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DATE DUE DATE DUE DATE DUE FFB 2 2 21553 TRANSCRIPTIONAL AND TRANSLATIONAL ANALYSIS OF MAREK’S DISEASE VIRUS GLYCOPROTEIN D By Xinyu Tan A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements ‘ for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1996 ABSTRACT TRANSCRIPTIONAL AND TRANSLATIONAL ANALYSIS OF MAREK’S DISEASE VIRUS GLYCOPROTEIN D By Xinyu Tan The Marek’s disease virus (MDV) unique short (Us) region encodes three glycoproteins, glycoproteins D (gD), I (g1), and E (gE). MDV g1 and gE were detected in MDV-infected duck embryo fibroblast (DEF) cells, but gD was not. The absence of a detectable level of gD expression could be explained by a defective open reading frame, inefficient antibody, inefficient transcription, or inefficient translation. When the gD, g1, and gE genes were subjected to in vitro coupled transcription and translation in the absence or presence of canine pancreatic microsomal membranes, their precursor polypeptides or glycoproteins were produced, respectively, and could be immunoprecipitated by respective antibodies. The results of Northern blot and RT-PCR analysis showed that the only gD-containing transcript is a low abundance 7.5 kb transcript that initiated two genes upstream of the gD gene, and it is unlikely to be translated into gD protein. Thus inefficient transcription is the main reason for absence of detectable gD expression in cell culture. When the gD, g1 and gE proteins were transiently expressed in 0087 cells, gD expression was not as efficient as expression of g1 and gE. After the N- terminal coding region of gD was replaced with N-terminal codons of gI, expression of the gI-gD hybrid was as efficient as expression of gI. Therefore, a negative regulatory element exists in the N-terminal coding region of the gD gene that may contribute to the absence of detectable gD expression in cell culture. DEF cells were infected with commonly used MDV strains. Although gI was detected in all of the infected cell cultures, gD expression was under the limit of detection. Therefore, absence of detectable gD expression in cell culture is a general phenomenon of MDV. MDV may be more closely related to varicella-zoster virus (V ZV), which does not have gD homolog, than to other alphaherpesviruses. It is known that VZV g1 and gE play important roles in virus infection. Thus, based on this research, expression of MDV g1 and gE will be a more important focus for future study. To My Mom and Dad iv ACKNOWLEDGMENTS I would like to thank Dr. Leland F. Velicer for his guidance, support, and patience in my research, writing, and many other things. Special thanks to members of my guidance committee, Dr. P. Coussens, Dr. R. Maes, Dr. R. Schwartz, and Dr. D. Salter for theoretical instruction and technical advice. I am grateful to Dr. Lucy Lee for providing lysates of CEF cells infected by the recombinant fowlpox gD. My special thanks to Dr. William MacArthur for helpful discussion and suggestion in my research, and careful review of my writing. Special thanks are also extended to Ruth Vrable for her excellent technical assistance. Finally, my sincere gratitude to my father and mother for their discipline, encouragement, and full support since I was born. TABLE OF CONTENTS List of Figures .................................................................................. viii Chapter I: Literature Review .......................................................... 1 1. Introduction to Herpesvirus ........................................................ 1 1.1 Distribution and classification ............................................ 1 1.2 Pathogenesis and pathology ................................................ 3 1.3 Replication ............................................................................ 4 1.4 Viral DNA ............................................................................ 5 1.5 Viral proteins ........................................................................ 6 2. Introduction to Marek’s Disease Virus ........................................ 8 2.1 History ................................................................................... 8 2.2 Pathogenesis ......................................................................... 9 2.3 Virus replication and virus-cell interactions ...................... 11 2.4 Viral DNA and proteins ....................................................... 12 3. Herpesvirus Transcription and Its Regulation ........................... 14 3.1 General aspects ..................................................................... 14 3.2 Correlation between viral DNA sequence and transcription maps ........................................................ 15 3.3 Induction of immediate-early genes .................................... 18 3.4 Process of DNA replication ................................................... 19 3.5 Regulation of late gene ......................................................... 20 3.6 Regulation of genes in the latent phase ............................... 22 3.7 Interaction between viral and cellular genes ....................... 23 4. Herpesvirus Glycoproteins ............................................................ 24 4.1 Entry of virus into cells ......................................................... 24 4.2 Glycoprotein B ....................................................................... 26 4.3 Glycoprotein C ....................................................................... 26 4.4 Glycoprotein D ....................................................................... 27 4.5 Glycoprotein I and glycoprotein E ........................................ 28 5. Immune Responses to Herpesviruses ........................................... 29 5.1 Natural resistance ................................................................. 29 5.2 T cell mediated immune response ........................................ 30 5.3 Antibody-mediated immune response .................................. 31 5.4 Vaccines .................................................................................. 32 5.5 Immune responses against MDV .......................................... 33 6. MDV Us region .............................................................................. 34 6.1 DNA sequence ........................................................................ 34 6.2 Proteins .................................................................................. 35 6.3 MDV mutants in Us region .................................................... 36 6.4 In conclusion .......................................................................... 37 References .......................................................................................... 38 Chapter II: Transcriptional Analysis of Marek’s Disease Virus Glycoproteins D, I and E: gD Expression is Down-regulated at the Transcription Level in Cell Culture ......................................................... 50 Abstract ........................................................................................... 51 Introduction ..................................................................................... 52 Materials and Methods ................................................................... 55 Results .............................................................................................. 60 Discussion ......................................................................................... 82 References ......................................................................................... 86 Chapter III: Expression of Marek’s Disease Virus Glycoprotein D ............................................................ 90 Abstract ............................................................................................ 91 Introduction ...................................................................................... 92 Materials and Methods .................................................................... 95 Results .............................................................................................. 102 Discussion ......................................................................................... 120 References ......................................................................................... 128 Chapter IV: Summary and Conclusions .......................................... 131 LIST OF FIGURES Figure Chapter II 1. Schematic representation of the MDV Us region DNA fragments used for antibody production and Northern blot analysis 2. MDV gD is not expressed in MDV-infected DEF cells 3. Expression of MDV gD and other MDV Us genes by in vitro coupled transcription/translation 4. Northern blot analysis of mRNA for MDV-infected cells using DNA probes 5. RT-PCR analysis of MDV gD transcription in MDV-infected cells 6. Northern blot analysis of mRNA from MDV-infected cells using riboprobes 7. RNAase protection assays of RNAs from MDV-infected cells 8. MDV transcript termini deduced from Northern blot analysis and RNAase protection analysis Chapter III 1. Schematic diagram of restriction sites that were used to construct expression plasmids 2. Generation of antibody to GSTgD fusion protein 3. Glycosylation of MDV gD, g1 and gE in vitro 4. Immunoprecipitation analysis of MDV gD produced by in vitro coupled transcription/translation, and glycosylation 5. Absence of gD expression is a general phenomenon for MDV 6. Expression of MDV gD, gI and gE in COS7 cells V111 Page 59 62 65 68 70 73 76 79 101 104 107 110 113 116 Comparison of gI and gE from the C087 cell expression system and from MDV GA-infected DEF cells 1 19 Expression of the gI-gD hybrid in COS7 cells 122 Expression of MDV gD by recombinant fowlpox virus 125 Chapter I Literature Review 1. Introduction to Herpesvirus 1.1 Distribution and classification Animal viruses include RNA viruses and DNA viruses. Herpesviruses are DNA viruses. A typical herpesvirus virion consists of a core containing a linear, double-stranded DNA, an icosadeltahedral capsid approximately 100-110 nm in diameter containing 162 capsomeres with a hole running down the long axis, an amorphous, sometimes asymmetric material that surrounds the capsid and is designated as the tegument, and an envelope containing viral glycoprotein spikes. (Roizman 1990). Herpesviruses are widely distributed in nature, and most animal species host at least one type of herpesvirus. Based on the biological properties, the herpesviruses have been classified into three subfamilies: the alphaherpesviruses, the betaherpesviruses, and gammaherpesviruses. With some exceptions, the studies of DNA sequence homology are generally consistent with this formal classification, and are particularly useful for the classification of viruses that are closely related. Characteristics unique to alphaherpesviruses include a variable host range, relatively short reproductive cycle, rapid spread in cell culture, efficient destruction of infected cells, and capacity to establish latent infections primarily, but not exclusively, in sensory ganglia. This subfamily contains herpes simplex l 2 virus 1 (HSV-l), herpes simplex virus 2 (HSV-2), varicella-zoster virus (VZV), pseudorabies virus (PRV), bovine herpesvirus 1 (BHV-l), equine herpesvirus 1 and 4 (EHV-l and EHV-4), feline herpesvirus 1 (FHV-l), Marek's disease virus (MDV), and herpesvirus of turkey (HVT), among others (Roizman 1990). The betaherpesviruses, on the other hand, have restricted host ranges. Their reproductive cycle is long, and the infection progresses slowly in culture. The infected cells frequently become enlarged, and carrier cultures are readily established. The virus can be maintained in latent form in secretory glands, lymphoreticular cells, kidneys, and other tissues. Cytomegalovirus (CMV) belongs to this subfamily (Alford et a1. 1990). The gammaherpesviruses replicate in lymphoblastoid cells in vitro, and some also cause lytic infections in some types of epithelioid and fibroblastic cells. The viruses are specific for either T or B lymphocytes. In the lymphocyte, infection is frequently either at a pre-lytic or lytic stage, but without production of infectious progeny. Latent virus is frequently demonstrated in lymphoid tissue. Epstein- Barr virus (EBV) and herpesvirus saimiri belong to gammaherpesvirus subfamily (Kieff et a1. 1990). MDV is the subject of this research. Since MDV belongs to alphaherpesviruses, the knowledge about alphaherpesviruses will be the focus of this review, and HSV-l will be used as the prototype in most cases. The distinguishing characteristics of other alphaherpesviruses will be discussed when necessary. 1.2 Pathogenesis and pathology Transmission of HSV-1 depends upon the intimate contact of a susceptible individual with someone excreting these viruses. At the start of infection, viruses first come in contact with cells at the mucosal surfaces or abraded skin, then replicate at the site of infection. After that initial replication, either intact virions or just capsids are transported by neurons via retrograde axonal flow to the dorsal root ganglia. At that site, another round of viral replication takes place before latency is established. Although replication can sometimes lead to disease and can infrequently result in life-threatening central nervous system infection, it is usually latency which predominates. After latency is established, an appropriate provocative stimulus will cause reactivation to occur; the virus will become evident at mucocutaneous sites, appearing as skin vesicles or mucosal ulcers (Roizman et a1. 1990). Another common alphaherpesvirus, VZV, produces two distinct clinical syndromes: chickenpox (varicella), and shingles (herpes zoster). In the pathogenesis of varicella, the virus apparently enters through the mucosa of the upper respiratory tract and oropharynx or, alternatively, via the conjunctiva. Viral replication begins at the primary inoculation site, and subsequently virus disseminates via the bloodstream and lymphatics (primary viremia). The virus is then taken up by cells of the reticuloendothelial system, where it undergoes multiple cycles of replication during the remainder of the incubation period. Viral replication is initially limited by nonspecific and developing virus-specific immune responses, but in most individuals these defenses are soon overwhelmed 4 and a more extensive secondary viremia develops. The latency and reoccurrence of herpes zoster is very similar to HSV—l (Gelb 1990, Whitley 1990, Harson et a1. 1995). The portals of entry of the betaherpesvirus CMV may include blood, or involve oral or genital contact. CMV is cytopathic and can produce tissue destruction of the cytomegalic inclusion-bearing cells. In the human, CMV characteristically infects ductal epithelial cells and seldom fibroblasts. This virus can be found in virtually all organ systems. The most commonly involved organs include the parotid gland, kidneys, liver, and lung (Stinski 1990, Alford et a1. 1990). Infection of the gammaherpesvirus EBV initiates in the oropharynx following salivary exchange. Thereafter, a prolonged persistent oropharyngeal infection with the virus ensues. B cells probably become infected in the vicinity of the oropharynx and then circulate in the blood and return to the marrow and various lymphoid organs. In common with the other herpesviruses, EBV can be associated with symptomatic primary infection (such as infectious mononucleosis) or as a latent infection. Thereafter, the virus usually persists for the remainder of the life of the host; occasionally reactivation occurs (Miller 1990, Kieff et a1. 1990). 1.3 Replication Among themselves, herpesviruses have a very similar pattern of replication. HSV-l will be used as an example here. To initiate infection, the virus must attach to cell receptors. Fusion of the virus envelope with the plasma membrane rapidly follows. The de-enveloped capsid is then transported through the 5 cytoplasm to the nuclear pores, where DNA is released into the nucleus. Transcription, replication of viral DNA, and assembly of new capsids takes place in the nucleus. Viral gene expression is coordinately regulated and sequentially ordered in a cascade fashion. The bulk of viral DNA is synthesized by a rolling circle mechanism, yielding concatemers that are cleaved into monomers and packaged into capsids. After packaging of DNA into preassembled capsids, the virus matures and acquires infectivity by budding through the inner lamella of the nuclear membrane (Roizman et al. 1990, Whitley 1990). 1.4 Viral DNA HSV-l DNA is linear and double-stranded. In the virion, the DNA is packaged in the form of a toroid. The ends of the genome are probably held together or are in close proximity. The HSV-l genome size is approximately 150 kbp. It consists of two covalently linked components, designated as L (long) and S (short). Each component consists of unique sequences (UL and Us) bracketed by inverted repeats (IR). The L and S components of HSV-1 can invert relative to one another, yielding four linear isomers (Whitley 1990). HSV-l and other herpesviruses contain more than 70 protein-encoding genes. Their genomic DNA sequences are widely divergent, but comparisons among the sets of amino acid sequences show that they have closely comparable and colinear sets of genes. However, the degrees of amino acid sequence similarity between individual gene pairs range widely, and indeed there are some equivalently positioned genes which are probably functional homologues but exhibit little or no sequence similarity. In addition, the contents and orders of 6 genes in the short region (Us plus the flanking copies of IRS) differ more among the viruses than those in UL, evidently as the result of recombination events (McGeoch et a1. 1993). Nonetheless, these comparisons showed definitively that alphaherpesviruses have a common ancestry, and belong to the same evolutionarily related group. Sequences for betaherpesviruses and gammaherpesviruses showed that these subfamilies are only distantly related to the alphaherpesviruses. The sequence evidence is thus generally compatible with the pre-existing classification, which was primarily based on biological properties (McGeoch et a1. 