3: Lu .. . .5 .A $3355 .. . 3.3.2., ...fz.fl..: c x . a 4.1.; n. . 5r ‘ . Vt» ...... 15:. 1L. , a? - ' 7F: 1 1:51. 1..., r i, «1.... S .35 2;. 2 km.» ‘22: ‘ , a 5.);OHVV.‘ , . in. x . L..r V a: 15:...“ .. £533.... 31.3.1. 1 it . :51). . 3 . 5.... If! .» . “tuna...- . y... .7 . t. attic f ‘.....-...Sz: . 2.5,. Q :x:..£1i .haw :E. J L.£ .. , .5... . ,. ‘ #25:? .E .15.. flux. . .... 4:1! 21.. .hhrtvfl 1A2. \ 3313, - w I 5 3?: i... .53 ‘ 511,... ‘1..ng Wang. - III IIIIIIIIIIIIIII LIBRARY Michigan State University IIIIIIIIIIIIIIIIIIIIII 3 1293 01044 This is to certify that the dissertation entitled IDENTIFICATION OF AN IMMEDIATE-EARLY GENE IN MAREK'S DISEASE VIRUS LONG INTERNAL REPEAT REGION AND CHARACTERIZATION OF ITS GENE PRODUCT. PP14 presented by Yu Hong has been accepted towards fulfillment of the requirements for PhD Animal Science degree in Paul M. Coussens Major professor Date q ‘ \“t‘qfi MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 meoMMouMVuxnootd. PLACE ll RETURN BOX dd. duo. To AVOID FINES «turn on or DATE DUE DATE DUE DATE DUE m ’7 usuuAnmuiv-Awafimdoppomml W1 7 _._—.—r~'-_ 7?. IDENTIFICATION OF AN IMMEDIATE-EARLY GENE IN MAREK’S DISEASE VIRUS LONG INTERNAL REPEAT REGION AND CHARACTERIZATION OF ITS GENE PRODUCT , PP14 By Yu Hong A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1994 ABSTRACT IDENTIFICATION OF AN IMMEDIATE-EARLY GENE IN MAREK’S DISEASE VIRUS LONG INTERNAL REPEAT REGION AND CHARACTERIZATION OF ITS GENE PRODUCT PP14 By Yu Hong Marek’s Disease Virus (MDV) is an oncogenic avian herpesvirus whose genomic structure is similar to herpes simplex virus and varicella-zoster virus. Repeat regions of the MDV genome have been intensively investigated due to a potential relationship to MDV oncogenicity and abundant expression of immediate-early transcripts. However, controversy regarding size, number, and direction of transcripts derived from these regions has been present. No gene product encoded by the transcripts was reported. By Northern hybridization analysis, a 1.6 kb immediate-early transcript was localized to the BamHI-I2 region. The viral genomic DNA of that region was subsequently sequenced without finding a continuous (>100 amino acids) open reading frame (ORF) in either direction. Through cDNA cloning and sequencing, two cDNAs of 1.4 kb (C1) and 1.35 kb (C2) originating from the BamHI I2 region were identified. Both cDNAs are derived from spliced mRNAs spanning the BamHI-H and I2 fragments. C1 and C2 use the same splice acceptors and 3’ ends, but differ at their 5’ ends and utilize different splice donors. With a combination of primer extension and sequence analysis, the upstream promoter-enhancer region of C1 cDNA has been defined as a bidirectional regulatory region shared by the MDV pp38 gene. Sequencing analysis shows two small open reading frames (ORF) within each cDNA (ORF 1a and ORF2 in C1, ORFlb and ORF2 in C2). Potential ORFS Yu Hong of the sequence have no significant homology with any known protein in the Swiss- Protein data base. To detect any protein product encoded by these two cDNAs, DNA fragments encoding ORFla and ORFlb were cloned into pGEX-BX vectors to produce CST-fusion proteins and induce antisera. In Western blot analysis of MDV infected cell lysates, a 14 kDa polypeptide (p14) was identified by antisera against both ORFla and ORFlb. This 14 kDa protein is expressed in cells which are lytically infected with MDV, strains GA passage 8 (oncogenic), Mdll passage 14 (oncogenic) and passage 83 (attenuated), as well as in the MDV latently infected and transformed MSB-l cell line. Furthermore, we demonstrate that p14 is MDV serotype 1 specific and highly phosphorylated (designated as pp14). Further analysis reveals that pp14 is predominantly found in the cytoplasmic fraction of MDV infected cell lysates and can be detected in cytoplasm of MDV infected cells by immunofluorescence. In memory of my mother. iv ACKNOWLEDGEMENTS I wish to express my sincere thanks to my advisor, Paul M. Coussens, for his continue guidance, encouragement, and financial support. 1 also wish to thank members of my guidance committee, Drs. S. Trienzenberg, L. Velicer, J. Ireland, and R. Schwartz for their valuable time and helpful suggestions. I gratefully acknowledge all the people I have worked with in Dr. Coussens’s lab, especially Mindy Wilson and Heidi Camp, for their friendship and helpful conversations. I especially thank R. Southwick for his expertise with computers and assistance with preparation of graphics. Finally, I wish to express my deepest appreciation to my husband Ben, my daughter Kari, my brothers and sisters for their infinitive love, support and encouragement. TABLE OF CONTENTS Chapter I. Literature review ............................................. 1 1. Introduction ........................................... 2 2. Biology of Marek’s disease virus (MDV) ....................... 3 A). MDV virions ..................................... 3 B). MDV serotypes ................................... 3 C). MDV isolation and cultivation ....................... 4 3. Pathology of MDV ...................................... 5 4. Molecular biology of MDV ................................. 8 A). MDV genome structure ............................. 8 B). MDV physical map and gene arrangement ............... 9 C). Temporal gene expression of herpesviruses ............... 13 i). HSV (alpha-herpesvirus) IE gene expression ......... 14 ICPO. ................................. 17 ICP4 .................................. 20 ICP27 .................................. 23 ICP22/47. .............................. 25 (ii) HCMV (beta-herpesvirus) IE gene. ............... 26 (iii) EBV (gamma-herpesvirus) IE genes. .............. 28 vi (iv) MDV IE genes ............................. 31 (V). Early and late genes. ........................ 34 5. MDV latency ......................................... 36 6. MDV tumorigenicity .................................... 41 Charter II. Identification of an Immediate-early Gene In The Marek’s Disease Virus Long Internal Repeat Region Which Encodes a Unique 14 kDa polypeptide ...... 47 Abstract ............................................... 48 Introduction ............................................ 49 Materials and methods ..................................... 52 Results ................................................ 58 Discussion ............................................. 67 Chapter III. A 14 kilodalton Immediate-early Phosphoprotein is Specifically Expressed in Cells Infected With Oncogenic Marek’s Disease Virus Strains and Their Attenuated Derivatives .................................................. 86 Abstract ............................................... 87 Introduction ............................................ 88 Materials and methods ..................................... 91 Results .......... 96 Discussion ............................................ 100 vii Chapter IV. Discussion ................................................. 109 1. Summary of results and conclusions ........................ 110 2. Future research directions ............................... 116 Appendix .................................................. 120 List of Reference ............................................ 131 viii LIST OF FIGURES Chapter I. Figure 1. Location of genes on MDV genome. ........................ 12 Figure 2. Comparison of the locations of IE genes on the HSV and MDV genomes. 16 Chapter H. Figure 1. Northern blot hybridization to identify immediate-early gene transcripts in MDV BarnHI-IZ. ............................................. 75 Figure 2. Schematic representation of the location, structure and primer extension analysis of C1 and C2 cDNAs. .......................................... 77 Figure 3. The complete nucleotide sequence of two cDNA clones. ........... 79 Figure 4. Northern blot hybridization to confirm the origin of cDNA sequence. . . 81 Figure 5. Schematic representation of the positions of ORFS in C1 and C2 cDNA clones Figure 6. Western blot analysis detection of MDV specific-proteins encoded by ORF 1a and ORF lb. ................................................. 85 Chpater 111. Figure l. Antigenic relatedness of ORF 1a and ORFlb. .................. 104 ix Figure 2. Characterization of p14. ................................. 106 Figure 3. Localization of pp14 by indirect immunofluorescence. ........... 108 APPENDIX Figure 1. Location of BamHI-Xbal subfragment in MDV genome and computer analysis of ORFS of the viral genomic DNA sequence ........................ 123 Figure 2. Nucleotide sequence of the BamHI-Xbal region in MDV BamI-II-I2 fragment. .................................................. 125 Figure 3. Fusion protein expression vector and fusion protein clone identification. .127 Figure 4. Western blot showing antibody specificity to ORF 1a protein. ....... 128 Figure 5. Western blot analysis of post-translation modification of pp14. ...... 129 Figure 6. Double-stained immnofluoresence on MDV infected CEF cells. ..... 