 1992b), lanes 1.2 or RaUSlO, lanes 3,4. D) MDV USlO is regulated as a late polypeptide. Infected cell lysates from untreated (lane 11) or PAA-treated (lane 12) cells were analyzed by immunoprecipitation with RaUSlO. PAA controls are described in Figure 40 below. 202 IMMUNOPRECIPlTATION/SDS-PAGE ANALYSIS OF THE POLYPEPTIDE ENCODED BY MDV U810 A. Initial B. Specificity of C. Phosphorylation oi D. MDV U810 is Identification RaUS10 (32.213) MDV U510 Regulated as a Late Gene ‘ + ‘T'PE nausmuusm nausm I 2 — — +TrpE-USIO —— —— —- —++—INF——++ lNF—+-—+ PAA—+ kDa .... 9"» 3'" “- I F. 038— "‘ 43— ' ' U U - O 29— . . _24 kDa— . 24 kDa 18- 123 456 78910 _ 1112 1 = rabblt 1 aUSIO "P-orthophosphals 2 = rabbit 2au310 labeled cell lysates 203 MDV "52. Nothing is known about the nature of U82 homologs other than the fact that they appear to be dispensable for replication in cell culture (Cantello et al., 1991; Weber et al., 1987). RaUSZ was found to specifically immunoprecipitate a 30-kDa MDV U82 homolog (Figure 3A, lane 2); an antibody-blocking experiment was similarly performed to demonstrate the specificity of RaUSZ (Figure 38). USS m. MDV codes for a product (Bnmovskis et al., 1992; Ross et al., 1991) which has a close relationship to the HSV U83-encoded protein kinase (PK) (McGeoch et al., 1986; McGeoch and Davison, 1986) and its PRV- (van Zijl et al., 1990; Zhang and Leader, 1990), VZV- (Davison and Scott, 1986; McGeoch and Davison. 1986), and EHV-l (Colle et al., 1992) counterparts. These products exhibit significant homology to the serine—threonine PK superfamily (Hanks et al., 1988; Leader and Purves, 1988). The MDV PIC-specific antisera, RaPKl, RaPKZ, and RaPK3 were directed against three different regions of the polypeptide as depicted in Figure 1. Under standard wash conditions (2-leLB washes) these antisera were found to irnmunoprecipitate several polypeptides, including a 47,49-kDa doublet present in infected cells (Figure 44!, lanes 2,4,6) and a 68-kDa polypeptide present in uninfected cells (Figure 4A, lane 1). Additional polypeptides, possibly less related serine-threonine PKs, were precipitated as well. To clarify the nature of the MDV-encoded PK product(s), higher stringency wash conditions were employed (Figure 43; see Materials 8: Methods). As a further test for specificity, an antibody-blocking experiment was conducted as before. U83 antisera 177-297 and 298-393 were found to specifically precipitate a 47,49-kDa doublet in MDV-infected cells (Figure 4B, lanes 6 and 10). With these wash conditions antiserum 89-176 was found to primarily precipitate a 68-kDa cellular polypeptide; once again, the reaction appeared specific, since trpE-US3/89-176 blocked this precipitation, while trpE 204 FIGURE 3. lmrmrnopreclfltatiorVSDS-PAGE analysis of the polypeptide encoded by MDV I132. A) Identification of the MDV U82 polypeptide. Lysates from mock (lane 1)- or MDV (lanes 2) -infected cells were analyzed by immunoprecipitation with RaUSZ. 3) Specificity of RaUSZ antiserum. Lysates from mock (lane l)- or MDV (lanes 2,3) -infected cells were analyzed by immunoprecipitation with RaUSZ (lane 1) or RaUSZ preincubated with either trpE (lane 2) or trpE—USZ (lane 3). 205 IMMUNOPRECIPITATION/SDS-PAGE ANALYSIS OF THE POLYPEPTIDE ENCODED BY MDV U82 A. Initial Identification B. Specificity of RaUSZ (22-87) — + — TrpE — - + TrpE-U82 — + INF - + + kDa in " kDa 68- .. a- 68 43 ~ 'I ' -43 29_ . 30 kDa \-. 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