1993). DNA sequence interpretations underlie proposed structures of herpesvirus genes. An example is the latency-associated transcripts (LAT). These are a set of RNAs produced in latently infected neurons, where they are the only viral transcripts detectable. LATs have two postulated main functions: first, one or more LAT species may be a functional, protein-expressing mRNA; and second, LATs could act as antisense inhibitors of viral gene expression (McGeoch 1991). 1.5 Viral proteins Viral proteins can be grouped according to their functions. Some of the functions have been proven experimentally, while others are inferred from sequence analysis (McGeoch 1991). The first group is the membrane-associated proteins. Twenty HSV-l genes have been predicted to encode proteins which possess an N-terminal hydrophobic signal sequence and one or more potential transmembrane domains. Some of them are glycoproteins (UL27, gB; UL44, gC; U86, gD; US7, gI; UL8, gE), 7 others are just membrane-associated species (UL10, UL20). Some of the membrane proteins are rich in serine and threonine residues (gC, gG). Some proteins have been heavily O-glycosylated, which can confer a highly extended semi-flexible rod conformation to these proteins. Four HSV-l proteins are predicted to be multiply-inserted into membranes (UL10, UL20, UL43 and UL53), and so could be involved in transmembrane channel formation or signal transduction. Comparisons with local amino acid sequence motifs specific for protein families, together with global searches of protein sequence databases, have found similarities of thirteen alphaherpesviral protein families to non- herpesviral proteins, which are so significant that they are confidently taken to represent evolutionary and functional relationships. This route played an important part in assigning functions to, 1) two protein kinase families (UL13 and US3), 2) two DNA helicase families (UL52 and UL9), and 3) thymidylate synthase encoded by VZV gene 13. Additional herpesvirus proteins of known function include members of newly recognized families: uracil-DNA glycosylase (UL2), DNA polymerase (UL30), ribonucleotide reductase (UL39 and UL40) and deoxyuridine triphosphatase (dUTPase; UL50). The thymidine kinases (TKs) of herpesviruses (UL23) resemble other nucleoside and nucleotide kinases in possessing a near N- terminal NTP-binding motif. However, they appear not to be directly equivalent to the nuclear TK of eukaryotic cells, but instead are more related to eukaryotic deoxycytidine kinase. 8 The regulatory proteins have been studied extensively. VP16, ICP4, ICPO and ICP27 play important roles in the virus-specified control systems. They will be discussed in detail later. 2. Introduction to Marek's Disease Virus 2.1 History First described in 1907, Marek's disease (MD) is one of the most common lymphoproliferative diseases of chickens. MD is characterized by a mononuclear infiltration of one or more of the following: peripheral nerves, gonad, iris, various viscera, muscle, and skin. Observations of the disease came from the USA, Netherlands, Great Britain, and many other countries (Calnek et a1. 1991). Outbreaks of acute MD with unusually high mortality and a preponderance of visceral lymphomas have been observed since 1949 and became quite common between 1960 and 1970. In the early 19603, regular experimental transmission of the disease was successful. In 1967, research uncovered a herpesvirus (MDV) as the etiologic agent. Proof that this herpesvirus was the etiologic agent of MD was acquired through circumstantial evidence and reproduction of the disease with cell-free herpesvirus obtained from feather follicle epithelium (FFE) of infected chickens. Virus culture and identification were followed by a more complete understanding of the pathogenesis of the disease. Perhaps the most important practical development was the attenuation of the virus and its successful application as a vaccine against MD. 2.2 Pathogenesis MDV is characterized as a cell-associated herpesvirus. Additional groups of nonpathogenic herpesviruses isolated from turkeys and chickens are considered part of the MDV group. Based on serologic classification, three distinct virus groups were identified that correlated with biological properties. Serotype 1 are virulent MDVs, serotype 2 are avirulent MDVs, and serotype 3 are HVT (Calnek et al. 1991). Direct or indirect contact between birds effects virus spread, apparently by the airborne route. Affected follicular epithelial cells are in the keratinizing layer of stratified squamous epithelium; they slough off to detach with molted feathers to serve as a source of contamination in the environment. Virus associated with feathers and dander is infectious, and contaminated poultry house dust remains infectious for at least several months. Four phases of infection in vivo can be delineated: 1) early productive- restrictive virus infection causing primarily degenerative changes, 2) latent infection, 3) a second phase of cytolytic infection coincident with permanent immunosuppression, and 4) a proliferative phase involving nonproductively infected lymphoid cells that may or may not progress to the point of lymphoma formation. Virus gains entry via the respiratory tract where it is probably picked up by phagocytic cells. Shortly thereafter, cytolytic infection can be detected in the spleen, bursa of Fabricius, and thymus, peaking at 3-6 days. The primary target 10 cells in all three organs are B cells. The necrotizing effects of this early infection provoke an acute inflammatory reaction with infiltration of various cells including macrophages, granulocytes, and both immunologically committed and uncommitted lymphocytes. At about 7 days post infection, a transient immunosuppression may occur due to the presence of suppressor macrophages. Coincident with the development of immune responses, the infection switches to latency. Cell-mediated immunity (CMI) has been shown to be important in the switch. Most latently infected cells are activated T cells, although B cells can also be involved. The latent infection is persistent and can last for the lifetime of the bird. Susceptible birds develop a second wave of cytolytic infections after 2 or 3 weeks post infection, coincident with permanent immunosuppression. The lymphoid organs are again involved, and localized foci of infection can be found in epithelial tissues in various visceral organs, and especially in the skin, where a striking infection of the feather follicle epithelium occurs. The latter is unique in that it is the only known site of complete virus replication. The extent of infection during this phase depends on factors known to govern incidence of tumors, and the most susceptible birds develop the most widespread and severe infections. Lymphoproliferative changes constituting the ultimate response in the disease may progress to tumor development, although regression of lesions can and commonly does occur either before or after frank lymphomas are apparent. 11 Death from lymphomas may occur at any time from about 3 weeks post infection onward. 2.3 Virus replication and virus-cell interactions Replication of MDV is typical of other cell-associated herpesviruses. In MDV- infected CEF cells, enveloped virions enter the cell by conventional absorption and penetration, which occur within 1 hour. Viral antigens appear by 5 hours, DNA synthesis occurs by 8 hours, nucleocapsid production occurs by 10 hours, and enveloped virion production by 18 hours. Viral synthesis peaks at about 20 hours. Viral DNA replication occurs during the S phase of the cell cycle (Calnek et a1. 1991). Most commonly, cells are infected by contact with other infected cells. Cell-to- cell transfer of infection in vitro is normally accomplished through formation of intracellular bridges, and presumed to be the principal mode of virus spread both in vitro and in vivo. Three general types of virus-cell interactions are recognized: productive, latent, and transforming. Productive infection occurs mainly in nonlymphocytes, but occasionally in lymphocytes. In productive infection, replication of viral DNA occurs, antigens are synthesized, and in some cases virus particles are produced. The number of genome copies per cell can exceed 1200. There are two types of productive infection. Fully productive infection with MDV in the FFE of chickens results in development of large numbers of enveloped, fully infectious virions (Banders et al. 1994). 12 A productive-restrictive infection can occur in some lymphoid and epithelial cells in the chicken and in most cultured cells. Antigens are produced, but most of the nucleocapsids produced are nonenveloped and thus noninfectious. However, a variable number of the nucleocapsids formed in cultured cells may become enveloped, and these virons can be recovered cell-free and infectious by disruption of cells in distilled water. Latent infections are nonproductive. Latency of MDV has been observed only in lymphocytes, predominantly in T cells, but also in some B cells, in which only about five copies of the viral genome are present. The viral genome is not expressed, therefore no virus- or tumor-associated antigens are found. A third type of interaction, transforming infection, is characteristic of most transformed cells from MD lymphomas and lymphoblastoid cell lines derived from such lymphomas. This type of infection occurs only in T lymphocytes of chickens and has been demonstrated only with virulent serotype 1 MDV. Transformed cells contain about 5-15 copies of viral genome. Viral antigens and virions are seldom observed, although a portion of the viral genome may be transcribed. Of the several viral antigens, only the phosphorylated proteins (pp36/39 and pp24) have been detected in transformed cells both in vitro and in vivo (Delecluse et a1. 1993). 2.4 Viral DNA and proteins The DNA of MDV is typical of alphaherpesvirus genome. The DNA is a linear, double-stranded molecule that has a base composition of 46% guanine 13 plus cytosine ratio, and a molecular weight of 108-120 X 106 Daltons, which is equivalent to a size of 166-184 kilobase pairs (Ono et al. 1992, Silva et al. 1991). The MDV genome consists of covalently joined long (L) and short (S) components. The S component comprises a unique segment (Us), flanked by a pair of extensive inverted repeat regions (IRs). The L component contains a unique segment (U L) that is flanked by similar extensive repeat regions (IRs). Two proteins of MDV, the A antigen and the B antigen, have been studied extensively. It is no surprise that their genes were sequenced before the disclosure of other MDV genes. The gene encoding A antigen was identified by hybrid selection and cell-free translation due to the availability of its specific antisera. Later comparative analysis found that this gene is the HSV—l gC homolog (Coussens et al. 1990). The gene encoding the B antigen was found to be the HSV-l gB homolog (Chen et al. 1992). Some genes unique to MDV have been cloned (pp38, meq, pp14) (Cui et al. 1991, Chen et al. 1992, Jones et al. 1992, Hong et a1. 1994). These genes are thought to play role in MDV tumorigenicity. With the maturation of molecular biology technology, tens of thousands of base pairs can be sequenced in a short time, and more and more MDV gene sequences have become available. The Us region has been entirely sequenced. Many genes in UL region have also been sequenced, which include ULl, UL2, UL3, UL39, UL40, UL45, UL46, UL47, UL 48, UL49, UL50, ICP27 and ICP4 (Anderson et al. 1992, Yanagida et a1. 1993, Ben et al. 1994, Koptidesova et al. 1995). 14 3. Herpesvirus Transcription and Its Regulation 3.1 General Aspects Herpesviruses share two properties of gene regulation. First, herpesvirus genomes are promoter rich; generally, the expression of a given protein is mediated by a specific promoter mapping at that gene. Thus, extensive transcription units expressing long multigene transcripts that must be processed into a variety of mRNAs are the exception rather than the rule. This means that there need be no strict constraint on precise genomic order of genes of genomic organization, only on genomic content and co-regulation of essential functions. Second, the latent phase of infection characteristic of all herpesviruses leads to a close and lengthy association between viral and host cell genomes. The attenuated or tightly controlled replication of the viral genome in specific tissue, during reactivation from latency, which persists throughout the life of the host, leads to evolutionary isolation between herpesviruses infecting the same host, such HSV and VZV (Wager 1991). As in the case of most groups of DNA viruses, herpesvirus genes are expressed in temporally regulated phases during the productive replication cycle. All herpesviruses share a general set of features of their lytic replication cycle: the lytic cascade. According to their kinetics of expression following infection, gene transcripts can be classified into groups of immediate early (IE), early (E), and late (L). 15 After viruses invade host cells, the virion structural protein VP16 activates immediate early gene transcription. Then, immediate early gene products turn on early and late gene expression. In the presence of these proteins, viral genomes are replicated and new virions are assembled. For any DNA virus undergoing a productive replication cycle, the commitment to viral DNA replication is an irreversible one leading to cell death. There is, however, the other pathway after infection, which is the establishment, maintenance, and reactivation from the latent phase. 3.2 Correlation between viral DNA sequence and transcription maps Detailed studies of DNA sequences and their transcription in HSV-1 has yielded the following general view. Each gene has its own promoter, which is recognized by RNA polymerase II under the control of transcriptional regulatory factors. Transcripts each contain a single functional protein-encoding open reading frame (ORF) flanked by 5' and 3' noncoding sequences, and bear a 3' polyA tract. In the simplest case, the gene's transcript polyadenylation (PA) site is right downstream of the coding ORF (McGeoch 1991). However, not every gene possesses a separate PA site. The transcription of a gene may extend through one or more similarly oriented distal genes before finally reaching a site for addition of poly(A). In such a case, the mRNA then contains additional coding sequences downstream of its primary ORF, but these are considered not to be translated from that mRNA species. Thus, a group of genes may make use of the same PA site, and their transcripts then form nested 16 sets, called 3' co-terminal families. Splicing of herpesvirus transcripts is rare, but has been detected in a few cases. For a precise description of transcript structure in relation to the DNA sequence locations of initiation and polyadenylation sites, the TATA box and AATAAA site are very helpful. The TATA box is often found immediately upstream of transcription initiation sites. However, this element often occurs frequently in the genome sequence, in positions not considered to have any relevance to control of RNA synthesis and processing. Prediction of locations of the 3' termini and poly(A) tracts in relation to the AATAAA element is relatively satisfactory. The DNA sequence AATAAA is generally found 5 to 30 bp upstream from the sites of 3'-polyadenylation of RNA polymerase II transcripts, so that it is regarded as partially indicative of a PA locus and is widely used in interpreting sequence data. However, the sequence is also observed to occur in locations where it is quite unlikely to indicate a PA sites. Thus, any prediction should ultimately be verified by direct experimentation on transcript structures. HSV-1 UL1, UL2 and UL3 are rightward oriented. There is an AATAAA sequence downstream of the UL3 ORF. Thus UL1, UL2 and UL3 RNAs would be a 3' co-terminal set. Genes UL11 to UL14 are expressed as a leftward oriented 3' co-terminal family. Transcripts for UL18 and UL19 were mapped as a leftward-oriented, 3' co-terminal set. However, there is an AATAAA sequence at the end of the upstream ORF, UL19, suggesting that UL19 transcripts might also terminate there. 17 HSV-1 UL24 to UL26 transcripts are a rightward-oriented, 3' co-terminal set. There is a possible PA site between UL24 and UL25, but no corresponding RNA species were identified. Between the leftward-oriented UL29 and the rightward UL30, lies a sequence which functions as an origin of DNA replication. It has been shown that two transcript species terminate immediately downstream of the UL30 ORF, while a much larger species may represent a readthrough of the PA signal just distal to the UL30. The leftward-oriented UL31 and UL32 are transcribed as a 3' co-terminal pair and an appropriately placed AATAAA signal is found. The coding sequences of UL30 and UL31 overlap on opposite strands by 19 codons and the PA signal of each lies within the other's coding sequence; this is the only example of such a tail-to-tail overlap found for HSV-1. UL33 to UL35 form a 3' co-terminal family. UL36 has its own transcript. Each of UL46 to UL51 also has its own mRNAs. Four separate species of transcripts of UL49 have been described, with two initiation codons and two termination sites. One initiation site is right at the upstream of the UL49 ORF, and the other further upstream within UL50. There is a PA site downstream of the UL49 ORF, and some transcripts evidently continue to the PA site downstream of UL48. UL52, UL53 and UL54 are rightward oriented and may be 3' coterminal. The long repeat region is more complex than the unique region with regard to transcription regulation. There are latency-associated transcripts (LATs), and a l8 spliced mRNA (e.g., ICPO transcript). Between U1, and LAT sequences, there are four AATAAA sequences. Three are rightward oriented, and one leftward oriented. The AATAAA sequences there might serve to terminate any "stray" readthrough transcriptions proceeding from UL toward the LAT region. USB transcripts have two separate initiation loci, just like UL49 and UL24. Downstream of the US5 is a potential PA site which is probably nonfunctional and thus apparently similar to the UL19 and UL24. The USS and U89 transcripts are 3' co-terminal, with an ATTAAA sequence in their PA site. US 10 and USll are similarly oriented and exhibit an out-of-frame overlap which is supported by protein mapping data. The other Us genes have their own transcripts (Fletcher et al. 1994). 