130 BHV BSA CAT CEF cDNA CHX CIP CMV CsCl Da DEF DR EBV EBNA-l ELISA FITC gB LIST OF ABBREVIATIONS base pairs bovine herpes virus bovine serum albumin chloramphenicol acetyltransferase chicken embryo fibroblasts complementary DNA cycloheximide calf intestinal phosphatase cytomegalovirus cesium chloride dalton duck embryo fibroblasts direct repeat early Epstein-Barr virus EBV nuclear antigen enzyme-linked immunosorbent assay fluorescein-5’—isothiocyanate glycoprotein B glycoprotein C xi GST HCMV HIV HP HSV HVT ICPO ICP4 ICP22 ICP27 ICP47 IE IgG IRL IRs kbp kDa IUdR LAT LP LTR glutathione-S-transferase human cytomegalovirus human immunodeficiency virus high passage herpes simplex virus herpesvirus of turkeys (HSV) infected cell protein No. 0 (HSV) infected cell protein No. 4 (HSV) infected cell protein No. 22 (HSV) infected cell protein No. 27 (HSV) infected cell protein No. 47 immediate early immunoglobulin G internal repeat long internal repeat short kilobase pairs kilodalton 5’-iodo-2’-deoxyuridine late latency -associated transcript low passage long terminal repeat xii MD M DV meq MIEP Oct- 1 ORF PAA PAGE PE SDS TK TM TRL TRS IS Marek’s disease Marek’s disease virus Marek’s EcoRI Q fragment encoded gene major IE enhancer-containing promoter-regulatory region octamer binding factor-1 open reading frame phosphonoacetic acid polyacrylamide gel electrophoresis phycoerythrin sodium dodecyl sulfate thymidine kinase tunicamycin terminal repeat long terminal repeat short temperature-sensitive unique long unique short (HSV)virion protein No. 16 very virulent marek’s disease virus varicella—zoster virus xiii Chapter I Literature review > 1. Introduction Marek’s disease virus (MDV) is an oncogenic avian herpesvirus which causes a highly contagious lymphoproliferative disease in chickens, named Marek’s disease (MD) (Churchill and Biggs,1967; Nazerian and Burmaster,1968). MD was first described by Joseph Marek, a Hungarian veterinarian in 1907 (Payne, 1985), but discovery of MDV as the etiologic agent of Marek’s disease (MD) did not occur until the late 1960’s (Churchill and Biggs,1967; Nazerian and Burmaster,1968). Accordingly, MDV became one of the first herpesviruses directly associated with a neoplastic disease. Prior to discovery of the etiologic agent and development of effective vaccines, MD constituted a serious economic threat to the worldwide poultry industry. MD has been effectively controlled by vaccination with attenuated MDV or an antigenically related but apathogenic herpesvirus of turkey (HVT) (Churchill et al., 1969; Okazaki,et al.,1970; Witter,1985). However, frequent vaccine breaks, attributed in part to the emergence of more virulent MDV strains, prompted significant interest in developing better methods for prevention and control of MDV. In addition to economic importance, MDV offers a superb model for herpesvirus oncology. MDV reproducibly induces neoplasms in its natural host and virus isolates representing a wide spectrum of oncogenic potential are available. The development of HVT or attenuated MDV as effective vaccines, which prevent the symptoms of MD, provides the first example of a naturally occurring malignant lymphomatous disease to be effectively controlled by vaccination. However, the molecular mechanism of MDV oncogenicity and immunoprotection by vaccines are still unclear. In recent years, a tremendous amount of information on MDV molecular biology has been published, 3 greatly enhancing our knowledge of MDV pathogenesis and tumorigenesis. 2. Biology of Marek’s disease virus (MDV) A). MDV virions MDV has a typical herpesvirus structure, which consists of 162 capsomeres arranged with icosahedral symmetry (Nazerian and Bunnaster, 1968; Schat, 1985). The nucleocapsid contains a core of double-stranded viral DNA and a protein shell (capsid) assembled in the nuclei of infected cells. The nucleocapsid is surrounded by an amorphous tegument and can be enveloped or not. Enveloped virions, 150-160 nm in diameter, are principally associated with nuclear membrane where the envelope is derived and buddcd to form nuclear vesicles in MDV infected cells. Naked virions are 85-100 nm in diameter and usually found in the nuclei of infected cells (Nazerian and Burmester, 1968; Nazerian, 1971; Hamdy et al., 1974). Naked and enveloped virions can sometimes be seen in the cytoplasm and, rarely, in the extracellular space. The only place where large numbers of cytoplasmic enveloped virus particles, 270-400 nm in diameter, can be detected is in the feather follicle epithelium (FFE) of infected chickens (Calnek et a1, 1970). MDV derived lymphoblastoid cell lines and tumor cells occasionally contain naked virus particles which are essentially similar to virions observed in infected cell cultures. B). MDV serotypes Marek’s disease virus is classified as three serotypes, based on agar gel precipitation (AGP) and indirect fluorescent antibody (IFA) assays (von Bulow and Biggs 1975; Schat and Calnek, 1978). Serotype classification has been further confirmed by 4 restriction enzyme pattern analysis of viral genomes (Ross et al., 1983). Oncogenic strains and their attenuated derivatives form serotype 1, while naturally occurring, nononcogenic strains of MDV are classified as serotype 2. Nononcogenic herpesvirus of turkey (HVT) is classified as serotype 3. Serotype 1 is further subdivided as very virulent (vaDV), virulent, mild or attenuated based on variable pathogenicity or oncogenicity in chickens. Very virulent strains, such as MD/5 and RB-lB, can cause high incidence of MD lymphomas in all chickens, except vaccinated birds from genetically resistant lines. Virulent strains including GA and JM can cause high incidence of MD in genetically susceptible but not in resistant birds. Mild strains can cause tumors in only a minority of very susceptible chickens (Schat, 1985). Repeat passage of very virulent or virulent serotype-1 MDV in cell culture leads to attenuation resulting in loss of oncogenicity (Churchill et al., 1969; Nazerian 1970; Schat, 1985). Attenuated serotype 1 strains, together with serotype 2 and 3, have been employed to produce monovalent, bivalent or trivalent vaccines against MD-induced tumors (Witter,1985). C). MDV isolation and cultivation MDV is a highly cell associated virus with the exception of feather follicle epithelium where cell-free infectious virions may be readily isolated (Calnek, et al., 1969; Schat, 1985). Cell-associated MDV can be isolated from viable lymphocytes and, in the case of serotype 1 virus, from lymphoma cells. After initial isolation, MDV can be propagated in primary fibroblast cells obtained from various avian embryos, such as chicken and duck embryos (Purchase et al., 1971). In cell culture, discrete foci can be observed 2 to 7 days post-infection with the characteristics of refractile rounded cells and 5 syncytia formation. Due to strict cell-association in vitro, little or no infectious cell free MDV can be recovered from medium (Calnek et al.,l970; Churchill and Biggs, 1967; Schat, 1985). 3. Pathology of MDV MDV is a highly contagious agent which spreads horizontally by direct or indirect contact with infected chickens and via an airborne route (Payne, 1985; Sevoian et al., 1963). Once MDV enters a chicken, there are three types of virus-cell interactions: 1) productive infection, 2) non-productive latent infection, and 3) non-productive neoplastic infection or transforming (Payne, 1985; Schat, 1985; Calnek and Witter, 1991). Productive infection is cytolytic, characterized by replication of viral DNA and synthesis of numerous viral antigens. Productive infection can be further divided into fully productive and productive—restrictive, or semi—productive infection. Fully productive infection with MDV only occurs in feather follicle epithelium (FFE) and characterized by production of large numbers of enveloped, fully infectious virions, accompanied by cell death. Semi-productive infection occurs in B lymphocytes, some epithelial cells and in most cultured cells. This type of infection, in which infectivity remains cell-associated, results in production of abundant naked nuclear virions, production of viral antigens and leads to cytolysis (Calnek and Witter, 1991). As a consequence of the production of viral antigens, T lymphocytes are activated and non-productive latent infection (latency) is established coincidently with development of host immune responses (Payne,1985; Calnek, 1985). Latent infection is detected primarily in lymphocytes and is characterized by persistence of the viral genome in cells without expression of most viral antigens or virions. Latent infection can persist for the 6 lifetime of the animal and virus can be rescued by co-cultivation with permissive cells or inoculation into chickens (Payne, 1985). Non-productive neoplastic infection (transformation) describes neoplastic lymphoma cells, transplantable lymphomas and lymphoma-derived cell lines (Payne, 1985). In this type of infection, viral genomes persist in cells with limited antigen expression, including Marek’s associated tumor surface antigen (MATSA: activated T- cell marker) (McColl et al, 1987). Transformed cells, in general, are activated T-helper cells which are CD4+ CD8' (Schat et a1, 1991). MDV transformed cell lines are used to investigate mechanisms of latent infection and oncogenesis of MDV, a more extensive literature review on the properties of these cell lines will be presented in the M_D_lf My section. Based on experiments with oncogenic MDV infection of antibody-free, genetically susceptible chickens, MDV infection can be generally divided into four stages: 1) early cytolytic infection, 2) latent infection, 3) permanent immunosuppression and 4) transformation. These stages are sequential but often overlapped (Calnek, 1985). Primary infection by MDV usually occurs via the respiratory tract, where MDV is picked up by phagocytes and then disseminated from lung to lymphoid cells (Payne, 1985; Calnek and Witter, 1991). Cytolytic infection of MDV initially affect lymphoid tissues, primarily B lymphocytes and a few T lymphocytes. Tissue changes which accompany initial infection are infiltration of macrophages and granulocytes and reticular cell hyperplasia, followed by degeneration of spleen, bursa, and thymus. From 5 to 7 days post infection, the cytolytic infection of primary B-lymphocytes changes to latent infection of predominantly T-lymphocytes coincidence with temporary recovery from 7 immunosuppression. Latently infected T-lymphocytes are dispersed to various organs and tissues via the circulatory system (Payne, 1985). Another common place involved in MDV infection is peripheral nerve system, characterized by two main pathological processes: 1) neoplastic lymphoproliferation and 2) segmental, cell-mediated demyelination. At 2 to 3 weeks post infection, transformation of T-lymphocytes occurs, accompanied by permanent immunosuppression. Massive lymphomas can be observed in visceral organs (including kidney, gonad organ, heart, lung, liver etc.), skin, muscle, and neural tissues are also involved (Payne, 1985). Association of atherosclerosis with MDV infection in the coronary, aortic, celiac, gastric and mesenteric arteries can be found as a late change with mildly pathogenic MDV (Payne, 1985). Clinical signs of MD usually appear at 3-4 weeks post-infection. Two pathological forms of Marek’s disease, classical and acute, have been defined. Classical MD predominantly affects peripheral nerves and is associated with asymmetricly progressive paralysis of one or more extremities. A very common characteristic attitude in MD is recumbency of chickens with one leg stretched forward and the other backward. Uneven gait, lameness, torticollis, droopy wings, or closed eyelids are also characteristics depending upon the locations of involved peripheral nerves (Purchase, 1985; Calnek and Witter,1991). While only a minority of cases in classical MD develop lymphomas, acute MD is more virulent and characterized by multiple lymphomatous of various visceral organs and tissues (Payne, 1985). The final consequence of MDV pathogenesis depends upon virus strain and dose, host genotype, age, sex, and immune status (Payne, 1988). 