3.3 Induction of immediate-early genes In an HSV-1 infection, the first event is activation of immediate early genes. The trans-activator of immediate early genes is a structural component of the virus, its VP16 protein. This protein is also named TIF, Vmw65 or ICP25 (Roizman et al. 1991). The VP16 protein is encoded by the UL48 ORF. The molecular weight of the protein is about 54 kDa. An extremely acidic region is found at the carboxyl half of the protein, which includes the domain responsible for the trans- activation. VP16 forms complexes with HSV DNA in the presence of cellular transcription factor OTF1, or under conditions in which OTF1 can participate in the reaction. As a transcription factor, OTF1 interacts with an octamer motif of 19 the cis-regulatory element in the promoters of ubiquitously expressed genes. The cis-acting site of immediate early genes is the sequence TAATGARAT. Together with OTF1, VP16 can bind to this motif of immediate early genes and induce transcription (Everett et al. 1991). There are five major immediate early genes: ICPO, ICP4, ICP22, ICP27 and ICP47. After the transcription is activated by VP16, these five gene products coordinate HSV-1 gene transcription and replication. 3.4 Process of DNA replication Of the many herpesviruses, the genome replication of HSV has been most extensively studied. Cis- and trans-acting elements involved in viral DNA synthesis have been identified. Shortly after infection, most of the viral DNA molecules lose their free ends as a result of circularization. The ends of the DNA genome are ligated together probably by a host cell enzyme. Two replication cis-elements have been found in HSV-1 genome. One has been located within the invert repeats flanking Us region, and called oriS. The other, oriL, has been localized to the middle of UL (Weller 1991). In addition to replication cis-elements, the HSVl genome encodes many trans- acting proteins involved in DNA synthesis. Of over 72 proteins encoded by HSV- 1, at least 13 have been shown to be involved in viral DNA synthesis. These factors can be divided into two classes: those involved in nucleotide metabolism and those directly involved in DNA synthesis (Olivo et al. 1991). 20 Several enzymes are involved in nucleotide metabolism. Thymidine kinase, ribonucleotide reductase, dUTPase, and uracil-DNA glycosylase have been studied extensively. In general, these enzymes are dispensable for viral growth in exponentially growing cells in culture, although many of these enzymes may be required in growth-arrested cells or during in vivo infection of animal hosts. UL5, UL8, UL9, UL29, UL30, UL42 and UL52 are required for viral DNA synthesis. UL30 encodes a DNA polymerase. UL29 encodes a major DNA binding protein. The UL42 gene product is a double-stranded DNA binding protein. The mutants of these genes exhibit alterations in DNA synthesis. After virus infection, immediate early genes are activated first. Their gene products activate the other viral genes, including those involved in DNA replication. Meanwhile viral DNA molecules are circularized. The proteins for DNA replication recognize cis-elements in viral DNA and initiate viral DNA replication. Large head-to-tail concatamers consisting of tandem repeats of the viral genome are then formed. Finally, the concatamers are cleaved and DNA genomes are packaged into capsids. Viral alkaline nuclease and several other gene products are required for this process (Everett et al. 1991). The genes involved in DNA replication are grouped as early genes. Their activation depends on immediate early gene products. In turn, DNA replication is required for some late gene activation, especially those that are strict late genes. 3.5 Regulation of late genes 21 Late genes are defined as those which have a stringent dependence on viral DNA synthesis for their expression. Under conditions where viral DNA replication has been blocked by drugs, early genes are expressed at similar levels with or without drugs, while late genes either are not expressed or significantly lower when drugs are added (Homa et al. 1991). The promoter elements that are required for the efficient expression of the three different classes of HSV genes are different. Immediate early genes require a TATA box, distal signals, and far-upstream TAATGARAT elements; early genes require only a TATA box and distal elements, and late genes need only a TATA box and its adjacent region for the landing of transcription factors. The ICP4 and ICPO play roles in activating late gene expression, however, some cellular factors may also be involved in the late gene activation. The 5' transcribed noncoding region of late genes are important, and may recruit some transcriptional factors. The mechanism for the requirement of viral DNA replication in late gene activation is still unknown. One of the hypotheses is based on the notion that transcription of true late promoters is blocked early during infection and that one role of replication is to remove that block. This block could be due to direct binding of a viral protein or a cellular protein, or the physical state of the viral DNA template in infected cells. Late genes resident in the viral genome may be physically restrained from being transcribed and the role of replication would be to relieve that constraint (Blair et al. 1991). 22 3.6 Regulation of genes in the latent phase Almost all of the alphaherpesviruses can establish latent infections in their natural hosts, primarily, but not exclusively, in nervous tissues, which include sensory and autonomic nerve ganglia, and the central nervous system (CNS). Latent infection is often divided into three distinct phases: establishment, maintenance and reactivation. Again, HSV-1 is the prototype of the alphaherpesviruses used in the research of latency (Feldman 1991). In acutely infected ganglia there are two types of virus-neuron interaction, one which results in lytic infection and a second whose outcome is latent infection. The variables which are responsible for selection of one pathway over the other in a neuron are currently unknown. However, the cellular factors involved in repressing immediate early gene expression may be paramount to this decision. Restricted viral transcription in latently infected ganglionic and CNS neurons has been observed with several alphaherpesviruses. With HSV-1, latency- associated transcripts (LATs) were detected in latently infected ganglia as two partially collinear abundant non-adenylated RNAs of approximately 1.2 and 2.0 kb. The predominately nuclear RNAs are related by splicing and are antiparallel to and overlap the 3' end of the RL2 gene, which encodes the ICPO gene. Available data suggest that LAT function is not an absolute requirement for viral latency and reactivation under experimental conditions, but rather appears to quantitatively enhance the efficiency of the latency and reactivation cycle. 23 Thus, a real LAT function in nature may be to confer a quantitative survival advantage to a virus within a host population over time. Additionally, LAT function may be situational; LATs may serve more than one latency-related function, albeit closely related or overlapping ones (the establishment and reactivation functions), depending on nerve cell type, animal model or reactivation stimuli. The molecular processes which underlie viral reactivation are poorly understood. Very possibly, a reactivating stimulus would lead, directly or indirectly, to the expression of a critical immediate early regulatory gene. That event would subsequently induce further lytic phase gene expression and replication. 3.7 Interaction between viral and cellular genes Infection of host cells by HSV-1 results in shutofi' of most host genes and activation of viral transcription. A simple explanation is that viral genes have virus-specific signals, which can be recognized by viral proteins. But more evidence suggests that viral proteins act more indirectly by complexing with or modifying cellular factors that interact with both HSV-1 and cellular promoters (Smiley et al. 1991). Viral host shutoff protein (VHS) is encoded in UL41. VHS destabilizes both viral and cellular mRNAs during infection. Therefore, the VHS-induced RNA turnover system displays little or no sequence specificity. The destablization of viral mRNA is tightly coupled with the rate of viral gene transcription, thus facilitating the transition from one phase of infection to the next. 24 Some cellular gene promoters can be either activated or repressed by HSV-1 infection under different circumstances. For example, endogenous globin gene transcription is repressed after virus-infection. But when the gene is newly introduced into cells, its promoter is susceptible to HSV-1 induced activation. It is also clear that the response to IE proteins is somewhat dependent on promoter sequence. Indeed, some evidence suggests that the precise sequence of the TATA box plays a major role in determining the temporal course of expression of HSV-1 genes. The selective activation of HSV1 genes relies not only on the primary sequence of individual HSV1 promoters, but also on additional structural features which distinguish newly introduced DNAs from the endogenous cellular genome. A complete description of the viral regulatory strategies will require a greater understanding of the role of the possible covalent modification, the intranuclear architecture and the higher-order packaging of genes in the transcriptional process. 4. Herpesvirus Glyc0proteins 4.1 Entry of virus into cells For enveloped viruses in general, entry into a cell requires binding of virus to receptors on the cell surface followed by endocytosis of the virion or by direct fusion of the virion envelope with the cell plasma membrane. Herpesviruses can enter cells by fusion with the plasma membrane. This fusion can be blocked‘by neutralizing antibodies and then enhanced by a chemical fusogen (Spear 1993). 25 The actual number of membrane glycoproteins present in the virion envelope is not yet known for any of the herpesviruses. To date, at least ten membrane glycoproteins specified by HSV-1 have been identified and partially characterized. The gB, gC, gD, gH, gI, gE and gG have been shown to be constituents of the virion envelope. The other three, gJ, gK and gL, may also be present in the virion envolope, along with other previously unrecognized membrane proteins. It seems likely that any of the proteins exposed on the surface of the virion could interact with the cell surface or with other virion constituents to influence the binding of virus to cells or the process of penetration. Deletion mutagenesis is a useful first step to reveal the essential roles of selected glycoproteins in virus infectivity. Deletions of several glycoproteins yield mutants that can be propagated in cell culture. However, some of the apparently dispensable envelope glycoproteins may play important roles in virion binding or viral penetration, particularly on certain cell types. Multiple molecular interactions are required for the entry of herpesviruses. The initial interaction of virion with cell is usually the binding of a member of the gC family to cell surface heparan sulfate. Then gD interacts with an unidentified cell surface component. Besides gD, several other virion glycoproteins are also required for steps that occur subsequent to the initial attachment of virus. These proteins include homo-oligomers of gB and hetero- oligomers of gH and gL. Consequently, viral penetration succeeds via fusion of the virion envelope with the cell plasma membrane. 26 4.2 Glycoprotein B The gB gene is highly conserved among herpesviruses. Its homologs have been found in all herpesviruses studied thus far. DNA sequence analysis of the HSV gB gene predicts a 904-amino-acid polypeptide, including a 30-residue, cotranslationally cleaved signal sequence, an N-terminal extracellular domain, a large hydrophobic transmembrane domain postulated to include three membrane-spanning helices, and a large cytoplasmic C-terminal domain. The functional gB molecule is an oligomer produced in high abundance in lytic infection. Virons lacking gB bind to host cells but fail to penetrate and initiate infection. Although wild-type HSV-1 elicits little cell-cell fusion, gB mutants induce polykaryocyte formation frequently. It has been shown that the essential function of the gB cytoplasmic domain is related to its role in membrane fusion. Moreover, the role of gB in membrane fusion likely involves some communication between the external and cytoplasmic domains. HSV-l-induced membrane fusion during virus penetration and between adjacent cells is predicted to be analogous. gB is essential in both pathways. However, the roles of the other individual proteins in each process are unknown, and their respective functional domains are poorly understood. 4.3 Glycoprotein C Glycoprotein C (gC) is one of numerous glycoproteins found on the surface of HSV1-infected cells and within the viral envelope. Although this glycoprotein is not essential for virus production in cell culture, gC serves multiple accessory functions, including the ability to bind the C3b component of complement and to 27 function in virus attachment to cells upon infection via heparan sulfate moieties on the cell surface membrane. Glycoprotein C is also a major viral antigen which elicits a strong humoral and cellular immune response during infection. Binding of gC to heparan sulfate moieties of cell surface proteoglycans is the principal component of the initial interaction of virion with cell, at least for a variety of cultured cell types. A feature shared in common by gC homologs is a cluster of basic amino acids in the vicinity of a very hydrophilic region near the N-terminus, which is good candidate for heparin-binding domain. Proteoglycans are ubiquitous molecules present as integral membrane proteins of cells and as components of the extracellular matrix. Several families of cellular genes encode their protein cores. The core proteins are modified by the addition of long unbranched sulfated polysaccharides, called glycosaminoglycans (GAGs), as well as by N-linked and O-linked oligosaccharides. The three most abundant GAGs on plasma membrane proteoglycans are heparan sulfate, chondroitin sulfate and dermatan sulfate. It has been concluded that presence of cell surface heparan sulfate is a specific requirement for the normal binding of HSV-1 to cells; that chondroitin sulfate cannot effectively substitute for heparan sulfate, and that the level of sulfation of heparan sulfate is important to its activity in HSV-1 infection. 4.4 Glycoprotein D Glycoprotein D is found in the envelope of HSV-1. This protein is essential for entry of virus into mammalian cells. gD has been implicated in receptor binding, cell fusion, and neuroinvasiveness. It is suggested that gD binds to the 28 mannose-6-phosphate receptor, but the significance of this binding remains to be elucidated. Immunization of animals with gD stimulates the production of virus-neutralizing antibodies and protects them from lethal challenge with HSV- 1 and the establishment of latency (Hassens et al. 1995). Cellular expression of HSV-1 gD, PRV gD, and BHV-l gD has been used to define a superinfection-interference function of this protein. Cells expressing these proteins are characterized by their ability to resist homologous and heterologous herpesvirus infection (Chase et a1. 1990, Johnson et al. 1989). Furthermore, it is shown that cellular expression of gD does not prevent viral adsorption, but does prevent the entrance of the virus into the cell (Heffner et al. 1993, Kauh et al. 1991). 4.5 Glycoprotein I and Glycoprotein E HSV-1 Glycoprotein I and glycoprotein E are not essential for virus infection and replication in cultured cells. However, these proteins may play important roles in vivo in infection of diverse cell types, movement of virus through tissues, or protection from the host immune responses. g1 and gE form a complex which acts as a receptor for the Fc domain of IgG. The gI-gE Fc receptor may protect HSV-l-infected cells from complement-mediated immune lysis by causing IgG aggregation or by reducing the ability of complement components to bind to virus-associated or cell-associated IgG. However, protective effects of this type have not been demonstrated in vivo in an animal model (Edson 1993, Mulder et al. 1994). 29 There is evidence that the gE-gI hetero-oligomer is important to facilitate virus spread in viva, a property that is apparently unrelated to the IgG Fc receptor activity. HSV-1 mutants lacking either gI or gE are inhibited in their capacity to spread between certain types of cultured cells by the direct cell contact route and the mutants spread poorly in epithelial tissues. However, g1 and gE mutants do not display defects in entry of extracellular virus particles into the same cells (Card et al. 1992, Dingwell et al. 1994, Zsak et a1. 1992). Thus, cell-to-cell spread of viruses, apparently across cell junctions, has features that are distinct from entry of extracellular viruses (Babic et al. 1995, Dingwell et al. 1995, Yao et al. 1993). 5. Immune Responses to Herpesviruses 5.1 Natural resistance The study on HSV-1 infection showed that nature killer (NK) cells, interferon, and macrophages are the primary immune elements after virus infection. In many virus infections, NK cell numbers increase in peripheral blood 2 to 3 days after infection and then decline. Apparently, NK cells form the first line of defense against HSV-1. Interferon production is one of the earliest indicators of a virus infection, with levels in serum peaking around 2 days after infection. Interferons are involved in inhibition of virus replication and are also important in activating NK cells and macrophages (Nash et a1. 1993). 30 Natural resistance is still a poorly defined phenomenon and involves resistance genes whose functions are still to be determined. The efficacy of these mechanisms depends upon the extent of initial infection, which is affected by age upon infection and site of infection. Neonatal infections are generally more severe due to immaturity of the host defense system. 5.2 T cell mediated immune response In mice infected by HSV-1, activated CD4+ T cells are observed which can effectively transfer delayed type hypersensitivity (DTH) to infected recipients. These T cells can also initiate rapid clearance of infectious virus from epidermal surfaces, probably by recruiting and arming macrophages. CD4+ T cells mediating this property produce interferon y, a potent activator of macrophages, and have properties typical of the Th1 subset of CD4+ T cells. Accumulation of macrophages at sites of infection can lead to elimination of virus or virus- infected cells by a number of mechanisms (Nash et al. 1993, Arvin et al. 1992, Bergen et al. 1991, Huang et al. 1992). CD8+ T cells also contribute to the control of virus infection. CD8+ T cells are efi‘ective at epidermal sites of infection, particularly when there is extensive viral replication. In most circumstances in the skin, however, they appear to be secondary to the CD4+ T cells, at least in the primary infection. In the nervous system of mice, CD8+ T cells do contribute to the control of infectious virus in the neuron. Probably CD8+ T cells exert antiviral immunity via cytokine release. 31 CD8+ cells from HSV-infected mice or humans recognize proteins produced at different stages of the infectious cycle. In particular, IE gene products are major antigens recognized in association with MHC I. In some situations, HSV may evade recognition by interfering with antigen presentation in the infected cell. In HSV-2 infected fibroblasts, MHC I expression is reduced, which interferes with recognition and destruction of the infected cell by specific T cells. 5.3 Antibody-mediated immune responses Once the antibody response to HSV-1 has been induced, it helps to prevent re- infection. However, the presence of neutralizing antibodies does not prevent virus being activated from latency. Antibody-dependent cell cytotoxicity (ADCC) is effective against target cells early in the infectious cycle. The cells mediating ADCC in the blood of human include macrophages, neutrophils and NK cells (Nash et al. 1993). Neutralizing antibodies are also active against cell-free virions, preventing spread to the nervous system and in the blood stream. Antibodies can also neutralize virus emerging from nerve endings, which would re-infect epithelial cells during a recurrence. It is possible to achieve a high titer of neutralizing antibodies in mice by selective immunization with gD. Although such animals are unable to prevent infection of epidermal cells when challenged with live virus 6 months after immunization, the immune response is able to inhibit establishment of a latent infection (Kimman et al. 1994). Although the humoral immune response has a role to play in countering HSV infections, the virus has evolved strategies to interfere with some antibody- and 32 complement-mediated effector functions. HSV-1 gC acts as a receptor for complement C3b, thus interfering with its interaction with C5 or C5b and subsequent components of the membrane attack complex. The gI/gE heterodimer is involved in binding to the Fc receptor of IgG. This interaction may not only prevent antibody-dependent complement-mediated lysis of infected cells and virions, but could also affect ADCC and phagocytosis by neutrophils and macrophages. 5.4 Vaccines Traditional virus vaccines, including those developed for HSV-1 prophylaxis or treatment, have been of two types: modified live or inactivated virus. The advent of recombinant DNA technology has expanded dramatically the possible approaches to vaccine development (Burke 1993, Bourne et al. 1996). The use of modified live-virus (MLV) vaccines has many theoretical advantages. The immune response is much broader, more durable, and a better mimic of that elicited by natural infection. MLV HSV vaccines can be developed through rational design. One of the strategies is inactivating or deleting genes likely to be involved in neurotropism, neurovirulence, latency or reactivation. A variation of the MLV vaccine strategy is the use of a replication-defective virus containing a mutation in an essential gene, so that infection is limited to a single round. Full-length and truncated derivatives of proteins (subunits) have been produced in alternative hosts using recombinant DNA technology. The gB, gC, gD, gI and gE have been proven to be useful as vaccine singly or in combination. 33 The use of viruses that have an established record of safety following administration to humans as vectors for expression of selected HSV genes represents a hybrid between the live virus and subunit protein approaches. Vaccinia virus and adenovirus are the current common vectors (Ludvikova et al. 1991, Vafai 1995). 5.5 Immune responses against MDV Infection of chickens with serotype 1 MDV results in a primary infecion of B lymphocytes resulting in lysis of these cells. As a consequence of the production of viral antigens, T cells become activated and susceptible to infection, and latent infection is established in the activated T cells. Further, a group of T cells, CD4+/CD8- T cells, may be transformed (Dandapat et al. 1994, Zelnik 1995). Both humoral and cell-mediated immune responses have been postulated to be important for protection against Marek’s disease, but many more experiments need to be done to dissect the importance of the different antigens for the development of immunity after infection or vaccination (Lee et al. 1991, Witter 1991, Schat 1991). To examine the importance of specific antigens for the production of protective antibodies, proteins have been highly expressed by either recombinant fowlpox virus or baculovirus. The expressed proteins were used to vaccinate chickens, followed by challenging these chickens with virulent MDV viruses. By this methods, gB has been confirmed to be the major glycoprotein for the induction of virus-neutralizing antibodies, while pp38 did not protect against challenge 34 (Davidson et al. 1991, Niikura et al. 1992, Yanagia et al. 1992, Yoshida et al. 1994). To. examine the importance of cytotoxic T lymphocytes (CTL) and viral antigens in cell-mediated immune responses, a chicken cell line is required that expresses MHC class I antigen and virus antigens. CEF and DEF cells do not express MHC class I antigen. Chicken kidney cells do express MHC I, but can not express enough MDV viral antigens. A new approach is to use reticuloendotheliosis virus (REV) -transformed B cell lines to express MDV antigens, but no significant results have been reported at this time (Pratt et al. 1992, Schat et al. 1994, Uni et al. 1994). 6. MDV Us region 6.1 DNA Sequence Part of the genome of the very virulent RBlB strain was sequenced (Ross et al. 1991). The sequence of the entire MDV Us region has been completed using the GA strain (Brunovskis and Velicer 1995). Comparing the common fragments of the two strains has revealed over 99% identity at both the nucleic acid and the predicted amino acid levels. The U, region is 11,160 bp in length, and is flanked at the 5’ and 3’ ends by a 63-bp stretch of IRs and TRs DNA, respectively, each inversely complementary to the other (Brunovskis et al. 1995). The overall guanine plus cytosine ratio of the region sequences was found to be 41%. This result agrees with those obtained from alphaherpesviruses, while sharply contrasting with the CpG 35 deficiencies associated with all gammaherpesviruses. Since MDV had been traditionally regarded as a gammaherpesvirus due to its lymphotropism, the sequence of Us region further confirms the argument that genetically MDV belongs with the alphaherpesviruses. The region sequenced contains at least 11 ORFs likely to code for proteins. Seven of them are HSV-1 homologs, and the other four, designated unique Short region Open Reading Frame (SORF), are unique to MDV. The MDV homologs of HSV-1 include U81 (ICP22), U82, U83 (a serine-threonine protein kinase), U86 (gD), US7 (gI) U88 (gE) and U810. In addition, SORF-2 is a fowl pox virus homolog, while SORFs- 1, -3 and -4 are MDV-specific. 6.2 Proteins To facilitate characterization of the proteins encoded by ORFs in the Us region, the antisera to U81, U810, U82, U83, U86 (gD), US7 (g1) and U88 (gE) were made against a panel of different bacterial-expressed TrpE fusion proteins (Brunovskis et al. 1992). The MDV U81 polypeptide was characterized as a 27 kDa phosphoprotein by immunoprecipitation with antibody to TrpE-U81 fusion protein. U82-specific antibody precipitates a 30 kDa polypeptide; US 10-specific antibody detects a 24 kDa phosphoprotein. Three different antisera to U83 fragments precipitate a 47,49 kDa doublet corresponding to the MDV protein kinase-related product (Brunovskis et al. 1992). MDV gE-directed antisera immunoprecipitates gE-specific 62 and 72 kDa glycoproteins and a 45 kDa precursor polypeptide from MDV-infected cells. 36 MDV gI-directed antisera immunoprecipitate a gI-specific 45 kDa glycoprotein and an unglycosylated 35 kDa precursor polypeptide. In contrast, no glycoprotein was found when the same analysis was performed with MDV gD- directed antisera. 6.3 MDV mutants in Us region Construction of MDV mutations in Us genes have been pursued mainly in Dr. Robin Morgan’s lab. The lacZ gene of E. coli has been inserted into the gD gene of GA strain (Parcells et al. 1994). The insertion site was positioned one-third of the way into the U86 gene (gD) from the 5’ downstream. Comparing the mutant with the parent virus, they had similar growth kinetics in cell culture. Furthermore, the US6lac mutant could be reisolated from the spleens and peripheral blood of infected chickens with a frequency comparable to that of the parent virus. Therefore the gD homologue is nonessential for growth in cell culture and in the chicken. Mutants that have lacZ insertions into U81, U82, US 10,and U83 have also been constructed. Of these mutants, only those lacking a functional U81 gene have a discernible phenotype in cell culture, namely, a growth impairment in established CEF (Cantello et al. 1991, Sakaguchi et al. 1993). MDV mutants having a large deletion that removes U81, U810, U82 and the neighboring SORF-2 and SORF-3 genes have been constructed on both GA high passage and RBlB low passage strains (Parcells et al. 1995). These mutants had a growth impairment in established CEF cells similar to that lacking a functional U81 gene. Although the large deletion mutants were still infectious 37 for the first few days postinfection, virus yield was decreased at 3 to 5 days postinfection. Plaques formed by mutants were notably smaller than their parental virus. 6.4 In conclusion The available knowledge about herpesviruses has been reviewed extensively, from biology to molecular biology, and from viral pathogenesis to host immune responses. Because the subject of our research, MDV, belongs to the alphaherpesviruses subfamily, the prototype of alphaherpesviruses, HSV-1, is the focus of this review. HSV-1 has been studied in great detail, and information of HSV-1 has provided clues for other alphaherpesviruses. In the case of MDV, the HSV-1 homologous genes were identified first, and the gene products were characterized with comparison to corresponding HSV-1 proteins. Later, MDV unique genes were found, and characterization of these proteins may lead to explanation of MDV-specific biological properties, such as lymphotropism and tumorogeneicity. 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Chapter II Transcriptional Analysis of Marek’s Disease Virus Glycoproteins D, I and E genes: gD Expression is Down-Regulated at the Transcription Level in Cell Culture 50 51 ABSTRACT The various alphaherpesviruses, including Marek’s disease virus (MDV), have both common and unique features of gene content and expression. The entire MDV Us region has been sequenced in our lab (Brunovskis et al., 1995, Virology. 206, 324-338). Genes encoding the MDV glycoprotein D (gD), glycoprotein I (gI) and glycoprotein E (gE) homologs have been found in this region, although no gG homolog was found. In this work, transcription of the tandem MDV gD, g1 and gE genes was studied, and found to have both unique characteristics and also features in common with other alphaherpesviruses. MDV gD could not be immunoprecipitated from MDV GA-infected duck embryo fibroblast cells by antisera reactive to its trpE fusion proteins, while gI and gE could be. When the gD gene was subjected to in vitro coupled transcription/translation, the precursor polypeptide was produced and could be immunoprecipitated by anti-gD. Northern blot, RT- PCR and RNAase protection analyses have shown that: 1) no mRNA initiating directly from the gD gene could be detected, 2) a large, but low abundance, 7.5 kb transcript spanning five genes, including the one encoding gD, was seen on longer exposure, and 3) transcription of the g1 and gE genes formed an abundant bicistronic 3.5 kb mRNA, as well as an abundant 2.0 kb gE-specific mRNA. Therefore, the MDV gD gene expression is down-regulated at the transcription level in MDV-infected cell culture, which may be related to MDV’s cell-associated nature in fibroblast cells. Compared to the highly gD-dependent herpes simplex virus, and the other extreme of the varicella- zoster virus which lacks gD gene, MDV is an intermediate type of alphaherpesvirus. 52 INTRODUCTION Marek’s disease virus (MDV) is a highly infectious herpesvirus which induces lymphomas in chickens. The apathogenic and antigenically related herpesvirus of turkey (HVT) is usually effective as a vaccine against Marek’s disease, and is the first successful vaccine against a naturally occurring tumor of any species. While a very interesting and valuable natural host animal model for oncogenesis, this cell-associated herpesvirus system is somewhat complex. Fully enveloped infectious virions are produced only in feather follicle epithelium (FFE) of the skin; they then detach with feather dander, contaminate dust, spread by the airborne route and infect new hosts via the respiratory tract. Four phases of infection in vivo can be delineated: 1) early productive-restrictive virus infection causing primarily degenerative changes, 2) latent infection, 3) a second phase of cytolytic infection coincident with permanent immunosuppression, and 4) a proliferative phase involving nonproductively infected lymphoid cells that may progress to the point of lymphoma formation (6). MDV and HVT have genome structures closely resembling that of the alphaherpesviruses, such as herpes simplex virus 1 (HSV-1) - the prototype alphaherpesvirus, varicella-zoster virus (V ZV), pseudorabies virus (PRV), bovine herpesvirus 1 (BHV - 1) and equine herpesvirus 1 (EHV-l). Alphaherpesvirus genome structure consists of covalently joined long (L) and short (8) components. The 8 component comprises a unique short (Us) segment, which is flanked by a pair of inverted repeat regions. There are four glycoprotein genes in the HSV-1 Us region, encoding glycoproteins G (gG), D (gD). I (g1) and E (gE) (12). HSV-1 gD is a virion envelope component which plays an essential role in HSV-1 entry into susceptible mammalian cells (18). HSV-1 gD has been 53 implicated in receptor binding, cell fusion, and neuroinvasiveness (14). Immunization of animals with HSV-1 gD stimulates the production of virus- neutralizing antibodies and protects them from both lethal challenge with HSV-1 and the establishment of latency (5). Homologs of HSV-1 gD have been identified in the genomes of PRV and BHV-l, among other alphaherpesviruses. The gDs of HSV-1, PRV and BHV -1 cause viral interference (19, 29, 8). Although PRV’s gD homolog is essential for penetration, its production is not required for cell to cell spread (28). The g1 and gE homologs of HSV-1, PRV and VZV are found to form complexes. HSV-1 gE and VZV gE act as IgG Fc receptors that may utilize an antibody bipolar bridging mechanism to protect virus-infected cells from antibody-dependent cellular cytotoxicity (17, 23). HSV-1 and PRV gE are involved in neurotropism and virulence during virus infection of animals (26, 7). The entire MDV Us region has been sequenced in our lab (3, 4). Genes encoding the MDV gD, g1 and gE homologs have been found in this region, although no gG homolog was found. Antisera to their TrpE fusion proteins have been produced (Figure 1). MDV g1 and gE have been identified in MDV- infected cells by immunoprecipitation with their respective antisera (2, 3, 10). In contrast, no gD was found in MDV-infected cells with antisera to its trpE fusion proteins (2). In this study, antiserum to TrpE-gD was used in a further attempt to detect MDV gD expression in MDV-infected cells, but no protein of the predicted size was found. When the Northern blot, RT-PCR and RNAase protection analyses were performed, an abundant 3.5 kb bicistronic mRNA including MDV g1 and gE genes was detected together with an abundant 2.0 kb monocistronic transcript specific for the gE gene. In contrast, there was 54 no specific message initiating from the gD gene promoter, although a low abundance polycistronic mRNA including MDV U83, SORF4, gD, g1 and gE genes was found. 