4. Molecular biology of MDV A). MDV genome structure. The genome of MDV is a linear, double-stranded DNA and contains nicks or gaps, a property shared with other herpesviruses. The molecular weight of MDV DNA is 110- 120 x 106 daltons, as calculated by sedimentation analysis and contour length measurements of electron microscopy. Total size of MDV DNA is approximately 180 kbp for serotype 1 and 167 kbp for HVT (Lee et al., 1971; Hirai et al., 1979; 1988; Cebrian et al., 1982; Fukuchi et al., 1984; Wilson and Coussens, 1991). The density of MDV DNA in CsCl gradients was determined to be 1.705 g/cm, indicating a 46-47% composition of guanine and cytosine (Lee et al., 1971; Hirai et al., 1979; 1985). This density is similar to that of chicken cell DNA and complicates the purification of viral DNA by gradient centrifugation. MDV was initially classified as a gamma-herpesvirus based on its biological properties, especially its lymphotrOpism which is similar to Epstein-Barr Virus (EBV) (Roizman et al., 1981). MDV genome structure, however, was found to be remarkably similar to that of human alpha-herpesvirus (e. g., herpes simplex virus and varicella-zoster virus) with a characteristic of Herpesviridae group B genome, as determined by electron microscopy and restriction enzyme mapping (Roizman and Sears, 1991; Cebrian et al., 1982; Fukuchi et al., 1984). The basic structure of MDV DNA consists of unique long and unique short regions (UL, Us) flanked by internal inverted repeats (IRL, IRS) and terminal repeats (TRL, TRS) (Cebrian et al., 1982; Fukuchi et al., 1984). In addition to extensive inverted repeats, several sets of direct repeats (DRl to DRS) have been found in MDV DNA. These DR sequences consisting of more than 100 bp are mostly located 9 in internal or terminal repeat regions (Hirai, 1988). Among them, a 132-bp direct repeat (DRl) is amplified from 1-3 copies to over 30 copies coincident with the attenuation of MDV oncogenicity (Bradley et al., 1989, b; Chen and Velicer, 1991). This region will be further discussed in MDV tumorigenicity section. By using Southern blot hybridization and DNA nucleotide sequencing, Kishi et al. (1991) have reported a-like sequence in serotype 1 MDV inverted repeat region with a similar structure to the a sequence of HSV—1. HSV-1 a sequences are located at the junction between the L and S components and at the termini of the HSV-1 genome. These a sequences are related to intramolecular recombination and cleavage/packaging of viral concatamers during rolling-cycle replication (Jacob et al., 1979; Roizman, 1979; Vlazny et al., 1982 ). Although the structure of the putative a-like region of serotype-1 MDV is similar to the HSV a sequence, there is no nucleotide sequence similarity to the HSV a sequence. Similarly, an a-like sequence has been identified in MDV serotypes 2 and 3 (Reilly and Silva, 1993; Camp et al., 1993). These data further support the similarity of MDV genome with that of alpha-herpesviruses. B). MDV physical map and gene arrangement Physical maps of the three MDV serotypes have been constructed using different restriction endonucleases (RE) (Fukuchi et al., 1985; Igarashi et al., 1987; One et al., 1992). Unexpectedly, viruses of serotype 1, 2 and 3 each have a unique restriction enzyme pattern, in spite of their antigenic similaity (Ross, et al., 1983; Hirai et al., 1979). Conflicting results on DNA sequence similarity between the three serotypes varied from 5% to 80% (Ross et al., 1983; Hirai et al., 1986; 1984; Gibbs et al., 1984). The 10 development of RE maps and availability of genomic clones of MDV DNA established a foundation for molecular studies on MDV. Although nucleotide sequence analysis of MDV has only recently begun, mapping data indicates that genes encoded in the unique long and unique short regions are collinear with varicella zoster virus and herpes simplex virus genes (Buckmaster et al., 1988; Ross et al., 1991; Brunovskis and Velicer, 1992). Two MDV glycoproteins, A and B antigen, have been characterized as HSV gC and gB homologs, respectively (Coussens and Velicer, 1988; Isfort et al.,1987; Ross et al., 1989; Chen and Velicer, 1992). Based on random sequencing analysis, thirty-five MDV genes were identified by comparison to varicella-zoster virus (VZV) (Buckmaster et al., 1988). In addition, extensive colinearity of alphaherpesvirus homologous genes in the MDV Us region has been recognized (Ross et al., 1991; Brunovskis and Velicer, 1992). Recently, homologs of HSV ICP4, ICP27, VP16 and gK have been identified and sequenced from serotype 1 MDV (Anderson et al., 1992; Yanagida et al., 1993, Ren et al., 1994). Together, these results have provided a basis for MDV reclassification as an oncogenic alphaherpesvirus (Roizman, et al., 1992). In contrast to the unique region genes, genes in repeat regions are more specific to individual viruses. A 38 kDa phosphoprotein (pp38) and a for/jun oncogene homolog (meq), both expressed in MDV transformed cell lines, were localized to the IRL of the MDV genome within BarnHI-H and BamHI-I2 fragments, respectively (Chen et al., 1992; Cui et al.,1991; Jones et al., 1992). Recently, a 14 kDa MDV specific protein encoded by a cDNA spanning BanHI—H and I2 regions has been identified (Hong and Coussens, 1994) and is expressed in transformed cell lines. The relationship between genes encoded in repeat regions and MDV tumorigenicity has attracted intensive investigation in these 11 regions. Genes within repeat regions will further discussed in the MDV ntmorigeniciry section. A brief illustration of MDV gene transcription is summarized in Figure l. 12 3mm EB mm. a w :2 92 was A". 4| Am. Va flv I‘ll R82 x» or; O» mm E. m I m 4 Al Al Iv Al Iv Iv ._ mp. ~= E ME. m3 43 9.55% >02 2:256 >92 .5 856 he E58...— .— 2:3..— 13 C). Temporal gene expression of herpesviruses As with other herpesviruses, MDV gene expression is coordinately regulated and sequentially ordered in a cascade fashion (Nazerian and Lee, 1976; Maray et al.,1988; Schat et al., 1989; Wagner, 1991). Generally, three major kinetic classes of genes are expressed: 1) immediate-early (IE, or alpha, (1 ), 2) early (E or beta, [3 ), and 3) late (L or gamma, 7 ). Immediate- early (IE) genes are expressed immediately upon infection and do not require de novo viral protein synthesis. Their gene products are required for subsequent activation of early and late virus genes, as well as autoregulation of IE genes. Metabolic inhibitors, such as cycloheximide (CHX) will cause accumulation of IE gene transcripts. Early (E) genes are the next class expressed and their synthesis requires the activity of at least one IE protein. Early gene expression is enhanced, rather than reduced, in the present of drugs that block viral DNA synthesis, such as phosphonoacetic acid (PAA). The expression of late genes requires both viral protein synthesis and viral DNA replication (Wagner, 1991). While early genes primarily encode proteins which are required for viral DNA replication, late genes encode structural proteins required for virion assembly and VP16, an IE gene transactivator (Roizman and Sears, 1991). Comparing with other herpesviruses, studies on MDV gene expression and regulation are still far behind, partly due to the cell associated property of MDV. Information from other herpesviruses, such as herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), are very valuable and greatly facilitate our studies on MDV. Thus, in this next section, the review will start from HSV, the most extensively studied herpesvirus, and focus on immediate- early gene products and their biological properties. 14 i). Herpes simples virus (alpha-herpesvirus) IE gene expression HSV is one of the most intensively investigated viruses. It has a double-stranded DNA genome of about 150 kb and encodes more than 70 genes (McGeoch et al., 1988; Wagner, 1991). HSV gene expression is regulated primarily at the transcriptional and post-transcriptional levels during the productive replication cycle (Weinheimer and McKnight, 1987; Godowski and Knipe, 1986; Roizman and Sears, 1991). Although immediate-early genes are the first group of genes expressed, IE genes are induced by a viral structural protein (late gene product), VP16 (or a-trans-inducing factor, (1—TIF, ICP25, UL48, Vmw65). VP16 is a component of the virion tegument which is transported into the nucleus different from that of viral DNA. VP16 Specifically stimulates viral IE gene expression through direct interaction with the cellular Oct-1 transcription factor system. Oct-/V P16 recognizes a specific cis-acting element (TAATGARAT) in IE gene promoters (Roizman and Sears, 1991). HSV DNA contains five immediate-early genes, IE-l (0(1), IE-2 ((127), IE—3 (0(4), IE-4 ((122), and IE-5 ((147), corresponding to gene products ICPO (VmwllO), ICP27 (Vmw63), ICP4 (Vmw175), ICP22 (Vmw68), and ICP47 (Vmw12), respectively (Everett et al., 1991; Roizman and Sears, 1990). All HSV IE genes map to near the termini of the L and S components (Figure 2). IE] and IE-3 are located within inverted repeats of the L and S components, respectively, and therefore are diploid in each genome. In the circular arrangement of viral DNA, IE genes form two clusters of [EL 3, 4, and IE-S, 3 and 1. Each cluster contains an origin of DNA replication (Orig) between IE3 and IE4 or IE3 and IE5 In spite of the clustering, each IE gene has its own promoter- regulatory region, as well as transcription initiation and termination sites (Roizman and 15 Sears, 1991). IE genes are transcribed by the host RNA polymerase 11. Synthesis of IE proteins can be detected 30 minutes post infection and reaches a peak at 2—4 hr post- infection. All HSV IE proteins, with the exception of ICP47, have been shown to have regulatory functions important for transcription of early and late HSV genes ( Roizman and Sears, 1991). 16 EB 3mm “V I 3.2 «i9 NEU— f Iv m5. T J; a”: _ SE. 4a EUH 80* N vLUH my I >>v fig 4>> mm; SE “ SE.— a: m: 8:859 >92 EB >m= 2: :e 850 m: .6 £553.: 2: he neg—3:80 .N 95w?"— 17 ICPO. ICPO is the gene product of IE-l which is the only spliced IE gene in HSV. The IE-l gene is located in the repeated sequences which flank the unique long region of HSV genomes. Nucleotide sequence analysis, coupled with $1 nuclease mapping and cDNA analysis revealed that [ED is 3,587 bp long and composed of three coding exons separated by two introns (Perry, et al., 1986; Everett, et al., 1991; Zhu et al., 1991). Based on the sequence of the coding region, ICPO is predicated to be composed of 775 amino acids with a molecular weight of 78,452. In SDS-polyacrylamide gels, however, the apparent molecular size of ICPO is about 110 kDa (O’Hare and Hayward, 1985; Perry et al., 1986). ICPO is a highly phosphorylated protein and predominantly located in the nuclei of infected cells (Ackermann, et al., 1984; Perry, et al., 1986; Wilcox et al., 1980 ). As deduced from the predicted amino acid sequence, ICPO contains a highly acidic N- terminus, adjacent to two zinc-finger motifs, which are in DNA binding domains of several transcription factors. Two proline-rich regions are in the middle of ICPO (Perry, et al., 1986; Everett, 1988; Freemont et al., 1991). Proline-rich regions have been shown to act as transcription activation domains in CTF/NF-l, GTE—2, and AP-2. Despite identification of these structural characteristics, the mechanism by which ICPO regulates gene expression is still unclear. In vitro transient assays demonstrate that ICPO is a potent and promiscuous trans- activator of gene expression. ICPO is able to activate promoters from HSV genes of all three kinetic classes (IE, E, L), as well as heterologous promoters, including cellular genes, the SV40 early promoter and the human immunodeficiency virus (HIV) long terminal repeat (LTR) (Everett et al., 1991). In spite of action on a wide range of promoters, ICPO does not require specific cis-acting sequences. However, specific 18 sequences in HIV LTR promoters that are required for response to ICPO are reported (Mosca, et al., 1987). ICPO transactivates promoters by itself, or in synergy with ICP4, a major regulatory protein encoded by IE-3. Synergistic activation is much greater than that found with either of protein alone (Everett, 1984; Gelman and Silverstein, 1986). Based on serial in-frame insertion or deletion mutations throughout the ICPO polypeptide, it has been found the carboxyl terminal and cysteine-rich zinc-finger domains (regions 3 and 5) are most crucial for both synergistic activation with ICP4 and normal intranuclear distribution of ICPO. Whereas region 1 in the second exon is required for ICPO activity in the absence of ICP4 (Everett et al., 1990) Therefore, ICPO may act through two different mechanisms depending upon the presence of ICP4 (Roizman and Sears, 1991; Everett et al., 1991). In vivo studies of HSV mutants with lesions affecting both copies of ICPO show that ICPO plays an important, but not essential role during lytic infection in cell culture. ICPO mutants replicate in a multiplicity-dependent fashion, i. e., they grow poorly following low-multiplicity infection but well following high-multiplicity infection (Sacks and Schaffer, 1987; Stow and Stow, 1986). In low multiplicity infection, ICPO deletion mutants exhibit substantial impairment in viral polypeptide synthesis, delay expression of early and late gene transcripts, are defective in replication and have reduced initiation of plaque formation (Chen and Silverstein, 1992; Everett et al., 1991). Observations on relative importance of ICPO regions are mostly agreeable between mutant virus in vivo experiments and in vitro transfection assays. The mechanism by which ICPO activates transcription, however, remains obscure. Several possible mechanisms have been suggested, including binding to DNA indirectly through cellular or other viral proteins; 19 acting at post-transcriptional level; and making DNA more accessible to the transcriptional machinery (Everett et al., 1991; Cai and Schaffer, 1992). Alternatively, ICPO could activate a cellular transcription factor, or suppress negative regulatory factors (Preston et al., 1988). ICPO has also been suggested to play a role in efficient establishment and reactivation of latency, in which viral gene expression is limited to transcription of the latency-associated transcript (LAT) and no infectious virus can be detected (Leob, et al., 1989). Viruses with mutations in both copies of ICPO do not reactivate as efficiently as wild type virus. In an in vitro latency system, the product of the IE] gene, cloned in an adenovirus expression vector is sufficient to reactivate latent virus (Zhu et al., 1988). When mutant IE-l genes were examined, the transactivation domains of ICPO were shown to be required for reactivation (Zhu et al., 1990). Difficulties associated with explanation of the effects of ICPO mutations are due to overlap of the ICPO and LAT genes, which are transcribed in an opposite direction and have been also implicated in reactivation (Feldman, 1991). Overlap of these two genes results in ICPO mutants also containing mutations in LAT transcripts and raises the question of how to separate effects of mutations in ICPO from effects of mutations in LAT transcripts. Cai et al. (1993) reported a mouse ocular model to study establishment and reactivation of latency by a series of ICPO nonsense, insertion and deletion mutant viruses. Among these mutations, all nonsense mutants can induce synthesis of near—wild type levels of the 2 kb LAT which means LAT transcription is not disrupted. These mutants exhibite less efficient replication and reduced reactivation. In addition, when a single copy of the ICPO gene is inserted into an ICPO‘ LAT double mutant virus genome, replication and reactivation of the mutant is 20 restored to one-half the wild-type level. These results demonstrate the role of ICPO is distinct from that of the LATs in establishment and reactivation of latency. The role of ICPO in establishment of latency is to increase efficiency of virus replication at the site of primary infection and in ganglionic neurons, the site of latent infection (Leib et al., 1989). In reactivation from latency, ICPO can boost viral gene expression in neurons at the onset of reactivation. Therefore, an alternative pathway independent to VP16—Oct-1 interaction may exist for activating IE gene expression, particularly in the early stage of reactivation from latency where VP16 is absent (Elshiekh et al., 1991). This idea is supported by an experiment in which Vero cells were transfected with infectious mutant and wild-type viral DNA (no virion protein VP16 is present) (Cai and Schaffer, 1992). The reduction of ICP4 expression in mutant viral DNA transfection can be reversed by cotransfection of an ICPO expression plasmid, suggesting ICPO may play a back-up role to VP16 in activating IE gene expression. However, the mechanism by which ICPO genes are regulated to exert a role in reactivation of latency is unclear. Multiple copies of the octamer consensus binding site in the ICPO promoter region have been reported. A 15-bp critical promoter region of ICPO is mutually bound by two cellular proteins and is required for efficient constitutive expression of ICPO (O’Rourke and O’Hare, 1993; Elshiekh et al., 1991). ICP4. ICP4, encoded by IE-3 (IE175, or (14), is the major trans-activator of HSV genes. IE-3 encodes 1,298 amino acids with a predicted protein molecular weight of about 133 kDa (Roizman and Sears, 1991). ICP4 polypeptide localizes to the nuclei of infected cells and is phosphorylated such that at least three modified species (4a, 4b, 4c) are observed on denaturing polyacrylamide gels with molecular weights of 160 kDA, 163 21 kDa, and 170 kDa, respectively. One of the phosphorylated species (170 kDa) is stable throughout the course of infection, whereas phosphate cycles on and off at least two other species (Wilcox et al., 1980). Through studies involving viral genetics, transient assays, and biochemical analysis, it has been established that ICP4 is essential for productive viral infection and is a complex multifunctional protein (Dixon and Schaffer, 1980; Roizman and Sears, 1991). Viruses carrying temperature-sensitive (ts) deletion, and nonsense mutations which impair expression or activity of ICP4 fail to synthesize both early and late gene products and exhibit an overproduction of ICP4 and other IE proteins (Dixon and Schaffer, 1980; Roizman and Sears, 1991; Shepard et al., 1989). Consistent with in vivo experiments, results of transient expression assays utilizing a cloned ICP4 gene and reporter genes containing promoters of IE, early and late viral genes demonstrate that ICP4 transactivates early and late viral gene promoters while repressing IE gene promoters (DeLuca et al., 1984; 1985; Everett, 1984; O’Hare and Hayward, 1985, a; b). From these observations, it was concluded that ICP4 has at least two major regulatory activities: (i) induction-enhancement of early and late gene expression, and (ii) repression of ICP4 and possibly other immediate-early genes. As described above, ICP4 has a synergistic effect with ICPO. Recently, Zhu et al. (1994) reported that ICP4 can enhance nuclear localization of ICPO. These results suggest cooperative regulation by these IE genes. Analysis of primary amino acid sequences of ICP4 in HSV and comparison with analogous proteins in other herpesviruses (VZV, PRV etc.) indicate existence of five highly conserved intramolecular domains (McGeoch et al., 1986). Using deletion mutations, partial polypeptides of ICP4 confen'ing different functions have been reported 22 (DeLuca and Schaffer, 1988; Shepard et al., 1989). The N—terrninal 774 amino acids of ICP4 are proficient for DNA binding, autoregulation, and transactivation of some viral genes based on genetic approaches. The ability of ICP4 to form specific protein—DNA complexes is correlated with bOth transactivation and autoregulation activities (DeLuca and Schaffer, 1988; Shepard et al., 1989). Although it has been well defined that ICP4 is a DNA-binding protein, controversies on whether it binds to specific DNA sequences exist. ICP4 was first shown to interact with the sequence ATCGTCNNNNYCGRC (where R=purine, Y=pyrimidine, and N=any base). One such ICP4-binding site overlaps the start site of ICP4 transcription, leading to the suggestion that ICP4 binding to the start site of transcription suppresses expression by preventing RNA polymerase from associating with the mRNA cap site (Faber and Wilcox, 1986; Muller, 1987). However, other immediate-early genes lacking similar ICP4 binding sites within their promoter do not support this hypothesis. It has been reported that ICP4 can utilize a common DNA binding domain to interact directly with