55 MATERIALS AND METHODS Cells and viruses. The preparation, propagation and infection of duck embryo fibroblast (DEF) and chicken embryo fibroblast (CEF) cells with cell- associated MDV was performed as described previously (9). The virulent MDV GA strain used in this study was at cell culture passage level six following isolation of cell-free virus from feather tips obtained from infected birds with symptoms of Marek’s disease. Generation of TrpE fusion proteins and antibodies to fusion proteins. The vector system used to express the MDV genes in E. coli consists of a group of plasmids (pATH vectors) encoding approximately 37 kDa of the bacterial trpE open reading frame (ORF) under control of the inducible trp operon promoter. A polylinker with multiple cloning sites at the 3’ end of the trpE ORF allows in-frame insertion of foreign ORFs. The TrpE fusion proteins were generated as described previously (3, 9). The fusion proteins were purified from preparative SDS-7.5% polyacrylamide gels and used to prepare antisera by injecting rabbits as described (9). In vitro transcription and translation. MDV SORF4, gD, g1 and gE genes were cloned downstream of T3 or T7 promoters. In vitro coupled transcription and translation was performed with Promega’s TNT Coupled Reticulocyte Lysate System. Radiolabeling of proteins and immunoprecipitation analysis. MDV- infected and mock-infected control DEF cells were labeled with [35S]- methionine at 72 hours postinfection for 4 hours. After labeling was complete, the culture medium samples and cell lysates were prepared as described. Immunoprecipitation analysis was carried out as previously described (9). Briefly, 400 pl of 35S-labeled cell lysates were pretreated with 56 normal rabbit serum for 2 hours, then protein A was added and incubated for 1 hour before centrifugation. After centrifugation, the supernatants were mixed with the respective antisera for 2 hours. Then protein A-sepharose was added and the suspension was incubated with gentle mixing for 2 hours, followed by centrifugation. Precipitate was resuspended in sample buffer, recentrifuged and subjected to SDS-PAGE. Protein markers (GIBCO) were used as molecular weight standards. RNA preparation, isolation and Northern blot analysis. RNAs were prepared from MDV-infected and mock-infected control cells at 72 hours postinfection by the guanidinium isothiocyanate-phenol extraction procedure. Poly (A)+ RNAs were prepared by oligo-dT cellulose spin columns. Northern blot analysis was performed as described (31). DNA probes were labeled by the random priming method. Riboprobes were prepared as described below. Transcript size determinations were based on a comparison with a GIBCO RNA ladder run in parallel. Riboprobe and RNA marker preparation. Antisense riboprobes and RNA marker were generated by in vitro transcription off linearized plasmid templates with RNA polymerase T3 or T7 (Promega). For riboprobes and RNA marker used in RNAase protection, the 20 pl reaction solution included 5mM of each GTP, ATP, CTP, in addition to 4.5 ul [Cl-321)] UTP (800 u Cilmmol). For riboprobes used in Northern blot analysis, 50 uM unlabeled UTP was added into the reaction. To determine the size of the protected fragments, a set of RNA markers was generated in the proper range. pBCSK+ plasmid (Stratagene) was cut by BamHI, XhoI, Hinfl or Pqu, and RNA fragments of 51, 102, 139 and 245 bases were produced, respectively. The RNA marker was a mixture of the four radiolabled RNA fragments. 57 RNAase protection analysis. 500,000 cpm of a riboprobe was hybridized with 40 pg of total RNA in 20 ul hybridization buffer (40 mM PIPES [pH6.4], 1 mM EDTA, 0.4 M NaCl, 80% formamide), and incubated at 42°C overnight. The hybridization solution was diluted with 300 pl digestion buffer (300 mM NaCl, 10 mM TrisHCl [pH=7 .5], 5 mM EDTA, 40 ug/ml RNAase A & 800U/ml RNAase T1), and incubated at 30°C for 60 min. After digestion, the samples were extracted with phenol/chloroform, and the RNAs were precipitated in ethanol. The pellets were then resuspended in loading buffer, and subjected to electrophoresis on a polyacrylamide/urea sequencing gel. RT-PCR Assays. Total RNA preparations were treated with RNAase-free DNAase I (10 units per mg of total RNA) for 30 min at 37°C. Reverse- transcription was performed with Superscript II RT (GIBCO) using the oligo- dT primer. This reaction (1/ 10th aliquot) was subjected to 30 cycles of PCR consisting of 94°C for 1 min, 50°C for 2 min, and 72°C for 3 min. This product (1/ 10th aliquot) was loaded onto agarose gels for electrophoresis, and DNA was visualized with ethidium bromide. Nucleotide positions of the primers are shown in Fig. 5A. Primer D sense (D8), 5’ TACGTGAATATGCCAACTGC 3’, corresponded to nucleotide positions 7340 to 7359. Primer D antisense (DA), 5’ AGTGAGTCCAGTGTAACCATCC 3’, corresponded to nucleotide positions 7875 to 7896. Primer I sense (18), 5’ TGGTATATGCTCAACCTCATGG 3’, corresponded to nucleotide positions 8574 to 8595. Primer I antisense (IA), 5’ ACCGATGTATATCCTACGATGG 3’, corresponded to nucleotide positions 8574 to 8595. Primer E sense (ES), 5’ GAATCGCTAAGTCTGAATGG 3’, corresponded to nucleotide positions 9791 to 9800. Primer E antisense (EA), 5’ CAGAATGTCAATGTTGGATGCG 3’, corresponded to nucleotide positions 10249 to 10270. 58 FIG. 1. Schematic representation of the MDV Us region DNA fragments used for antibody production and Northern blot analysis. The nucleotide position numbers are as previously published (3). Each bar represents an MDV U. region fused to trpE protein for antibody production. Arrows indicate those fragments also used as DNA probes specific for gD, g1 and gE regions of the transcripts in Northern blot analysis. 59 Tl III .I. 089 Km? $5 63 8% 8% I .I._ II. I1 is: a $3 53 some 3:152 83 8mm mRo Sow nmmv mvmv Bow 03m I II .II.. 1 TI. .II. II II I 28 28 881st 52 8; 8mm 88 83.. 2mm 8: N2: 7 A j A Z A :Cr A :v__ v _rAJ_A_ 69 mm: :9 5: 59 em: EmOm mm: mm: 9E8 Sm: Pm: w_m>..OOm_._.Z< m0”. 0mm: whims—06in. <20 20.0wm 3 >05. Figure 1 60 RESULTS Immunoprecipitation analysis failed to detect MDV gD in MDV- infected cell culture. Among the eleven MDV Us genes, seven. are HSV-1 homologs: U81, U810, U82, U83, U86 (gD), U87 (gI) and U88 (gE). Fragments from each gene have been cloned in-frame with trpE in order to make TrpE fusion proteins (Figure 1). Antisera to each trpE fusion protein have been produced. With these antisera, the proteins encoded by MDV U81, U810, U82, U83, U87 (gI) and U88 (gE) have been identified in extracts from MDV-infected DEF cells, but no gD (U 86) protein was found (2, 3). In further attempts to identify a possible lower level of MDV gD expression, immunoprecipitation and SDS-PAGE analysis were performed with rabbit anti-gD antibody and radio-labeled proteins from MDV GA-infected DEF cells. Anti-g1, anti-gE and anti-gB were used as positive controls. Normal rabbit serum (NR8) was used as a negative control. Anti-gE immunoprecipitated the two glycosylation forms of MDV gE (gp62 and gp72) (Figure 2 lane 8) , and anti-g1 immunoprecipitated both MDV g1 and gE (Figure 2 lane 6), as reported (2, 10). Anti-gB immunoprecipitated MDV gB (Figure 2 lane 10) (9). The MDV gD precursor polypeptide was predicted to be about 43 kDa (4). Based on its predicted amino acid sequence, gD has four potential N-linked glycosylation sites, resulting in a mature glycoprotein predicted to be about 53 kDa. However, no protein in the range of 40 kDa to 60 kDa was immunoprecipitated with anti-gD (Figure 2 lane 4). Four-fold longer exposure of the same gel still did not show any MDV-specific protein in that range (data not shown). When the same experiment was done in MDV GA- 61 FIG. 2. MDV gD is not expressed in MDV-infected DEF cells. Lanes 1, 3, 5, 7, 9: CON, mock-infected DEF cells. Lanes 2, 4, 6, 8, 10: INF, MDV GA-infected DEF cells. Lanes 1, 2: Immunoprecipitation analysis with NR8 (normal rabbit serum). Lanes 3, 4: Immunoprecipitation analysis with anti-gD. Lanes 5, 6: Immunoprecipitation analysis with anti-gI. Lanes 7, 8: Immunoprecipitation analysis with anti-gE. Lanes 9, 10: Immunoprecipitation analysis with anti-gB. Four day exposure. 62 Figure 2 MDV gD IS NOT EXPRESSED IN MDV GA-INFECTED DEF CELLS "assesses: C crcucrcn ONONONONON kDa NFNFNFNFN 200— b t' . c-‘g; I H“ 3(100) 97_ 999 i O - 3979 39E - 43— “.: ". 33:0”) 29— 12345678910 CON: Mock-infected DEF cells. INF: MDV-infected DEF cells. NR8, orgD, orgl, orgE, and 0:98: Immunoprecipita- tion analysis of infected cells with normal rabbit serum, anti-gD, anti-9|, anti-9E and anti-QB. 63 infected CEF cells instead of DEF cells, the same result was observed (data not shown). An MDV-specific protein of about 79 kDa was nonspecifically trapped by anti-gD, anti-gI, anti-gE and anti-gB (Figure 2 lanes 4, 6, 8, 10). Four-fold longer exposure showed that it also could be trapped by NR8 (data not shown). Thus, the 79 kDa protein seen in this gel is most likely the p79 reported by this lab previously (16). It was recognized as a sticky protein which could be nonspecifically trapped by either normal rabbit serum or specific pathogen-free chicken serum from MDV- or HVT-infected cells, but not from mock-infected cells. The gene encoding p79 has been sequenced (11), and is not the gD gene. Similar experiments have been done with extracts from MDV Md5 stain- infected, Md11 strain-infected, and JM/102w strain-infected DEF cells, and no gD expression was detected (manuscript in prepare). Therefore, the absence of a detectable level of gD expression in MDV-infected cell culture is not GA strain specific. MDV gD gene encodes a functional open reading frame. The failure of detecting gD expression could result from a defective open reading frame (ORF); the presumed gD ORF may not encode an authentic protein. To address this question, the MDV gD gene, as well as SORF4, g1 and gE genes as controls, were cloned downstream of T3 or T7 promoters, and in vitro coupled transcription and translation was performed with T3 or T7 RNA polymerases and rabbit reticulocyte lysate (Promega kit). Each of the SORF4, gD, g1 and gE genes was transcribed and translated into a precursor polypeptide (Figure 3A) (3). The gD precursor polypeptide was about 43 kDa, close to that predicted from its amino acid sequence. 64 FIG. 3. Expression of MDV gD and other MDV Us genes by in vitro coupled transcription/translation. (A) Direct SDS-PAGE analysis. 12 hours exposure. Lane 1: Without DNA template. Lane 2: MDV SORF4 gene template. Lane 3: MDV gD gene template. Lane 4: MDV gI gene template. Lane 5: MDV gE gene template. Lane 6: Luciferase gene template. (B) Immunoprecipitation analysis prior to SDS-PAGE. Three days exposure. Lane 1: precursor polypeptide of glycoprotein gD was immunoprecipitated by anti-gD. Lane 2: precursor polypeptide of glycoprotein gI was immunoprecipitated by anti-g1. Lane 3: precursor polypeptide of glycoprotein gE was immunoprecipitated by anti-gE. Figure 3 EXPRESSION OF MDV 90 AND OTHER MDV U. GENES BY in vitro COUPLED TRANSCRIPT ION/TRANSLATION A. Direct SOS-PAGE Analysis 8. Immunoprecipitation Pi t D -PA E S M M M ror as S G - O D D D + C R V V V C o F 9 9 9 O 9 kDa N 4 D I E N E p 200- 97- 68- 43- 29— 18- -- 123456 123 66 Failure to detect gD expression in MDV-infected cells by immunoprecipitation could result from failure of antibody to recognize gD. To confirm the anti-gD antibody is reactive to gD protein, immunoprecipitation analysis was conducted with the gD, g1 and gE precursor polypeptides produced by in vitro coupled transcription and translation, and their respective antisera. While three antisera to different gD fragments were used (Figure 1), only antibody against the gD fiagment encoded by nucleotides 7501-8154 was able to react with the gD precursor polypeptide (Figure 3B, negative data not shown). This antibody also recognizes the glycosylated form of gD (manuscript in prepare). This anti-gD antiserum had been used in the previous immunoprecipitation analysis (Figure 2). Successful detection of the gD precursor polypeptide produced by in vitro coupled transcription and translation demonstrated that the MDV gD gene encodes a complete gD protein. Therefore, absence of detectable MDV gD expression in cell culture may due to inefficient gD transcription, ineffective translation, or both. Northern blot analysis of mRNA from MDV-infected cells using DNA probes. Like other alphaherpesvirus homologs, MDV gD (US6), gI (U 87) and gE (U 88) genes are clustered in that order within the unique short region of the viral genome, spanning nucleotides 6943 to 10960 (Figure 1) (4). One poly(A) terminator sequence, AATAAA, is located right upstream of U86 (gD), and another AATAAA sequence is located downstream of U88 (gE). There is no such sequence downstream of U86 (gD) or U87 (gI). In eucaryotic systems, protein is most likely translated from the first favorable ATG of an mRNA. Therefore, for efficient expression of MDV gD or gI genes, mRNA which starts just upstream of the gD gene or the g1 gene should exist. 67 FIG. 4. Northern blot analysis of mRNA from MDV-infected cells using DNA probes. Three days exposure. Lanes 1, 3, 5: Uninfected DEF. Lanes 2, 4, 6: MDV GA-infected DEF. Lanes 1, 2: Hybridized with an MDV gD-specific probe. Lanes 3, 4: Hybridized with an MDV gI-specific probe. Lanes 5, 6: Hybridized with an MDV gE-specific probe. *The 7.5-8 kb band was of very low abundance and was seen only after 10-12 days exposure (Data not shown) . Figure 4 NORTHERN BLOT ANALYSIS OF POLY (A)+ RNA FROM MDV-INFECTED CELLS USING DNA PROBES SPECIFIC FOR: 90 9| 9E C I C I o l o N o N o N M Kb N F N F N F Kb ‘7' ‘9'! III 3'. — 9.5 ~7.5 — . ' . _ 75 — 4.4 ~3.5 — . en ; I — 2.4 '1 .- ~2.0 — ' . l — 1.4 .' ' ' , —o.24 69 FIG. 5. RT-PCR analysis of MDV gD transcription in MDV - infected cells. (A) Nucleotide positions in the MDV U. region encompassing gD, g1 and gE. D8: Primer gD sense. DA: Primer gD antisense. IS: Primer gI sense. IA: Primer gI antisense. ES: Primer gE sense. EA: Primer gE antisense. The nucleotide position numbers are as previously published (3). (B) Gene- specific primer pairs. Lanes 1, 3, 5: Reaction in the absence of reverse transcriptase. Lanes 2, 4, 6: Reaction in the presence of reverse transcriptase. Left marker: MHindIII marker. Right marker: 123 bp ladder. (C) Primer pairs spanning two or more genes. Lanes 7, 9, 11: Reaction in the absence of reverse transcriptase. Lanes 8, 10, 12: Reaction in the presence of reverse transcriptase. Left marker: MHindIII marker. Right marker: 1 kb ladder. 70 Figure 5 To: ‘4 <<05. on. E 95:69.). 2:50.032 .< mime “in 853235 >92 2. zo_E_momz92 “.0 m_m>._5 Son 2385 30 0:0 CU Ca 28. 9.3 $8 9.8 $8 89. v Wv WI w I .1 I II 38 36 ~80 38 99. 2% III I - I --I :1 T A L w A .2 m A flLHIIIHHIVHr IiVII 88. $3 mmmm 6mm 35 9.8 m E 98%.. 28 89. 2.3 22 9%.. Q9 mm: :9 Am: 59 em: 3E8 mm: 93 Exam 74 from the g1 gene are designated of IS and IA, and the primers from the gE gene are designated of E8 and EA (Figure 5A). When gene-specific pairs of primers were used in the RT-PCR reaction, all of the gD, g1 and gE cDNA fragments were amplified to the sizes expected from their DNA sequences (Figure 53). Reaction with primers DS/DA produced a DNA fragment of about 550 bp. Reaction with primers IS/IA produced a fragment of about 600 bp. Reaction with primers ES/EA produced a fragment of about 480 bp. Since no product was found in each control experiment (Figure 5B lanes 1, 3, 5), the positive RT-PCR data did not come from carry-over of viral DNA. This result indicated that there is a low abundance transcript which includes the gD gene. Instead of gene-specific primer pairs, primers from two different genes were used in pairs in the following RT-PCR reaction. When primer pair IS/EA, which spans the g1 and gE genes, was used, a DNA fragment of about 1700 bp was produced (Figure 5C lane 8). This result was consistent with a I gI-gE bicistronic transcript. When primer pair DS/IA, spanning the gD and g1 genes, was used, a DNA fragment of about 1800 bp was produced. Further, when the DS/EA primer pair, spanning the gD, g1 and gE genes, was used, a DNA fragment of about 2900 bp was produced. These results were consistent with a polycistronic transcript including the gD, g1 and gE genes Northern blot analysis of mRNA from MDV-infected cells using riboprobes. To further characterize the low abundance large transcript, more sensitive Northern blot analysis was performed with riboprobes and larger amount of mRNA. MDV-infected CEF cells were used instead of DEF cells in this experiment to determine if the transcription pattern was different between these two cell culture systems. 75 FIG. 7. RNAase protection assays of RNAs from MDV- infected cells. (A) Location of probes used for RNAase protection. The number adjacent to each probe indicates the distance in the number of nucleotides away from the first nucleotide of each ORF. (B) CON: RNA from mock-infected DEF cells. INF: RNA from MDV GA-infected DEF cells. M: RNA markers. Flgure 7 A. 90 9| 95 403-“ an 355-99 ORF 497-“ car [AATAAA r I 1 F I 1 I I l l g gl5’ gES' 3’ -183.__.I_.+32 -108.__I__.+118 -45|_.l__a+164 215 bs 226 bs 209 bs 015' £8 3 c I c I o N o N Beeee M N F Beeee M N F Beeee II 2425—. 245— 245. . Q—zze P O O C 139— . .442 ISO—I . - . u—Ize ' - " I 102— . 102— O a O 102— . 