many different DNA sequences found in or near HSV gene promoters with different affinity, depending on target sequences (Shepard et al., 1989; Imbalzano and DeLuca., 1992; Michael et al., 1988). ICP4 can stimulate transcription of minimal promoters in which the only recognizable cis-acting element is a TATA homology. Therefore, it was suggested that ICP4 may operate though the transcriptional machinery acting at TATA boxes (Imbalzano and DeLuca, 1992; DiDonato and Muller, 1989). Recently, Smith et al. (1993) reported that ICP4 forms a tripartite complex with TFIIB and either the TATA-binding protein (TBP) or TFIID by using gel retardation and footprinting assays. Formation of the 23 complex was not simply a result of tripartite occupancy of DNA, but a consequence of protein-protein interactions. In the presence of all three proteins, the affinity of ICP4 and TBP for their respective binding sites was substantially increased. The ability of ICP4 to bind to DNA is necessary but not sufficient for the formation of the tripartite complex, and the ability of ICP4 to form this complex correlates with its ability to activate transcription (Simth et al., 1993). ICP27. ICP27 is encoded by the IE-2 gene, which is located in the unique long region of HSV-1 genomes with an unspliced transcript of 1.8 kb (Sandri-Goldin, 1991). The coding sequence of 1,536 bp encodes a protein of 512 amino acids. Like other IE gene products, ICP27 accumulates in the nucleus and is highly phosphorylated, resulting in the observed molecular weight 63 kDa larger than the predicted weight of 55 kDa (Sandri-Goldin, 1991). ICP27 contains both stable phosphate groups and phosphate groups which cycle on and off during infection (Rice et al., 1993). The analysis of viral mutants containing temperature-sensitive mutations and deletion mutations demonstrate that ICP27 is an essential regulatory protein in viral infection (McCarthy et al., 1985; Sacks et al., 1985). Transient-expression (chloramphenicol acetyltransferase CAT assay) experiments provide evidence of the regulatory activities of ICP27 (Block and Jordan, 1988; Everett 1986). Several studies suggest that ICP27 exerts both positive and negative effects on ICPO- and ICP4-induced gene expression (Rice and Knipe, 1990; Sekulovich et al., 1988; Su and Knipe, 1989). In the presence of ICP4 and ICPO, ICP27 represses IE and E gene expression, whereas L gene expression is enhanced. However, in the absence of ICP4 and ICPO, ICP27 has little or no trans-regulatory effect on target genes. Cis-elements in 24 reporter genes which mediate positive and negative regulation have not been definitively identified, but recent studies have indicated that mRNA processing signals are critical to regulation of a reporter gene by ICP27 (Chapman et al., 1992; Sandri-Goldin and Mendoza, 1992). Specifically, 3’ sequences involved in pre-mRNA cleavage and polyadenylation mediate positive regulation by ICP27, while intron sequences appear necessary for transrepression. These studies suggest that ICP27 can regulate gene expression at a post-transcriptional level in transfected cells, possibly by affecting mRNA processing (Rice et al., 1993). ICP27 is also partly responsible for post-translational modification of ICP4. ICP4 migrates faster in wild-type virus infected cells and in cells cotransfected with ICP4- and ICP27-expressing plasmids than in cells infected with ICP27 null mutants or cells transfected with an ICP4-expression plasmid only (McMahan and Schaffer, 1990; Rice and Knipe, 1988; Su and Knipe, 1989). In contrast to ICP4, ICP27 inhibits nuclear localization of ICPO. The inhibitory effect depends on ICP27 expression levels (Zhu et al., 1994). Assessment of the predicted amino acid sequence of ICP27 reveals that the protein can be divided into two halves with a hydrophilic amino terminal half and hydrophobic carboxyl terminal half (Sandri-Goldin, 1991). Mutational studies show the C—terminal half is required for both activation and repression functions (Hardwicke et al., 1989; McMahan and Schaffer, 1990). Although this region is not similar in nature to those of other trans- activator proteins, it does contain a consensus sequence resembling metal binding domains or "zinc-finger" motifs. The N-terminal half contains a high proportion of acidic amino acid residues (25 of the first 64 residues) and nine serine residues which are potential phosphate acceptors (Sandri-Goldin, 1991). Recently, Rice et al. (1993) have reported that 25 the acidic N-terminal half of ICP27 is required for efficient transrepression in transfected cells and is required for an essential lytic function. It appears ICP27 is a multifunctional protein which contains multiple functional domains to mediate distinct activities. ICP22/47. ICP22 and 47 are encoded by HSV IE gene 4 and 5 (U81 and U812), respectively, and are present in one copy per genome (Roizman and Sears, 1991). The predicted translated and apparent molecular weights of ICP22 are 46.521 and 72.0 kDa, respectively (Roizman and Sears, 1991). Although ICP22 is regulated as an IE gene (Post and Roizman, 1981), the specific function(s) of ICP22 remains to be elucidated. Sears et al. (1985) demonstrated that ICP22 affects expression of some late genes. ICP22 deletion mutants can be propagated in some cell lines but not others, suggesting that permissive cells may complement the ICP22 protein by utilizing a host cell factor. ICP47 is the only IE protein without an obvious regulatory function on viral gene expression. Deletion mutants of ICP47 grow as well as wild-type virus in cell culture (Mavromara-Nazos et al., 1986). Based on characteristics of wide host range of HSV, it is conceivable, by analogy with the studies on the ICP22 mutant, that the function of ICP47 is to complement certain functions missing in some cells infected by HSV in its natural host. However, no direct evidence for this hypothesis has been reported. Therefore, the function of ICP47 remains to be determined. (ii) Human cytomegalovirus virus (beta-herpesvirus) IE gene. Cytomegaloviruses are species-specific viruses that have the largest genome (230- 240 kb) in the herpesvirus family (Stinski, 1991). In HCMV, three distinct segments of genomes are expressed at IE times, including major IE genes, UL36-38, and US3. The 26 major IE region is the most extensively studied, consisting of two transcription units, IE region 1 (IE1, UL123) and IE region 2 (IE2, UL122) (Stenberg et al., 1984; 1985). These two genes are driven by a single promoter regulatory region, referred to as the major IE enhancer-containing promoter—regulatory region (MIEP), located upstream of IE1 (Stinski, 1991; Boshart et al., 1985; Thomsen et al., 1984). This regulatory region is unusually strong and contains multiple sets of highly conserved repetitive elements. The repeat elements are in two regions designated region I and II. Region I contains a potential serum response element (SRE) and a consensus binding sequence for NFl/CBP which overlaps a 13-bp repeat element Region 11 contains five GC-boxes for SP1 binding, an API site, and four different repeat elements of 16-, 18-. 19-, and 21-bp which have consensus binding sites for CRE, ATP, and NF-KB. It is likely that one repeat element interacts with more than one nuclear factor or presence of one repeat element influences binding to one or more other repeat elements. MIEP containing many cis-acting sites for eukaryotic cell transcription factors makes it highly efficient during virus infection. However, MIEP itself is also positively and negatively regulated by virus-specific proteins which will be discussed later. By alternative splicing, IE1 and IE2 yield a family of mRNAs which encode a series of unique, but related proteins (Stinski, 1991; Stenberg et al, 1989). The IE1 transcription unit consists of the first four exons with a translation initiation codon in exon 2. IE1 codes for a 1.95-kb mRNA that translates into a 72-kDa protein (IE72). IE2 includes exon 1 through 3 and exon 5. Transcripts derived from this region encode several mRNAs of 2.25-, 1.70-, and 1.40-kb, resulting in of translation proteins 86- (IE86), 55- (IE55), and 40-kDa, respectively (Stenberg et al., 1989; 1985; 1990). All of the major IE 27 proteins Share a common N-tenninus of 85 amino acids derived from exons 2 and 3, with the exception of the 40 kDa protein that originates solely from exon 5 (Stenberg et al., 1985). Most of the proteins derived from these two IE regions are phosphorylated with apparent molecular weights larger than that calculated from primary sequence data. The functional characteristics of different HCMV IE proteins differ dramatically, although they share many domains. While 86-kDa IE2 protein can activate most promoters of HCMV assayed to date, IE1 only augments transactivation activity of IE2 on viral promoters (Klucher et al., 1993; Malone et al., 1990). Both IE1 and IE2 are capable of transactivating heterologous promoters, such as HIV LTR or the heat shock protein 70 (HSP70) promoters. In addition to its function as a transactivator, the 86-kDa IE2 protein negatively autoregulates its own expression. This 86-kDa IE2 is a sequence specific DNA-binding protein that interacts directly with a DNA sequence, termed the cis repression signal (CRS), that is located between the TATA box and the transcriptional start site of the MIEP of HCMV (Arlt et al., 1994; Cherrington et al., 1991; Lang et al., 1993; Liu et al., 1991). In contrast, the 72- kDa IE1 and the 55-kDa IE2 proteins can transactivate MIEP (Barachini et al., 1992; Cherrington et al., 1989). Analysis of amino acid sequences of HCMV IE proteins reveals some characteristics similar to those of other transcription factors, including three amphipathic helices at the N-terminus of all IE proteins; single zinc finger motifs in 86-kDa IE2 and 72-kDa IE1; a leucine zipper in the 72-kDa IE1 protein; and a leucine-rich region in the 86-kDa IE2 protein. However, mechanisms whereby HCMV IE proteins execute their functions remain largely undefined. In principle, two main mechanisms of action are 28 possible: 1) A direct interaction with DNA could occur, thereby tagging an activation domain close to the basal transcription machinery; 2) Alternatively, protein-protein interaction could play a role (Stinski, 1991). (iii) Epstein-Barr virus (gamma-herpesvirus) IE genes. Studies on EBV IE genes are different from other herpesviruses due to lack of appropriate in vitro culture systems. However, three immediate-early genes, BMLFl (Mta), BRLFl (Rta), and BZLFl (Zta) are identified after induction of the EBV lytic cycle in lymphocytes (Hayward and Hardwick, 1991). The BZLFl protein is the key immediate-early trans-activator of early lytic gene expression and its expression is sufficient to disrupt viral latency. BZLF] is located just downstream of BRLFl gene, therefore, BZLF] gene expression can be derived from two promoters (Hayward and Hardwick, 1991; Kieff and Liebowitz, 1991). The more 3’ BZLF promoter drives transcription of a 1.0-kb mRNA which encodes only the BZLFl gene product, whereas an upstream BRLFl promoter drives transcription of a 2.8-kb bicistronic mRNA which encodes both BZLFl and BRLFl gene products (Hayward and Hardwick, 1991). BZLF] gene yields a spliced transcript which contains three exons. It encodes a 34- to 38- kilodalton nuclear protein which resembles a c-fos leucine zipper protein, containing a basic DNA-binding domain adjacent to a coiled-coil dimerization domain (Hayward and Hardwick, 1991; Farrell et al., 1989; Kouzarides et al., 1991). The BZLF] protein not only binds to an AP-l site, like c-fos, but also binds to additional non-AP-l sites referred to as Z response elements (ZREs) (Chang et al., 1990; Urier et al., 1989). Thus, BZLF 1 can activate a number of early EBV promoters by a direct binding mechanism (Kenney 29 et al., 1989; Roony et al., 1989). BZLFl also binds directly to the EBV lytic origin of replication (Ori-lyt) and is required for lytic replication (Fixman et al., 1992; Schepers et al., 1993). Some cellular proteins, such as NF-KB and p53 can physically and functionally interact with BZLF 1, resulting in inhibition of BZLF] and consequently an inhibition of the switch from latent to productive infection (Gutsch et al., 1994; Zhang et al., 1994). BRLF 1 protein is also a transcription activator and sequence-specific DNA-binding protein (Chevallier-Greco, et al., 1986; Hardwick et al., 1988; Kenney et al., 1989; Gruffat et al., 1990; Manet et al., 1989). Three EBV promoters (the BMLFl IE promoter, the BHRFl early promoter, and the DR early promoter) have been reported to be transactivated by BRLFl protein (Chevallier-Greco, et al., 1986; Hardwick et al., 1988; kenney et al., 1989). Interestingly, all three promoters have upstream enhancer elements, and the BRLFI response region in each promoter is mapped to these enhancer elements. Consequently, BRLF] gene has been referred to as an enhancer factor (Manet et al., 1989; Urier et al., 1989). However, it was reported that BRLF] can transactivate a HIV type-1 long terminal repeat (LTR) with a deleted enhancer element (Quinlivan et al., 1990). BRLF] also positively autoregulates its own promoter through a nonbinding mechanism (Zalani et al., 1992). Thus, the BRLF 1 gene product may activate promoters by more than one mechanism, involving certain enhancer elements or enhancer independent; direct binding or indirectly through modulation of cellular transcription factors . Both BZLFl and BRLF] transactivate independently, but they also act synergistically (Chevallier-Greco, et al., 1986; Cox et al., 1990; Giot et al., 1991; Quinlivan et al., 1993). In these cooperative functions, both Z and R binding sites are required, suggesting BZLFl and BRLFl both bind to DNA directly (Quinlivan et al., 30 1993). In addition, BZLFl functions as either an enhancer or repressor of R-induced transactivation, depending upon the presence or absence of functional Z binding sites (Quinlivan et al., 1993). The third EBV IE transactivator called BMLF 1, is encoded by the BLF 1-BSLF2 ORF. It contains several mRNAs generated by alternative splicing and driven by two promoters of promoter M (PM) and more upstream promoter Ml (PMl) (Buisson et al., 1989). The BMLFI gene product analyzed on denaturing polyacrylamide gene migrates as a polypeptide family (molecular weights, 45, 000 to 70,000), with the major product being a phosphorylated 60-kilodalton nuclear protein (Hayward and Hardwick, 1991). In transient assays, BMLFl stimulates chloramphenicol acetyltransferase (CAT) expression controlled by several different EBV promoters. However, this BMLFl-induced increase of CAT activity is not accompanied by a significant increase in steady-state-level CAT mRNA, suggesting a post-transcriptional mechanism (Buisson et al., 1989; Kenney et al., 1988; 1989). In addition, when the CAT reporter gene is changed to growth hormone, transactivation by BMLFI is lost (Kenney et al., 1989). Reporter-gene dependence of BMLFl activation suggests that BMLFI is not directly involved in promoter activation, but instead may function to increase level of some unknown protein(s) required for EBV infection (Kenney et al., 1989). In addition, the PM promoter of BMLFl is activated by both BZLFl and BRLFl IE gene products (Buisson et al., 1989). (iv) MDV IE genes Studies on MDV IE genes have been hampered due to the cell-associated nature of MDV in vitro. Characterization of RNA transcripts isolated from MDV-infected cells 31 treated with a metabolic inhibitor, such as cycloheximide (CHX) indicate that transcripts from IE genes are mainly clustered in repeat regions similar to locations of other herpesvirus IE genes (Maray et al., 1988; Schat et al., 1989). IE Transcripts, ranging from 0.6 to 4.4 kb, derived from BamHI fragments-A, D, H, I2, 13, L, and M regions are reported by different groups (Maray et al., 1988; Schat et al., 1989). However, all these reports were based only on Northern hybridization analysis, without exact gene and gene product identification. Recently, two HSV IE homologs were identified with similar localization in MDV (Figure 2) (Anderson et al., 1992; Ren et al., 1994). The MDV ICP4 gene is located in BamHI-A fragment within the inverted repeat region. The gene is 4,245 nucleotide long with an AUG translation start site located at position 1,264. The predicted protein structure of MDV ICP4 is similar to its counterparts in VZV and HSV. MDV ICP4 contains five regions in which regions 2 and 4 are more conserved than others, and a serine-rich tract located towards the amino terminus (McGeoch et al., 1986; Anderson et al., 1992). However, the MDV serine run differs from those of other alpha-herpesvirus ICP4 homologs in that it is flanked on both sides by regions enriched in prolines and basic amino acids, whereas the HSV, VZV, and PRV serine runs are preceded by regions enriched in prolines and basic amino acids but are followed by strongly acidic regions. Also, a number of potential transcriptional regulatory sites are identified within or adjacent to the MDV ICP4 sequence, including ICP4 binding site, Oct-l site, and TAATn3A sequence similar to VP16 recognizing sequence of TAATGARAT (Anderson et al., 1992). However, neither a protein product nor a regulatory function is known. The HSV ICP27 homolog of MDV is mapped to the EcoRI-B fragment of MDV 32 DNA (Ren et al, 1994). The MDV ICP27 gene is 1,419 nucleotide long and encodes 473 amino acids with a predicated molecular weight of 54.5 kDa. Comparison of the predicted amino acid sequence of MDV ICP27 with that of HSV ICP27 and VZV ORF4 (HSV ICP27 homologue) shows a strikingly similarity (37.3% identity between MDV ICP27 and HSV-1 ICP27; 32.7% identity between MDV ICP27 and VZV ORF4) within the C-terminal region, which is the functional domain for HSV ICP27 and VZV ORF4. In addition, a conserved zinc finger motif is in the C-tenninus of MDV ICP27. The zinc finger motif in HSV ICP27 is involved in DNA, RNA and protein-protein interaction (Sandri-Goldin, 1991; Smith et al., 1991). Using antibody raised against TrpE-ICP27 fusion protein, a 55 kDa gene product was identified by western blot analysis (Ren et al., 1994). Information regarding functional assays of ICP27 of MDV is not yet available. Recently, a 1.6 kb immediate-early transcript is localized to the MDV BamH I2 region by Northern hybridization analysis of RNA isolated from MDV infected cells treated with CHX (Hong and Coussens, 1994). By cDNA cloning and sequencing, two cDNAs of 1.4 kb (Cl) and 1.35 kb (C2) are identified as spliced transcripts spanning MDV BarnHI-H and I2 fragments. C l and C2 use the same splice acceptors and 3’ ends, but they differ at their 5’ ends and utilize different splice donors. Despite abundant transcription detected in Northern blot analysis, sequencing analysis shows only two small open reading frames (ORFS) within each cDNA. C1 cDNA contains ORF 1a (83 amino acids) and ORF 2 (107 amino acids), while C2 cDNA contains ORF 1b (76 amino acids) and the same ORF 2 in C1. All potential ORFS within C1 and C2 were searched against the Swiss data base without finding any significant homology, suggesting that any protein encoded by these ORFS would be MDV specific. A 14 kDa polypeptide (p14) is detected 33 by Western blot analysis using antisera raised against ORFla- and ORFlb-GST fusion proteins (Figure 2). This 14 kDa protein is expressed in cells which are lytically infected with MDV oncogenic strains (GA, mdl l), and their attenuated derivatives, as well as in the latently MDV-infected, transformed MSB-l cell line. Like most IE gene products in other herpesviruses, p14 is a phosphoprotein and therefore, designated as pp14. However, both Western blot analysis of subcellular fractions and immunofluorescence detection reveal that p14 is predominately a cytoplasmic protein (Hong et al., 1994). Whether pp14 has a regulatory function on other MDV gene expression is still unknown at this point. In contrast to most other herpesvirus, MDV IE gene transcripts and gene products are identified in latently infected and transformed lymphoblastoid cell lines (Maray et al., 1988; Schat et al., 1989; Hong and Coussens, 1994). Therefore, it is tempting to speculate that MDV IE gene products are involved in induction and/or maintenance of the latency or transformed state. If true, this would make MDV latency similar to EBV. (V). Early and late genes. Early genes of herpesviruses are defined as genes which start transcription and protein synthesis at approximately 3 hr post-infection, peak by about 5-7 hr post-infection, and decline thereafter. Early proteins are involved in nucleotide precursor metabolism and viral DNA synthesis. Most early gene products, therefore, are metabolic enzymes and proteins for virus replication (Roizman and Sears, 1991). In HSV, the most well characterized early proteins include viral ribonucleotide reductase, major DNA-binding protein, thymidine kinase (TK), and