9 6 I 9 - . O 51— 77 In the MDV Us region, the SORF3 and U82 genes are leftward oriented, while U83, SORF4, gD, g1 and gE genes are rightward oriented (Figure 6A) (3). DNA fragments including each gene were cloned between T7 and T3 promoters so that riboprobes could be synthesized. All of the riboprobes were generated as leftward oriented. Thus, they were in antiparallel to the U83, SORF4, gD, gI or gE genes, but parallel to the U82 and SORF3 genes (Figure 6A). Because riboprobes are much more sensitive than DNA probes, and because four times more mRNA was used in these experiments than in the previous Northern blot analysis with DNA probes, the low abundance 7 .5 kb transcript was now more readily detected and analyzed (Figure 6B lane 8). Comparing this Northern blot experiment with previous one, both the gI-gE bicistronic 3.5 kb transcript and the gE-specific 2.0 kb transcript were detected (Figure 4, Figure 6B), as expected. A less abundant gE-specific 4.4 kb transcript was also detected by this more sensitive method after 1 day exposure (Figure 6C lane 12). However, 12 hours of exposure of the same blot clearly showed only the very abundant 3.5 kb and 2.0 kb transcripts (data not shown). The low abundance species would appear to have little significance in the synthesis of gE. Thus, the transcription pattern is very similar between the MDV infected-CEF cells and DEF cells. After four fold longer exposure, the 7.5 kb transcript was detected by U83, SORF4, gD, g1 and gE riboprobes, but not by the U82 probe (Figure 6C). Therefore, the low abundance large transcript detected by the gD probe does not initiate immediately 5’ of the gD gene, but from two ORFs upstream of the gD gene. The 7 .5 kb transcript is unlikely to be translated into gD protein. 78 FIG. 8. MDV transcript termini deduced from Northern blot analysis and RNAase protection analysis. Each rectangle represents protected fragment. Dotted lines indicate the 5’ end and 3’ end of each transcript. There are two possible 3’ termini for each transcript. 79 Figure 8 _ _ _ L _ Lo .9529» 8. 3 IL _ _I L _ “ " "wt 3.596: 8. 0m IL _ _ _ _ _ _ _ . _ _ _ LM.-I;_.AL__.3€; 8. 3 L _ _ _ _ L L n _ _m 28 _ _ LU " u _ _ 3mm, _ _ LHHU _ mVI mDNv; _ _ _ _ _ LIIIU _ _ .m _ _ 88m _ _ _ 9....IILImoT " _ .mmo _ _ re _ _ mam—N _ _ N9ILIST _ ma _ I _ L L L L L L -L L L L I: EIL _ $9 $3 :3 Am: 53 em: Knew mm: 3.: <.L._05. 80 RNAase protection analysis of MDV g1 and gE transcripts. In contrast to the gD gene, the g1 and gE genes are transcribed efficiently in MDV-infected cells. To determine the promoter regions and 3’ termini of these transcripts, RNAase protection analysis was performed. A series of subgenomic fragments inclusive of ATG sites or AATAAA sequence were subcloned into pBCSK+ plasmids. A series of riboprobes complementary to mRNA were generated (Figure 7A), hybridized to RNA obtained from mock- infected and MDV-infected cells at 72 hours post-infection, digested with RNAase, and analyzed on sequencing gels. The size of each fragment was calculated based on comparison with the synthesized RNA marker. To determine the initiation site proximal to the gI gene, plasmid pBCBl was constructed. This plasmid contains a 215 bp insert spanning sequences 183 bp upstream to 32 bp downstream of the gI ATG site (Figure 7A). After digestion with EcoRI, this plasmid could be used to generate a 215 bases antisense probe. RNAase protection produced an abundant fragment of 55 bases (Figure 7B gI5’). The size of this product was consistent with a initiation site located 23 bases upstream of the g1 ATG site. The result suggests that this transcript is the 3.5 kb gI-gE bicistronic mRNA (Figure 8). Another larger fragment of 215 bp was also protected, and was likely due to protection from the low abundance 7.5 kb transcript. To map the initiation site proximal to the gE gene, plasmid pBCKVl was constructed. The plasmid contains a 226 bp insert spanning the gE ATG site. After digestion with BanI, this plasmid could be used to generate a 226 bases antisense probe initiating 118 bases within gE gene and extending to 108 bases upstream of the gE ATG codon (Figure 7A). The full-length fragment from RNAase protection might result from both the 3.5 kb gI-gE bicistronic transcript and the low abundance 7 .5 kb polycistronic transcripts. In 81 addition, a smaller fragment of 142 bases was also protected (Figure 7B gE5’). The size of this fragment indicates a transcription initiation site 24 bases upstream of the gE ATG site. The result suggests that the gE specific transcript is the 2.0 kb mRNA (Figure 8). Because a AATAAA sequence is located downstream of the gE gene, but not of the g1 gene, only one riboprobe was needed to map the 3’ termini of these transcripts. Plasmid pBC8m4, which contains a 209 bp insert spanning sequences 45 bp upstream to 164 bp downstream of the AATAAA sequence, was constructed (Figure 7A). After digestion with EcoRI, this plasmid could be used to generate a 209 base antisense probe. The probe protected two fragments of 53 bases and 129 bases (Figure 7B 3’). The 129 bases fragment was consistent with transcripts terminating at 94 bases downstream of the AATAAA site. The 53 base fragment indicated a transcript terminus at eight bases downstream of the AATAAA site (Figure 8). It is likely that both the 3.5 kb and the 2.0 kb transcripts can terminate at either site. 82 DISCUSSION This study confirmed that MDV gD expression is under the limit of detection in MDV-infected cell culture, and showed that it is because of inefficient gD gene-specific transcription. The only transcript clearly detected by the gD gene probe initiates two ORFS upstream of the gD gene. Research on HSV-1 has revealed that each viral gene has its own promoter, and each transcript contains a single functional ORF. Although some of HSV-1 mRNAs contain additional protein coding sequences downstream of their primary ORFS, proteins are not translated from the downstream mRNA region. Instead, abundant proteins are translated from the mRNAs initiating immediately 5’ to their genes (25), consistent with the overwhelmingly monocistronic nature of eukaryotic genes. Thus, a priori, the possibility of significant gD gene translation from the 7.5 kb transcript is very unlikely. HSV-1 gD gene transcription initiates at about 85 bp upstream of its ATG codon. Two TATA motifs are located at 108 bp and 126 bp 5’ to the ATG codon. Furthermore, DNA within the 168 bp upstream is required for regulated expression of the HSV-1 gD gene, and the consensus ICP4 binding motif ATCGTC is found in this region (24). In the upstream region of MDV gD gene, three TATA motifs are located at 27, 36 and 50 bp 5’ to the ATG codon. No ICP4 binding motif is found there. A consensus poly(A) signal AATAAA sequence is located 60 bp 5’ to the gD start codon, which is very close to the TATA motifs. The proximity of the AATAAA sequence may destabilize the transcription initiation complex formed on the TATA sequences, and abort the transcription initiation from the gD gene. MDV gI gene transcription is initiated from 23 bp 5’ to its ATG Site, in contrast to its homolog, the HSV-1 gI transcript, which is initiated 91 bp 5’ to 83 its ATG Site. Similarly, the MDV gE untranslated region is 24 bases long, only one third the length of its HSV-1 homolog of 73 bases. Considering that the region between MDV gD and g1 genes, and between the g1 and gE genes, is much shorter than the corresponding regions among the HSV-1 gD, g1 and gE genes, the relatively shorter untranslated regions of MDV g1 and gE transcripts are expected. Either the TATATA sequence or the TATAG sequence upstream of the g1 gene CAP site may serve as the TATA box for transcription initiation. Similarly, the TATAT sequence, the TTTAAA sequence, or the TATAA sequence upstream of the gE initiation Site may serve as the TATA box for gE transcription initiation. Having mapped the 5’ termini of the gI and gE transcripts with RNAase protection assay, their promoter regions have been identified and can be studied. The VZV gpIV (gI homolog) promoter has a very low basal level of transcription, but viral factors or virus-induced factors can significantly activate its promoter activity (22). Whether the MDV gI and gE promoters share Similar features remains to be determined. The 2.0 kb and 3.5 kb transcripts of g1 and gE appear to coterminate at a typical AATAAA motif. It is very common among alphaherpesvirus transcription that one polyadenylation signal is shared by several genes, each initiating from their own promoter (20, 25). Two possible 3’ termini of g1 and gE transcripts were identified in the RNAase protection assay. Heterogeneous 3’ ends have been described previously for VZV gpIV (gI homolog) transcript (22). Very likely both 2.0 kb and 3.5 kb transcripts can terminate at either site. Because Figure 6 is a composite that results from the development of 12 different films, those that had been fixed somewhat longer had darker background (Figure 6B lanes 7, 8; Figure 6C lanes 1, 2, 9, 10). Longer 84 exposure of the Northern blot showed several very low abundance fragments smaller than the low abundance 7.5 kb transcript (Fig. 6C lanes 4, 6, 8). These signals may represent nonspecifically hybridized RNA, or, may represent transcripts in extremely low abundance. Translation from these uncharacterized transcripts, and leaky scanning translation from the 7.5 kb transcript, may be able to produce very few gD molecules. Nonetheless, even if these rare translations take place, the few gD molecules could play no significant role in MDV infection. This notion is supported by a study by Morgan’s group in which an MDV gD lacZ- insertion mutant was made which had similar growth kinetics to the wild type MDV in cell culture (27). Among the other alphaherpesviruses, HSV-1 has an essential gD gene; VZV, a highly cell-associated virus, lacks a gD homolog altogether; PRV and BHV-l are cell-free viruses, but their gD mutants become cell-associated, although their infectivity is greatly impaired (15, 21, 30). In keeping with these observations, MDV, a highly cell-associated virus in cell culture, lacks detectable gD expression in cell culture. Feather follicle epithelium of MDV- infected chicken is the only known tissue producing cell-free virus, and whether MDV gD is expressed there iS a very interesting question. HSV-1 is commonly used as the prototype for alphaherpesvirus research. Pioneer work in HSV-1 gene characterization laid the foundation for studying their homologs in other alphaherpesviruses. However, HSV-1 appears to be somewhat unique with respect to its dependence on gD. Consequently, analysis of a specific gene product such as gD, should be done on an individual basis. The gD homologs of many alphaherpesviruses play important roles in virus infection. That is especially true for HSV-1, whose gD is crucial in its pathogenesis and has been targeted as a subunit vaccine for prevention of 85 virus infection. Since the MDV gD gene was sequenced, many efforts have been made to assess its potential as subunit vaccine. However, it is now very clear that MDV gD does not play the same role as HSV-1 gD. Alphaherpesvirus g1 and gE homologs play important roles in cell to cell transmission of virus (13, 32). For the highly cell-associated virus VZV that does not have gD gene at all, its gE homolog is one of the most abundant viral protein in infected cells, and has been characterized as one of the major target in the host immune responses (1). Perhaps it is time for MDV vaccine research to switch focus from gD to gE and other proteins. 86 REFERENCES 1. Arvin, A. M. 1992. Cell-mediated immunity to Varicella-zoster virus. J. Infectious Diseases. 166 (Suppl 1): 835-41. 2. Brunovskis, P., X. Chen, and L. F. Velicer. 1992. Analysis of Marek’s disease virus glycoproteins D, I and E, p. 118-122. In Proceedings 19th World’s Poultry Congress, vol. 1. 4th Int. Symp. Marek’s Dis. Part, Posen and Looijen, Wageningen, The Netherlands. 3. Brunovskis, P., and L. F. Velicer. 1992. Genetic organization of the Marek’s disease virus unique Short region and identification of Us-encoded polypeptides, p. 74-78. In Proceedings 19th World’s Poultry Congress, vol. 1. 4th Int. Symp. Marek’s Dis. Part, Posen and Looijen, Wageningen, The Netherlands. 4. Brunovskis, P., and L. F. Velicer. 1995. The Marek’s disease virus (MDV) unique short region: alphaherpesvirus-homologous, fowlpox virus- homologous, and MDV-specific genes. Virology. 206: 324-338. 5. Burke, R. L. 1993. HSV vaccine development, p. 367-380. In B. Roizman and C. Lopez (ed.). The herpesviruses. Raven Press, New York. 6. Calnek, B. W., and R. L. Witter. 1991. Marek’s disease, p. 342-385. In Calnek, B. W. (ed.). Diseases of poultry. Iowa State University Press, Ames, Iowa. 7. Card, J. P., M. E. Whealy, A. K. Robbins, and L. W. Enquist. 1992. Pseudorabies virus envelope glycoproteins gI influences both neurotropism and virulence during infection of the rat visual system. J. Virol. 66: 3032- 3041. . 8. Chase, C. C. L., K. Carter-allen, C. Lohff, and G. Letchworth III. 1990. Bovine cells expressing bovine herpesvirus 1 glycoprotein IV resist 87 infection by BHV-l, herpes simplex virus, and pseudorabies virus. J. Virol. 64: 4866-4872. 9. Chen, X., and L. F. Velicer. 1992. Expression of the Marek’s disease virus homolog of herpes Simplex virus glycoprotein B in Escherichia coli and its identification as B antigen. J. Virol. 66: 4390-4398. 10. Chen, X., X. Tan, P. Brunovskis, and L. F. Velicer. Identification and characterization of the Marek’s disease virus homologs of the herpes Simplex virus glycoproteins I and E. (Paper submitted). 11. Cui, Z., Y. You, and L. F. Lee. 1992. Identification and localization of the Marek’s disease virus group-common antigen p79 gene, p. 89-92. In Proceedings 19th world’s poultry congress, vol.1. 4th Int. Symp. Marek’s Dis. Part, Pasen and Luoijen, Wageningen, The Netherlands. 12. Davison, A. J. (ed.). 1993. HSV and other alphaherpesviruses. Seminars in Virology. Vol 4. Issue 3. Academic Press, San Diego, CA. 13. Dingwell, K. 8. et al. 1994. Herpes simplex virus glycoproteins E and I facilitate cell-to-cell spread in vivo and across junctions of cultured cells. J. Virol. 68: 834-845. 14. Fuller, A.O., and W.-C. Lee. 1992. Herpes Simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration. J. Virol. 66: 5002-5012. 15. Heffner, 8., F. Kovacs, B. G. Klupp, and T. C. Mettenleiter. 1993. Glycoprotein gp50-negative Pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine. J. Virol. 67: 1529-1537. 16. Isfort, R. J., I. Sithole, H. Kung, and L. F. Velicer. 1986. Molecular characterization of Marek’s disease herpesvirus B antigen. J. Virol. 59: 411- 419. 88 17. Johnson, D. C., M. C. Frame, M. W. Ligas, A. M. Cross, and N. G. Stow. 1988. Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and g1. J. Virol. 62: 1347-1354. 18. Johnson, D. C., and M. W. Ligas. 1988. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors. J. Virol. 62: 4605-4612. 19. Johnson, R. M., and P. G. Spear. 1989. Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection. J. Virol. 63: 819-827. 20. Kinchington, P. R., J. Vergnes, P. Defechereux, J. Piette, and S. E. Turse. 1994. Transcriptional mapping of the varicella-zoster virus regulatory genes encoding open reading frames 4 and 63. J. Virol. 68: 3570- 3581. 21. Liang, X., C. Pyne, Y. Li, L. A. Babiuk, and J. Kowalski. 1995. Delineation of the essential function of bovine herpesvirus 1 gD: an indication for the modulatory role of gD in virus entry. Virology. 207: 429- 441. 22. Ling, P., P. R. Kimchington, M. Sadeghi-zadeh, W. T. Ruyechan, and J. Hay. 1992. Transcription from varicella-zoster virus gene 67 (glycoprotein IV). J. Virol. 66: 3690-3698. 23. Litwin, V., W. Jackson, and C. Grose. 1992. Receptor property of two varicella-zoster virus glycoproteins gpI and gpr homologous to herpes Simplex virus gE and g1. J. Virol. 66: 3643-3651. 24. McGeoch, D. J., A. Dolan, 8. Donald, and F. J. Rixon. 1985. Sequence determination and genetic content of the Short unique region in the genome of HSV1. J. Mol. Biol. 181: 1-13. 89 25. McGeoch, D. J. Correlation between HSV-1 DNA sequence and viral transcription maps. 1992. In Wagner, E. K. (ed). Herpesvirus transcription and its regulation. CRC Press, Boca Raton, Fla. 26. Neidhardt, H., C. H. Schroder, and H. C. Kaerner. 1987. Herpes simplex virus type 1 glycoprotein E is not indispensable for viral infection. J. Virol. 61: 600-603. 27. Parcells, M. 8., A. 8. Anderson, and R. W. Morgan. 1994. Characterization of a Marek’s disease virus mutant containing a LacZ insertion in the U86 (gD) homolog gene. Virus Genes. 9(1): 5-13. 28. Peeters, B., J. Pol, A. Gielkens, and R. Moormann. 1993. Envelope glycoprotein gp50 of pseudorabies virus is essential for virus entry but is not required for viral spread in mice. J. Virol. 67: 170-177. 29. Petrovskis, E. A., A. L. Meyer, and L. E. Post. 1988. Reduced yield of infectious pseudorabies virus and herpes simplex virus from cell lines producing viral glycoprotein gp50. J. Virol. 62: 2196-2199. 30. Rauh, I., and T. C. Mettenleiter. 1991. Pseudorabies virus glycoproteins gII and gp50 are essential for virus penetration. J. Virol. 65: 5348-5356. 31. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning. Cold Spring Harbor Laboratory Press, NY. 32. Zsak, L., F. Zuckermann, N. Sugg, and T. Ben-Porat. 1992. Glycoprotin g1(gE homolog) of Pseudorabies virus promotes cell fusion and virus spread via direct cell-to-cell transmission. J. Virol. 66: 2316-2325. Chapter III Expression of Marek’s Disease Virus Glycoprotein D 90 91 ABSTRACT In this study, antibody to Marek’s disease virus (MDV) glycoprotein D (gD) was made against a GST-gD fusion protein. This antiserum reacted with both the precursor polypeptide and with the glycosylated form of gD produced by in vitro coupled transcription and translation. Duck embryo fibroblast cells were infected with several commonly used MDV strains: GA strain, Md5 strain, low passage and high passage Mdll strain, JM/102w strain, and an antigenically related HVT FC/126 strain. Lysates of the infected cells were subjected to immunoprecipitation analysis with the anti- gD antibody, and anti-gI antibody was used as control. Although expression of gI was detected in all of the infected cells, gD expression was below the limits of detection. Therefore, absence of a detectable level of gD expression in cell culture is not GA strain-specific, but is a general phenomenon of MDV. When the gD gene was cloned into a eucaryotic expression vector, the gD protein was expressed. However, in comparison to gI and gE expression in the same system, gD expression was less efficient. To assess intragenic control elements, the N-terminal region of gD was replaced with that of gI. The expression of the hybrid gI-gD was significantly higher than the expression of gD. Previous work has shown that absence of gD expression in MDV-infected cell culture is due to inefficient gD gene transcription. This study suggests that negative regulatory elements in the gD gene may also be involved in the downregulation of gD expression. 