DNA polymerase. Some MDV early genes homologs of HSV have been identified, such as TK, DNA polymerase, DNA-binding protein etc. 34 (Buckmaster et al., 1988). Also, pp38, a MDV specific gene is expressed in infected cells treated with PAA, suggesting it may be an early protein (Chen and Velicer, 1992). Appearance of early proteins signals onset of viral DNA synthesis, and subsequently induction of late gene expression. Based on dependence for viral DNA replication, late genes can be subdivided into 71 and 72. Transcription of 71 genes occurs prior to initiation of viral DNA synthesis and thus does not depend stringently on viral DNA replication. As viral DNA synthesis begins, high level expression of 71 genes occurs and Y2 gene expression begins. Both 71 and Y2 protein synthesis continues throughout the remaining replication cycle (Wagner, 1991; Roizman and Sears, 1991). Most late gene products are structural proteins for virion capsid, tegument, and envelope. Eight glycoproteins, including gB, gC, gD, gE, gG gH, gI and g] are well characterized in HSV (Roizman and Sears, 1991). The functions of HSV glycoproteins are defined and involve virus attachment, penetration and cell fusion. In MDV, six HSV glycoprotein homolog genes (gB, gC, gD, gE, g1 and gK) are identified and sequenced (Buckmaster et al., 1988; Chen and Velicer, 1992; Coussens and Velicer, 1988; Isofort et al., 1987; Ross et al., 1989; 1991; Brunovskis and Velicer, 1992, Ren et al., 1994). Among them, gB and gC (also designated as A and B antigen, respectively), have been extensively studied at antigen level. MDV gB is located in BamHI-K3 and 13 regions and is processed into a family of proteins known as gplOO, gp60 and gp49 (Chen and Velicer, 1991; Ross et al., 1989). Antibodies against MDV gB can neutralize MDV in cell culture (Ikuta et al., 1984). Recombinant fowl pox virus (FPV) expressing an MDV gB homolog in vivo shows complete protection against Marek’s disease (Nazerian et al, 1992). 35 MDV gC gene is located in BamHI-B fragment (Isfort et al., 1986; Coussens and Velicer, 1988). It encodes a N-linked glycoprotein of 57-65 kilodalton with a precursor of 44 kDa (Isofort et al., 1986). MDV gC is a secreted protein and its expression is significantly reduced in attenuated MDV strains (Bulow and Biggs 1975; Ikuta et al., 1983; Isfort et al., 1986). However, the mechanism leading to reduced gC expression and its relationship with MDV oncogenicity or attenuation is still unclear. Based on comparison of the protein level, gene structure, steady-state RNA and transcription rates of MDV gC between oncogenic and attenuated MDV isolates, Wilson et al. (1994) suggest that reduced expression of MDV gC is directly related to a reduction in transcription rate of MDV gC genes in attenuated strains. Lack of DNA sequence alternations in gC gene coding regions and promoters in attenuated strains, Wilson et al. further suggest that reduced transcription of gC is due to alternation of viral or cellular proteins which regulate gC promoter activity in attenuated MDV (Wilson et al., 1994). 5. MDV latency An important biological property of all three subfamilies of herpesviridae is their ability to establish latent infections in their natural hosts. Alpha-herpesvirus latency occurs primarily in nervous tissue, including sensory and autonomic nerve ganglia and the central nervous system. Beta-herpesviruses can establish latent infection in secretory glands, lymphoreticular cells, kidney and other tissues. Garnma-herpesvirus latency is confined to lymphoid tissues (Roizman, 1991). Although MDV genomic structure is similar to that of alpha-herpesviruses, latent infection of MDV occurs predominantly in lymphocytes, similar to gamma-herpesviruses (Payne, 1985). As described previously, MDV infection 36 switches from lytic infection of primary B-lymphocytes to latent infection of predominantly T-lymphocytes at about one week post-infection, coincident with a temporary immune recovery (Payne, 1985). Most latently infected T cells are activated, CD4*CD8' T helper cells (Schat et al., 1991). Studies on MDV latency are mostly conducted on MDV transformed lymphoblastoid cell lines which are considered as latently infected. Virus can be rescued from most of these tumor cell lines by co-cultivation with permissive cells, such as CEF, DEF, or by inoculation of tumor cells into chickens (Schat, 1985). However, the ability to rescue virus can be lost after prolonged in vitro passage of MDV tumor cell lines. Based on the capability of virus to be recovered and viral antigen expression, MDV transformed cell lines are distinguished as producer cell lines and non-producer cell lines. Producer cell lines are those cell lines from which virus can be rescued after in vitro co- cultivation or following innoculation into susceptible chickens. Producer lines can be further classified as expression or non-expression lines based on proportion of cells which spontaneously express viral antigens. Expression lines, such as MDCC-CU36, MSB-l, contains a high proportion of antigen expression cells which can be detected by immunofluorescence (IF) tests using conventional anti-MDV sera. While non-expression cell lines, such as MDCC-CU41, MKT-l, contain none or only a few cells expressing viral antigen. Treatment of non-expression cell lines with 5-iodo-2-deoxyuridine (IUdR) can induce viral antigen expression detectable by anti-MDV sera. In non-producer cell lines, such as MDCC-HPI or MDCC-RPI, viral antigens are not detectable and virus can not be rescued by co-cultivation. All MDV lymphoblastoid cell lines are free of infectious virion particles, but contain multiple copies of the viral genome, typically between 5 to 37 15 copies per cell (Akiyama and Kato, 1974; Nazerian, 1977; Schat et al., 1989; Silver et al., 1979). The status of MDV genomes in transformed cell lines has been reported as closed circular DNA by several investigators using ethidium bromide-CSCI equilibrium centrifugation to separate viral and cellular DNA (Lee et al., 1971; Tanaka et al., 1980; Hirai et al., 1979). Recently, Delecluse and Harnmerschmidt (1993) observed MDV DNA integration into the host cell chromosomes in all six MDV transformed cell lines by Gardellar gel electrophoresis and in situ hybridization techniques. MDV integration sites are preferentially located at telomeres of large- and mid-size chromosomes or on mini- chromosomes. Only a minor pOpulation of MDV genomes were detected as linear or covalently circular DNA. Based on these observations with MDV and a similar phenomena of EBV DNA integration in a number of Burkitt’s lymphoma cell lines, the authors proposed that herpesvirus DNA integration is common and may provide clues for understanding virus tumorigenicity (Delecluse and Hammerschmidt, 1993). Similar to other herpesvirus latency, MDV transformed cell lines only have limited viral gene expression compared with many viral transcripts in lytically infected cells (Silver et al., 1979; Sugaya et al., 1990). Since MDV latency and oncogenicity are closely related, genes expressed in transformed cell lines may be important to initiate or maintain latency and/or transformation by MDV. However, contradictory results are reported by several groups. Maray et al. (1988) reported 29 transcripts detected in Northern hybridization dispersed over almost the entire MDV genome in MSB-l cells. Schat et al. (1989) compared transcription of MDV genomes in lytically infected cells and different types of lymphoblastoid cell lines. Between 4 and 7 transcripts are detected in MDCC- HPl (non-producer cell line) and MDCC-CU41 (non-expression cell line), respectively. 38 These RN As are transcribed from IE genes located mainly in repeat regions flanking UL and Us and in Us. Additional transcripts are identified when IUdR is added to culture medium. In MDCC-CU36, an expression cell line with a high percentage of antigen- positive cells, most of the transcripts in lytically infected cells are detected (Schat et al., 1989). Sugaya et al. (1990) reported 32 viral transcripts in a non—producer cell line, MKT- 1. These transcripts are clustered in the short and long repeat regions, similar to the observation of Schat et al. (1989). The most abundant transcripts were encoded in BamHI- I2 region. Together, these data suggest that repeat regions of MDV encode genes which may play important roles in MDV latency and transformation. Consistent with transcripts identified in Northern blot analysis, three genes and their gene products have recently been identified in MDV transformed cell lines. All of them are located in repeat regions. Phosphoprotein 38 (pp38) was first identified as one of three viral proteins (41, 38, and 24 kDA) detected by a monoclonal antibody against a lgtll fusion protein containing MDV BamHI-H fragment sequence (Silva and Lee, 1984). Based on Northern blot analysis and DNA sequencing, the gene encoding pp38 is mapped to the BamHI-H fragment region and spans the junction of MDV long unique (UL) and long internal repeat (IRL) regions. The pp38 gene is translated from unspliced mRN A in a leftward direction. The gene product, phosphoprotein 38, is 290 amino-acids in length and is phosphorylated primarily at serine residues (Nakajima et al., 1987). The protein is relatively rich in acidic residues with glutamic and aspartic acid composing 15% of the predicted amino acid sequence (Nakajima et al., 1987; Cui et al., 1991; Chen et al., 1992). Pp38 is only expressed in serotype-1 (oncogenic and attenuated) MDV infected cells, but not in nononcogenic serotype-2 and serotype-3 infected cells. It also 39 has been shown that pp38 is abundantly expressed in the latently infected MSB-l lymphoblastoid cell line without IUdR induction (Chen et al., 1992). Another MDV phosphoprotein, pp14, was recently identified and is also located in the long internal repeat (IRL) region. Encoded by spliced RNA(S) which span the BamHI-H and I2 fragments, pp14 is transcribed in a rigth direction, opposite to that of pp38 (Hong and Coussens, 1994). On the basis of transcriptional mapping and alignment with published BamHI-H viral genomic DNA sequence (Bradley et al., 1989 b), the upstream promoter regulatory region of pp14 is defined to be a bidirectional promoter-enhancer region shared by pp38 (Hong and Coussens, 1994). Similar to pp38, pp14 is expressed in MDV transformed cell line, MSB-l, and is MDV serotype-1 specific antigen. While pp38 is insensitive to phosphonoacetic acid inhibition, suggesting