92 INTRODUCTION Marek’s disease virus (MDV) causes severe lymphomas in infected chickens. The apathogenic and antigenically-related herpesvirus of turkey (HVT) has been used as an infected cell vaccine against MDV. However, HVT is not 100% protective against infection by the very virulent MDV strains, and cell culture vaccine production, storage and shipment are expensive (5). Thus, an alternate, inexpensive, and efficacious vaccine is needed. Herpes simplex virus 1 (HSV-1) gD is an essential structural protein for virus infection. Immunization of animals with HSV-l gD stimulates the production of virus-neutralizing antibodies and protects them from both lethal challenge with HSV-1 and the establishment of latency (4). Homologs of HSV-1 gD have been found in the Us region of pseudorabies virus (PRV), bovine herpesvirus 1 (BHV-l), and equine herpesvirus 1 (EHV-l). Vaccination of animals with a recombinant PRV gD homolog or a BHV -1 gD homolog was shown to protect them against lethal infection (12, 8). Similarly, animals inoculated with the recombinant EHV-l gD develop neutralizing antibody responses against EHV-l (11). The entire MDV Us region has been sequenced in our lab, and seven HSV-1 gene homologs, including the gD gene, have been found (2, 3). Antisera to their TrpE fusion proteins have been produced, and immunoprecipitation analyses on lysates of MDV GA-infected cells were performed. No gD 93 expression was found, although all of the other six HSV-1 homolog gene products were detected (1, 7). Extensive transcriptional analyses have uncovered the absence of effective gD gene transcription (14). However, the discovery of inefficient transcription could not exclude other factors that may also be involved in the failure of detection gD expression. The anti-gD antibody may not recognize the glycosylated form of gD protein, the absence of detectable gD expression may be GA strain-specific, or the gD translation may be inefficient. In this study, new antiserum was raised against a GST-gD fusion protein, and reacted with glycosylated form of MDV gD produced by in vitro coupled transcription/translation and glycosylation. Using this antibody, the absence of detectable gD gene expression was confirmed in MDV GA-infected cells, and that conclusion was extended to the other commonly used MDV strains Md5, Mdl 1, JM/102w, and HVT FC/126. The gD, gI and gE genes were cloned into a eukaryotic expression vector, and transfected into COS7 cells for a transient expression assay. Although gD expression was demonstrated, it was not as good as the expression of gI and gE. The N-terminal codons of the gD gene were then replaced with the N-terminal coding region of the gI gene. When the gI-gD hybrid was transiently expressed in C087 cells, the expression was much better than the native gD expression. Therefore, negative regulatory elements in the N- 94 terminal coding region of gD may contribute to the absence of detectable gD expression in MDV-infected cell culture. 95 MATERIAL AND METHODS Cells and viruses. The preparation, propagation and infection of duck embryo fibroblast (DEF) cells with cell-associated MDV was performed as described previously (6). The virulent MDV GA strain used in this study was at cell culture passage level Six following isolation of cell-free virus from feather tips obtained from infected birds showing signs of Marek’s disease. The low passage Md11 strain was at cell culture passage 19, and the high passage Md11 strain was at cell culture passage 88. The very virulent Md5 strain was at cell culture passage level five. The JM/102w strain was at cell culture passage 50. The HVT FC/126 strain was at passage 14. Generation of GST-gD fusion protein and antibody to GST-gD fusion protein. The gD gene fragment from nucleotides 7169 (NdeI) to 8025 (SpeI) was end-blunted and ligated into the SmaI site of pGEX3X (Pharmacia). The constructed plasmid, named pGEX3XgD, was sequenced, confirming the in-frame ligation. A bacterial culture harboring pGEX3XgD was induced with IPTG, incubated an additional 2 hours, and the bacterial pellet was collected following centrifugation. The bacterial pellet was then resuspended in 1% Triton X-lOO/PBS, sonicated, and Shaked gently for 30 minutes to aid in solubilization of bacterial proteins. Inclusion bodies were collected by centrifugation, resuspended in SDS-PAGE loading buffer (62.5 mM TrisCl pH 6.8, 2% SDS, 2% Mercaptoethonal, 0.01% Bromophenol blue, 96 10% Glycerol), incubated in boiling water for 10 min, and loaded on a preparative gel of SDS-PAGE. The gel was stained with 0.05% Commassie blue water solution for 10 min. Since more than half of the total amount of protein was GST-gD fusion protein, a band corresponding to the 56 kDa GST- gD was easily identified after the staining. This band was excised, and electroeluted. A 0.4 ml mixture of 50 pg GST-gD fusion protein and Titermax adjuvent was injected into a rabbit. The rabbit was boosted with the same mixture every four weeks. After three boosts, antiserum was collected, tested by Western blotting, and used as anti-gD antibody. Western blotting. Protein samples of GST and GSTgD were run on SDS- PAGE, and electro-transferred onto nitrocellulose. ECL western blotting analysis system (Amersham) was used to detect the protein signal. Kaleidoscope prestained protein standard (BioRad) was run in parallel to determine the efficiency of Western transfer and the molecular weight of the proteins. Plasmids for gene expression. The DNA fragment from nucleotide positions 6922 (BanI) to 8195 (HincII), which includes the gD gene, was cloned into the expression vector pCDNA3 (Invitrogen), and named pC3gD. The DNA fragment from nucleotides 8195 (HincII) to 9354 (KpnI) was cloned into pCDNA3 and named pC3gI. The DNA fragment from nucleotides 9443 (NspI) to 10971 (DraI) was cloned into pCDNA3 and named pC3gE. The plasmid pC3gID for expression of gI-gD hybrid included nucleotides 8195 97 (HincII) to 9016 (EcoRI) and 7501 (MluI) to 8195 (HincII). Several steps of subcloning had been performed before the final expression plasmids were obtained. Large scale preparation of these plasmids was performed with QIAGEN Maxi kit, and purified plasmids were dissolved in distilled water at concentration of 0.5-1 mg/ml. In vitro transcription, translation and post-translational modification. Gene expression in vitro was performed with TNT T7 Coupled Reticulocyte Lysate System (Promega). To synthesize a precursor polypeptide, the reaction solution included TNT lysate, TNT buffer, T7 RNA polymerase, amino acid mixture minus methionine, 35S- Methionine, RNaSin, and the expression plasmid. To produce a glycosylated protein, canine microsomal membranes were added to the reaction solution. The reaction was incubated at 30°C for 90-120 minutes. Products were then analyzed by SDS-PAGE and fluorography. Transfection of C087 cells. COS7 cells were transfected by either the electroporation or LIPOFECTAMINE method. For electroporation, 5><106 cells and 20 pg of plasmid DNA were resuspended in 0.8 ml PBS, transferred into a 0.4 cm chamber cuvette, and eletroporated at 230V, 960 pF using a BioRad electroporator. For LIPOFECTAMINE transfection, 2><105 cells were seeded in a 35 mm plate one day before transfection. For transfection, solution A, which includes 2 pg of DNA diluted in 375 pl OPTI-MEM medium (GIBCO), and solution B, which includes 6 pl LIPOFECTAMINE (GIBCO) 98 diluted in 375 pl OPTI-MEM medium, were combined and incubated at room temperature for 45 min. Then 750 pl OPTI-MEM medium was added to the mixture of solution A and B, and the total 1.5 ml mixture was added onto C087 cells. After the cells were incubated for 5 hours in a C02 incubator, 1.5 ml fresh OPTI-MEM with 20% fetal bovine serum was added. Thirty hours after transfection, cells were labeled with 35S-methione. Protein expression was analyzed by immunoprecipitation and SDS-PAGE. Radiolabeling of proteins and immunoprecipitation analysis. Mock-infected, and MDV-infected DEF cells were labeled with [35S]- methionine at 72 hours postinfection, and transfected C087 cells were labeled at 30 hours post-transfection. After a 4 hour labeling period was complete, cell lysates were prepared and immunoprecipitation analysis was carried out as previously described (6). Briefly, 400 pl of 35S-labeled cell lysates were pretreated with normal rabbit serum for 2 hours, then protein A was added and incubated at 4°C for 1 hour before centrifugation. After centrifugation, the supernatants were mixed with the respective antisera for 2 hours. Then protein A-sepharose was added and the suspension was incubated with gentle mixing for 2 hours, followed by centrifugation. The precipitate was resuspended in sample buffer, recentrifuged and subjected to SDS-PAGE and flurography. Protein markers (GIBCO) were used as molecular mass standards. 99 Immunofluorescence analysis. Cells were grown on coverslips, washed with PBS, and fixed by a mixture of 50% acetone and 50% methanol for 2 min. The fixed cells were then treated with anti-gD antibody, followed by fluorescence-conjugated anti-rabbit antibody. The stained cells were observed under a fluorescence microscope. 100 Fig. 1. Schematic diagram of restriction Sites that were used to construct expression plasmids. NdeI/SpeI fi'agment for pGEX3XgD. BanI/HincII for pC3gD. HincII/KpnI for pC3gI. NspI/DraI for pC3gE. HincII/EcoRI fi'agment for N-terminal of pC3gID, and MluI/HincII for C-terminal of pC3gID. 101 Fugue 1 U86 [gD] U37 [9'] U33 1951 6943 8154 8281 9328 9487 10960 > > > I | Ndel Spel EcoRI 7169 8025 9018 Banl Mlul Hincll Kpnl Nspl Dral 6922 7501 8195 9354 9443 10971 102 RESULTS Production of antibody to GST-gD fusion protein. The predicted gD amino acid sequence was analyzed by GENEPRO program (Riverside Scientific). A highly antigenic fragment from amino acids 77 to 362 was selected (Figure 1), and cloned in-frame with GST protein. A large amount of GST-gD protein was found in the inclusion body of the bacteria, but none was found in the soluble fraction of bacterial proteins. Lower temperature (30°C) growth, lower concentration of IPTG (0.01mM), and Shorter time induction had been tried, but still no soluble GST-gD was found. Consequently, the fusion protein was eluted from the inclusion body. The quality and quantity of the eluted protein were assessed by SDS-PAGE (Figure 2A) (5, 6). Blood was collected from the rabbit injected with GST-gD fusion protein, and tested by Western blotting against proteins prepared from bacteria harboring plasmid pGEX3X and pGEX3XgD. Both the 26 kDa GST protein and the 56 kDa GST-gD protein were detected by this antiserum. Some bacterial proteins of about 56 kDa could also be detected (Figure 2B). The new anti-gD antibody was made to compensate for the short supply of the Previous antibody (1), to confirm the previous results, to prevent bias from one antibody, and hopefully to increase the sensitivity of detection. Glycosylation of gD in vitro. The gD gene that was cloned downstream 0f T7 promoter has been transcribed and translated into an unglycosylated POIYPeptide in vitro. This precursor polypeptide had been previously 103 Fig. 2. Generation of antibody to GSTgD fusion protein. (A) Purified GSTgD fusion protein. M: protein markers. (B) Western blotting analysis of GST and GSTgD proteins with antiserum against GSTgD fusion protein. 104 Figure 2 (A) Purified GSTgD (B) Western Blotting G G S S T G T 9 S 9 kDa M D kDa kDa T D 105 immunoprecipitated by antibody against the TrpE-gD fusion protein (14). However, a fusion protein synthesized in bacteria does not include eucaryotic modification, such as glycosylation. Hence, it was not known how well the antibody would recognize the authentic glycoprotein. To synthesize the glycoprotein in vitro, canine pancreatic microsomal membranes were added to regular coupled transcription and translation reaction together with expression plasmids encoding the gD, gI or gE genes. The 43 kDa precursor of gD was processed to a 53 kDa glycoprotein (Figure 3 lanes 1 and lane 2, respectively). Similarly, the 37 kDa gI precursor was processed to a 50 kDa glycoprotein (Figure 3 lane 3 and lane 4, respectively), and the 44 kDa gE precursor was processed to a 62 kDa glycoprotein (Figure 3 lane 5 and lane 6, respectively). The in vitro glycosylation was not complete, and unglycosylated precursors were found together with glycosylated proteins. Both precursors and glycoproteins of gI and gE produced in vitro match the sizes of those found in MDV GA-infected DEF cells. The glycosylated gD was then subjected to immunoprecipitation analysis with different sera. While three negative controls, preimmune serum, anti-gl antiserum and anti-gE antiserum, did not recognize gD, both antibody against TrpE-gD and antibody against GST-gD reacted with both the glycosylated 56 kDa and unglycosylated 43 kDa of gD (Figure 4). Because antiserum to the GST-gD was more effective than antiserum to the TrpE-gD 106 Fig. 3. Glycosylation of MDV gD, g1 and gE in vitro. Lanes 1,3,5: Precursor polypeptides. Lanes 2,4,6: Glycoproteins after addition of canine pancreatic microsomal membranes. 107 Figure 3 GLYCOSYLATION OF MDV 9D, 91 and 9E in vitro 90 9' 9E n I_l l_l 3 4 kDa 5 6 r 'I II 200 '- 97 — . '. —::/——9E 43 — 5" . —gD . - ——9| 29 — - 18 _ Lanes 1,3,5: Precursor polypeptides Lanes 2,4,6: Glycoproteins after addition of canine pancreatic microsomal membranes. Direct SOS-PAGE Analysis 108 (Figure 4), the former will be used as anti-gD antibody in the following experiments. In summary, anti-gD antibodies against fusion proteins are useful in detection of glycosylated gD protein. Absence of detectable gD expression is a general phenomenon for MDV. MDV-related herpesviruses have been classified into three groups by serological analyses. This classification is consistent with their biological properties, and these groups are virulent MDVS (serotype 1), avirulent MDVS (serotype 2), and HVT (serotype 3). Although distingushable by serologic tests, the three serotypes also share many commom antigens (5). Based on their pathogenic potential, virulent MDVS were further classifed as mild (mMDV), virulent (vMDV): and very virulent (vaDV). Several strains of MDV serotype 1 have been adapted to growth in cell culture, and distributed among research groups all over the world. Although each group only use the strains that are convenient to them in their research, they usually draw conclusions to MDV serotype 1 in general, regardless of the strains. The assumption was that different strains behave generally the same and that occasional differences at the molecular level would be viewed as incidental. Based on the same rationale, we could assume that the absence of gD expression should hold true in cells infected with other MDV serotype 1 strains, and may also be true with other serotypes. To test this hypothesis, DEF cells were infected with several commonly used strains: very virulent Md5 strain, Mdll strain low passage and high passage, JM/102w strain, and 109 Fig. 4. Immunoprecipitation analysis of MDV gD produced by in vitro coupled transcription/translation, and glycosylation. Preserum: Rabbit serum before the rabbit was injected with GSTgD. GSTgD: Antibody against GSTgD fusion protein. TRPEgI: Antibody against TrpEgI fusion protein. TRPEgE: Antibody against TrpEgE fusion protein. TRPgD: Antibody against TrpEgD fusion protein. 110 Flgure 4 LITRPEQD TRPEQE TRPE 9.1 Antibody I GSTQD 200— ' 97— 68— 43— 29— 111 HVT FC I126 strain. Infected cells were labeled with 35S-methione, and cell lysates were subjected to immunoprecipitation with anti-gD, while anti-g1 was used as a positive control. Although gI expression was found in all of the infected cells (7), no gD expression was detected (Figure 5). In summary, the absence of detectable gD expression in MDV-infected cells is not restricted to specific MDV strains or cell passages, but is a general phenomenon of MDV. Transient expression of gD in COS7 cells. Although gD is not expressed in MDV-infected cell culture, it may be expressed in MDV-infected chickens, especially in feather follicle epithelial cells where cell-free viruses are produced. Since no gD was detected in MDV-infected cells, expression of the gD gene in cell culture will be a valuable tool thus far. Characterization of the expressed gD will enrich the knowledge about native MDV gD, and may be beneficial to further research with MDV-infected chickens. The gD, g1 and gE genes were cloned into pCDNA3, an expression vector with the CMV IE promoter. The plasmids were transfected into C087 cells, and protein expression was detected by immunoprecipitation and SDS-PAGE analysis (Figure 6). Expressed gD was about 53 kDa, which is approximate the size expected fi'om the predicted amino acid sequence and predicted glycosylation sites, and similar to the product of the in vitro glycosylation experiment (Figure 3). The gI was 50 kDa, and the gE was 62 kDa (Figure 6). When the expressed gI and gE were compared with the proteins from MDV-infected cells, anti-g1 antibody immunoprecipitated 45 kDa protein 112 Fig. 5. Absence of gD expression is a general phenomenon for MDV. DEF: Control, DEF cells. GA: MDV GA-infected DEF cells. Md5: MDV Md5-infected DEF cells. Md11(L): MDV Md11 (low passage)-infected DEF cells. Md11(H): MDV Md11(high passage)-infected DEF cells. JM: MDV JM/lOZw-infected DEF cells. HVT: HVT FC/126-infected DEF cells. Anti-gD: Immunoprecipitation analysis with anti-gD antibody. Anti-gI: Immunoprecipitation analysis with anti-gI antibody. 113 Figure 5 anti-9D anti-9| I l I R M M M M D M d d H D M d d H E G d II II J V E G d II II J V kDa F A 5 (L) (H) M T F A 5 (L) (H) M T ~ N IV 97— ‘ ' 68— u . 4r deer .8. ,1 29— 114 from both transfected cells and infected cells, and anti-gE antibody immunoprecipitated 62 kDa protein from both transfected cells and infected cells, but it also recognized another 72 kDa protein in infected cells (Figure 7 ). Comparing expression of gD with that of g1 and gE (Figure 6), the gD expression was not as efficient as that of the other two, which suggested that negative regulatory element may exist in the gD gene. To test this assumption, DNA encoding amino acids 1 to 188 out of total 403 amino acids of gD was replaced with DNA sequence encoding amino acids 1 to 253 out of total 355 amino acids of g1, and the hybridized 468 amino acids of gI-gD about 65 kDa was expressed in C087 cells, with gD and gI used as controls (Figure 8). Although natural gD expression was detectable with anti-gD antibody, expression of the gI-gD hybrid detected by anti-gD was Significantly higher (Figure 8). Thus, in MDV-infected cell culture, an element in the N terminal coding region of gD may also contribute to the undectable gD expression Characterization of gD expressed by recombinant fowlpox virus. The gD gene has been inserted into fowlpox virus (FPV) by Nobo Yanagida in Dr- Lucy Lee’s lab. When CEF cells infected by the recombinant FPVgD were subje<=ted to immunofluorescence analysis with our anti-gD antibody, Significant expression was seen. Immunoprecipitation analysis also detected the gD protein (Figure 9). Thus, it was further confirmed that anti-gD 115 Fig. 6. Expression of MDV gD, g1 and gE in C087 cells. CON: Control, C087 cells. gD: C087 cells transfected with the gD expression plasmid described in the materials and methods. gI: C087 cells transfected with the gI-expression plasmid described in the materials and methods. gE: C087 cells transfected with the gE-expression plasmid described in the materials and methods. Immunoprecipitation analysis was carried out with respective antibodies prior to SDS-PAGE. ' 116 Figure 6 EXPRESSION OF MDV gD, gl AND gE in vivo IN COS? CELLS C C C O 9 O 9 0 9 N o N I N E .1 200- ' 9" ! 97— 68- . -9E 90 . Figl 43— 29— 18— 90. gl and gE: The respective genes were cloned into the pRcCMV expression vector Control: No insert in pRcCMV . Immunoprecipitation Prior to SDS- PAGE 117 antibody recognizes the authentic gD, and gD can be expressed in a highly efficient eucaryotic system. 118 Fig. 7. Comparison of g1 and gE from the C087 cell expression system and fiom MDV GA-infected DEF cells. CON: Mock-infected DEF cells. INF: MDV GA-infected DEF cells. EXP: C087 cells transfected with expression plasmids. gI: immunoprecipitation analysis with anti-g1 antibody. gE: immunoprecipitation analysis with anti-gE antibody. 119 Figure 7 COMPARISON OF gl AND gE FROM THE COS? CELL EXPRESSION SYSTEM AND FROM MDV GA-INFECTED DEF CELLS I_—fil_—_I I N F 200 'oxrn 200 kDa 200— .' "'; I. 97— 'DXI'I'I es— ' :. 398 43— .I 29— CON: Mock-infected DEF cells INF: Infected DEF cells EXP: Expressed by the pRcCMV vector 120 DISCUSSION As expected, absence of detectable gD expression is a general phenomenon among different MDV strains. Unexpectedly, a negative regulatory element was found in the N terminal coding region of gD gene. The element could be a transcription Silencer, or an unfavored secondary structure for translation. Kozak has suggested a scanning model for initiation of translation in eukaryotes (9). The basic scanning model postulates that a 408 ribosomal subunit, carrying Met-tRNAmet and a set of initiation factors, enters at the capped 5’ end of the mRNA and migrates linearly until it reaches the first favorable AUG codon, whereupon a 608 subunit joins and first peptide bond is formed. The favorable AUG codons are further subdivided into optimal AUG codons and suboptimal AUG codons. Regarding the A of AUG is at position +1, an optimal AUG codon must have a purine at -3 and a G residue at +4; and a suboptimal AUG codon has either a purine at -3 or a G residue at +4, but not both. An unfavored AUG codon lacks both -3 purine and +4 G residue, and is very unlikely to initiate translation. Usually translation strictly initiates fi'om the first AUG codon if it is an optimal AUG. However, when the first one is a suboptimal AUG, leaky scanning may occur and second translation may initiate at the AUG downstream of the first one. But if the second AUG is far away fi‘om the first one, the second translation will 121 Fig. 8. Expression of the gI-gD hybrid in C087 cells. CON: Control, C087 cells. gD: C087 cells transfected with the gD expression plasmid. gI: C087 cells transfected with the gI expression plasmid. gID: C087 cells transfected with plasmid expressing the gI-gD hybrid. 122 9D' ". CON' 9 _ ma .9 w a Figure 8 123 not be initiated (10). In addition, unfavored AUG codons upstream of the first favored AUG can impair the translation initiation (9). The MDV gD transcript has a suboptimal AUG codon, UUCQGUEQA, which has a purine at -3 but no G residue at +4. A second suboptimal AUG codon, ACAQAUALGA, is located at +17, and a third suboptimal AUG codon, ACGAGAMA, is located at +55. Because neither of the first two AUG codons is optimal, and the three codons are very close to one another, leaky scanning can skip the first, even the second AUG and go down to the third AUG. Probably the three AUG codons will interfere with one another, and lead to an inefficient translation overall. In the MDV gI transcript, a second AUG, CGAUGUA__U_G_rU, is located at +5, but it is an unfavorable initiation site. No downstream AUG is found near the gE AUG codon. Hence, although MDV g1 and gE AUG codons also have suboptimal context, AUUGCGALLQU for g1 and GUCAUAA_UGU for gE, no leaky scanning can take place. Therefore, replacement of gD N-terminal coding region with gI N-terminal codons may enhance translation initiation by preventing leaking scanning. Synthesis of two proteins from one ORF as a result of leaky scanning has been found primarily in viruses (9). In 8V40 virus, human hepatitis B virus, and foot-and-mouth disease virus, translation initiates from two in-frame AUGS and generates long and short protein isoforms. In adenovirus, HIV and sendai virus, initiation from AUGS in different, overlapping reading frames produces two unrelated proteins. In MDV gD gene transcript, the 124 Fig. 9. Expression of MDV gD by recombinant fowlpox virus. CON: CEF cells infected with wild type fowlpox virus. gD: CEF cells infected with gD recombinant fowlpox virus. Immunoprecipitation with anti-gD antibody. 125 Figure 9 126 first AUG and the third AUG are in the same reading frame, but the second is in another frame, and translation from the second AUG will hit a stop codon shortly after the initiation. Thus, slightly different Sizes of gD proteins may be translated from the first and third AUG. The second AUG reduces the distance between the other two AUGS, and increases chances of the leaky scanning. The three close suboptimal AUG codons in gD transcript may be the negative regulatory element for inefficient gD expression. On the other hand, internal transcription silencer may exist. Previous work showed that there is not an eficient gD transcription in MDV-infected cells, although three TATA- like sequences are found upstream of gD ATG codon. This inefficiency could be explained by the proximity of a AATAAA motif. However, it is also possible that the gD gene itself includes an internal transcription Silencer. Detailed site-directed mutagenesis and transcriptional analysis are required for further conclusion. Many MDV strains have been isolated, and used interchangeably by researchers, although few direct comparison between strains has been done at the molecular level. This study directly compared the gD and g1 expression pattern among several commonly used strains, and confirmed they are basically identical. Different passages of Mdll strain were analyzed, although gI was detected in both high and low passages, expreSsion 127 in high passage was higher than expression in low passage, which is contrary to MDV gC expression (15). MDV gD was the primary focus of this research, and gI and gE were used as controls, because HSV1 gD plays a crucial role in virus infection, and purified gD proteins can protect animals from virus infection. However, increasing evidence shows that MDV gD is not as important as HSV1 gD in virus infection and host immune response(13), and finding the possible subunit vaccine of MDV may lie on study of the other gene products such as g1 and gE. Expression and characterization of MDV g1 and gE protein in this study will be the framework for high level expression of g1 and gE in either baculovirus or fowlpox virus systems, and for estimation of their potential as vaccines. 128 REFERENCES 1. Brunovskis, P., X. Chen, and L. F. Velicer. 1992. Analysis of Marek’s disease virus glycoproteins D, I and E, p. 118-122. In Proceedings 19th world’s poultry congress, vol.1. 4th Int. Symp. Marek’s Dis. Part. Posen and Looijen, Wageningen, The Netherlands. 2. Brunovskis, P., and L. F. Velicer. 1992. Genetic organization of the Marek’s disease virus unique short region and identification of Us encoded polypeptides, p. 74-78. In Proceedings 19th world’s poultry congress, vol.1. 4th Int. Symp. Marek’s dis. Part, Posen and Looijen, Wageningen, The Netherlands. 3. Brunovskis, P., and L. F. Velicer. 1995. The Marek’s disease virus unique short region: alphaherpesvirus-homologous,fowlpox virus-homologous, and MDV-specific genes. Virology. 206: 324-338. 4. Burke, R. L. 1993. HSV vaccine development, p. 367-380. In B. Roizman, and C. Lopez (ed.). The herpesviruses. Raven Press, New York. 5. Calnek, B. W., and R. L. Witter. 1991. Marek’s disease, p. 342-385. In B.W.Calnek (ed.) Disease of poultry. Iowa State University Press, Ames, Iowa. 6. Chen, X., and L. F. Velicer. 1992. Expression of the Marek’s disease virus homolog of herpes simplex virus glycoprotein B in E.coli and its identification as B antigen. J .Virol. 66:4390-4398. 129 7. Chen, X., X. Tan, P. Brunovskis, and L. F. Velicer. Identification and characterization of the Marek’s disease virus homologs of the herpes Simplex virus glycoproteins I and E. (Paper submitted). 8. Hurk, D. L., M. D. Parker, D. R. Fitzpatrick, T. J. Zamb, J. V. Hurk, M. Campos, R. Harland, and L. A. Babiuk. 1991. Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties. J .Virol. 65:263-271. 9. Kozak, M. 1991. An analysis of vertebrate mRNA sequences: intimations of translational control. J .Cell Biol. 115:887-903. 10. Kozak, M. 1995. Adherence to the first-AUG rule when a second AUG codon follows closely upon the first. Proc.Natl.Acad.Sci. 92:2662-2666. 11. Love, D. N., C. W. Bell, D. Pye, S. Edwards, M. Hayden, G. L. Lawrence, D. Boyle, T. Pye, and J. M. Whalley. 1993. Expression of equine herpesvirus 1 glycoprotein D by using a recombinant baculovirus. 12. Marchioli, C. C., R. J. Yancey, Jr., E. A. Petrovskis, J. G. Timmins, and L. E. Post. 1987. Evaluation of pseudorabies virus glycoprotein gp50 as a vaccine for Aujeszky’s disease in mice and swine: expression by vaccinia virus and Chinese hamster ovary cells. J .Virol. 61:3977-3982. 13. Parcells, M. 8., A. S. Anderson, and R. W. Morgan. 1994. Characterization of a Marek’s disease virus mutant containing a LacZ insertion in the U86 (gD) homolog gene. Virus Genes. 9(1): 5-13. 130 14. Tan, X., P. Brunovskis, and L. F. Velicer. Transcription analysis of Marek’s disease virus glycoproteins D, I and E genes: gD expression is undetectable in cell culture. (Manuscript in preparation). 15. Wilson, M. R., R. A. Southwich, J. T. Pulaski, V. L. Tieber, Y. Hong, and P. M. Coussens. 1994. Molecular analysis of the glycoprotein C- negative phenotype of attenuated Marek’s disease virus. Virology. 199:393- 402. Chapter IV Summary and Conclusions The focus of this research is MDV gD, with gI and gE used as positive controls. It is well known that the HSV-1 gD gene is essential for virus infection, and that purified gD proteins stimulate a strong immune response in vaccinated animals which protect them from virus infection. Hence, when MDV was grouped as an alphaherpesvirus, people were willing to search for and work on its gD homolog. Since the MDV gD gene was sequenced by Norman Ross’ group and by Peter Brunovskis in our lab, however, the progress in an understanding of MDV gD expression has been more surprising than exciting. With the sequence of entire Us region in hand, Peter made antibodies against TrpE fusion proteins of each of the seven HSV-1 Us homologs. All of them were detected in MDV GA-infected DEF cells with these antisera, except the gD protein. Then, Morgan’s group made a gD mutant by inserting the LacZ gene into MDV gD gene, and found that the mutant had similar growth kinetics to the wild type virus. Thus, the role of MDV gD became a mystery, which stimulated my curiosity. I repeated Peter’s experiment with anti-g1, anti-gE, and anti-gB antibodies as positive controls. In MDV GA-infected DEF cells, although gI, gE and g3 were detected, no gD was found, even after long exposure of the gel. Meanwhile, there was another puzzling result. When I was a rotation student in Dr. Salter’s group, we tried to express gD in CEF by RCAS, a 131 132 retrovirus-based expression vector. Although protein encoded by RCAS itself was detectable, and Southern and Northern blotting of the gD‘gene were positive, no gD expression was detected by antibody to TrpE-gD. Taken together, a straightforward explanation was that either the proposed gD gene was not a functional protein-coding open reading frame, or that the antibody to TrpE-gD could not recognize the gD protein. These doubts were quickly dispelled when the gD precursor polypeptide was produced from this ORF in vitro, and was immunoprecipitated by antibody to TrpE-gD. Furthermore, the anti-gD antibody also reacted with the glycosylated form of gD produced by in vitro coupled transcription and translation. The absence of detectable gD expression in cell culture must have a more complicated and fundamental explanation, and my bet was on regulation at the level of the transcription. Preliminary Northern blot analysis with a DNA probe did not find an abundant transcript containing gD. Although a 7.5 kb band was detected with a gD probe after longer exposure of the blot, it was not clear whether the 7 .5 kb band was areal transcript or just viral DNA that had copurifed. I then pursued RT-PCR analysis with the expectation to disprove existence of the 7 .5 kb mRNA, and to conclude the gD story by saying there is no gD transcript at all. Unfortunately, RT-PCR instead confirmed that the 7 .5 kb transcript was a polycistronic mRNA that included the gD gene. At that time, more sensitive and demanding Northern blot analysis was needed to characterize the low abundance, large 7.5 kb 133 transcript. Riboprobes were substituted for the DNA probes, and five-fold more mRNAs were used in each reaction. The effort was rewarded by finding that the 7 .5 kb transcript starts two genes upstream of the gD gene, and was unlikely to be translated into gD protein. The preceding research was on the GA strain of MDV alone, so one reasonable assumption was that the GA strain may carry a unique gD mutation. After a more efficient anti-gD antibody was made against GST-gD, searching of gD expression was extended to several commonly used strains, Md5, Mdl 1, JM/102w, and HVT FC/126, and the same conclusion was reached with these strains. I didn’t have access to the RBlB strain, because it is owned by Cornell University. However, from the available sequence data, the regions between SORF4 and gD gene are 100% identical between the RBlB and GA strains, thus absence of a detectable level of gD expression is expected of the RB 1B strain also. The absence of a detectable level of gD expression may be restricted to the cell culture, because Ross’ group claimed that they found gD expression in MDV-infected chickens. If gD has a role in virus-infected chickens, expression of gD in cell culture would be a useful system to characterize the protein and help further work in chickens. A transient system was chosen because construction of the expression plasmid is relatively simple. In this system, although gD expression was detectable, it was not as efficient as expression of g1 and gE. Since I assumed the anti-gD was not as efiicient as anti-gI and anti-gE, I was reluctant to perform the experiment suggested by 134 Dr. Coussens in which the N-terminal coding region of gD was replaced with the N-terminal codons of gI. When I finally did that experiment and examined the level of expression with anti-gD antibody, however, the expression of the gI-gD hybrid was higher than expression of gD protein. This surprising finding suggested that negative regulatory element exists in the N-terminal coding region of gD, and may also contribute to the absence of detectable gD expression in cell culture. The reason that MDV gD was not expressed by RCAS vector in CEF cells iS still unknown, but another failed experiment may be related to it. I tried to construct a stable OU2 cell line expressing gD by G418 selection, although I got resistant cells after transfection with pC3gD, no gD expression was found. It has been reported that BHV-l gD-expression cells lost the gD expression after certain passages in cell culture, which was due to the gD toxicity to the cells. Therefore, the failure to construct cells by RCAS or drug selection to stably express MDV gD may due to the vulnerability of chicken embryo fibroblast cells to MDV gD toxicity, and any surviving cells may carry mutations to downregulate gD expression. More and more evidence shows that MDV gD is not as important as its HSV-1 homolog in virus infection. One example is that a chicken injected with fowlpox gD was not protected from virus infection (Dr. Lucy Lee, personal communication). Therefore, MDV gI and gE, the controls of this study, hear more hopes for a subunit vaccine. The preliminary study of these 135 glycoproteins in this work has paved the way to highly expressing g1 and gE in other conventional systems. "‘IIIIIIIIIIIIIIIIIIIII“