that pp38 is an early viral protein, pp14 is derived from an immediate-early gene. Western blot analysis of subcellular fractions and indirect immunofluorescence reveal that both proteins are cytoplasmic (Cui et al., 1991; Hong and Coussens, 1994). The functions of pp38 and pp14 are yet to be determined. A third gene, designated as meq, is highly expressed in MDV infected lymphoblastoid cell lines and is located in BamHI-I2 and EcoRI-Q regions (Jones et a1, 1992). The meq gene encodes a protein of 362 amino acids, which has a leucine zipper repeat and an upstream domain rich in basic amino acids, both characteristics of the fos/jun family of transcriptional activators. The C-terminal region of meq contains a proline-rich domain (26% proline) and is rich in acidic amino acids, which are features of another class of transcription factors, such as AP-2, C/EBP, OCT -2. Using antiserum raised against a synthetic peptide corresponding to the leucine zipper region of meq, a 40 40 kDa protein is detected in the MDV transformed RP4 cell line. Although EcoR-Q derived transcription can be detected in MDV (strain JM) acutely infected DEF by Northern blot analysis, the 40 kDa protein is not present in cell lysates of DEF lytically infected with MDV (Jones et al., 1992). Based on DNA-protein binding and DNA-protein immunoprecipitation, Wen et al. (1988) reported a 28 kDa nuclear protein, MDNA, which is expressed in MDV transformed cell lines (MKT-l and MSB-l), but not in lytically infected cells. MDNA is bound to two loci in the BamHI-A fragment of the MDV genome. By DNase footprint, Wen et al. (1988) demonstrated that MDNA protect regions containing AT-rich and palindromic sequences. Therefore, the authors suggest that MDNA may function analogous to EBNA-l of EBV in immortalized cells. However, there has been no functional analysis to confirm this hypothesis. 6. MDV tumorigenicity The discovery of DNA tumor viruses was several decades later than that of RNA tumor viruses. Unlike RNA tumor viruses which all belong to a single group of retrovirus, DNA tumor viruses are distributed among six major families. Among them, polyomavirues, adenoviruses, and papillomaviruses are best understood, perhaps due to their small genomic size and ease of manipulation. Herpesviruses are associated with tumor induction, such as a link between EBV and Burkitt’s lymphoma, or nasopharyngeal carcinoma in humans, MDV with Marek’s disease in chickens. However, it was considered that herpesvirus in tumors might be a passenger rather than a causal agent. The successful recovery of infectious MDV virions from feather follicle epithelium of Marek’s 41 disease infected chickens and reproduction of lymphomas in specific-pathogen-free chickens provided the first solid evidence of herpesvirus as a oncogenic agents in their natural host (Churchill and Biggs, 1967; Nazirian and Burmester, 1968). Further proof came with discovery of live virus vaccines, made from an antigenically related but apathogenic herpesvirus of turkey (HVT), which can protect chickens against MD tumors (Churchill et al., 1969). Although MDV is the first tumor virus effectively controlled by vaccination, the mechanism of tumorigenicity and vaccine protection is poorly understood. After a brief cytolytic infection and short latency, MDV induces lymphoma within three weeks, suggesting MDV is an acutely oncogenic herpesvirus. Studies of MDV oncogenicity have focused on several aspects, including comparison of gene expression in oncogenic MDV with their attenuated derivatives or with nononcogenic serotype-2 and -3; identification of genomic structure changes during attenuation; characterization of gene(s) specifically expressed in MDV transformed tumor cell lines. Among them, repeat regions of the MDV genome have been intensively investigated because of speculation that transcripts within this region may be important in MDV-induced tumorigenicity. Several investigators have shown that serial in vitro passage of oncogenic MDV results in loss of MDV tumorigenicity. Attenuation is strongly correlated with an expansion in two particular regions (BamHI-H and -D), present in MDV TRL and IRL regions, respectively (Fukuchi et al., 1985; Silva and Witter, 1985). It was later discovered that expansion was due to amplification of a specific 132-bp direct repeat sequence located within BamHI-H and -D regions (Maotani et al., 1986). Tumor induction studies in chickens shows that cloned virus populations which exhibit an amplification in this region have decreased tumorigenic capability. By contrast, viruses which do not 42 contain amplified BamHI-H or -D regions efficiently induce tumors in chickens (Fukuchi et al., 1985). However, in all cases, changes in other regions of the genome are present. These results led to the hypothesis that the existence of one or more genes within the expanded regions is responsible for initiation or maintenance the tumorigenic state of MDV. Bradley et al. (1989) reported that a 1.8-kb gene family is transcribed rightwardly from the expanded region in MDV BamHI-H fragments. Nucleotide sequencing and transcriptional mapping data suggest the presence of a group of spliced transcripts (1.4 to 1.8 kb) which are composed of two exons. The first exon has two species with the same 5 ’end and different 3’ ends near or within the expanded region. The second exon contains five species with identical 3’ ends and various 5’ ends mapped outside of the expanded region. Therefore, this gene family shares a 5’ initiation Site and a 3’ termination site, but contains different introns. Furthermore, the 1.8-kb RNA family is only expressed in CEF infected by oncogenic strains of MDV and in cell lines established from MDV-induced tumors, but not in CEF infected with an attenuated strain of MDV (Bradley et al., 1989). Later, Bradley et al. (1989) demonstrated that disappearance of the 1.8 kb transcript is due to truncation of the 1.8-kb transcript into a 0.4-kb transcript. These data suggest that the 1.8 kb gene family is associated with MDV tumorigenicity. This hypothesis is supported by antisense inhibition of proliferation of MDV-induced lymphoblastoid cell lines using an oligonucleotide complementary to the putative splice donor sequence of the 1.8 kb gene family (Kawamura et al., 1991). Chen and Velicer (1991) reported four groups of transcripts derived from BamHI- H and D region based on cDNA analysis and SI nuclease protection assay. These 43 transcripts can be either initiated or terminated within or near the expanded region of the 132-bp direct repeat at multiple sites and are transcribed in both rightward and leftward directions. Therefore, these RNAs contain a partial copy or one or more full copies of the 132-bp repeat at either their 5’ or 3’ end. Each 132-bp repeat contains one TATA box and two polyadenylation consensus sequences in each direction. The authors hypothesized that the 132-bp repeat may serve as a bidirectional promoter region to generate a diversity of transcripts (Chen and Velicer, 1991). Since these four groups are transcribed in opposite directions and complementary RNAs function as antisense RNA, a function in regulation of gene expression is possible (Chen and Velicer, 1991). In agreement with Bradley’s paper, Chen and Velicer also speculate that the transcripts derived from the repeat regions are associated with the tumorigenic potential of MDV. Iwata et al. (1992) also reported a cDNA library derived from MDV strain Md5- infected cells. Twelve clones, containing 1.1- to 1.8-kb insertions, localized to the BamHI- H fragment. All these cDNAs are derived from unspliced mRNAs transcribed rightwardly. Iwata et al., therefore, concluded that the majority of mRNA from the BamHI-H region is composed of unspliced transcripts. Another cDNA library was constructed from MDV strain RBIB-infected cells by the group that identified the 1.8 gene family (Peng et al., 1992). Three classes of cDNA (four cDNAs) from the long inverted repeat region are identified, and all are transcribed rightwardly. Class I is represented by two cDNAs, one is a nonspliced 1.69-transcript which is completely located in BamHI-H fragment and contains two copies of the 132-bp repeat sequence; while another one is a spliced 1.5-kb transcript which share the same initiation site with the nonspliced 1.69-kb cDNA, but spanning BamHI-H and I2 region. 44 Class II represents the 1.9-kb spliced transcript which shares the same splice acceptors and 3’ ends as the 1.5-kb class I cDNA, but differs at the 5’ end and at splice donors. Class III cDNAs are represented by the 2.2-kb cDNA which is derived from a nonspliced mRNA spanning the BamHI-H and BamHI-I2 fragments of MDV DNA. Sequence analysis demonstrats two potential open reading frames in each of the cDNAs. According to the authors, two cDNAs of class I, the nonspliced 1.69-kb transcript and the spliced 1.5-kb transcript, belong to the 1.8 -kb BamHI-H gene family. The putative 63-amino-acid protein encoded by the first ORF (ORF-A) in the 1.69-kb cDNA and the putative 75- arnino-acid protein encoded by the first ORF (ORF-B) in the 1.5-kb cDNA shows limited homology with the mouse T-cell lymphoma (T LM) oncogene and the fer/fps family of kinase-related transforming proteins (Peng et al., 1992). Recently, Peng et al. (1994 a) reported that the 1.69-kb and 1.5-kb cDNA can induce prolonged proliferation and reduced serum dependence of primary CEF cells in transfection assays. In addition, Peng et al. (1994 b) synthesized a polypeptide corresponding to the C-terrninus of ORF-A in the 1.69-kb cDNA to raise antibody. A 7-kDa protein is detected in CEF cells infected with MDV oncogenic strain RBIB and MDV—induced tumor cell line, MSB-l. As described above, a cDNA library constructed by Hong and Coussens (1994) also identified two spliced cDNAs (C1 and C2) which span BamHl-H and -I2 fragments. These two cDNAs are derived from IE transcripts. One cDNA (C2) has the same splice donor and acceptor as the 1.9-kb cDNA reported by Peng et al. (1992). Therefore, there are at least three cDNAs, which utilize different splice donors but share a common splice acceptor site, in the BamHI-H and I2 fragment of MDV. Each cDNA contains only two small ORFS. Protein Similarity searches of putative C1 and C2 encoded polypeptide 45 against the Swiss-protein data base reveal only limited homology with a zinc-finger protein and myc proto-oncogene. However, these similarities are not in conservative or functional regions and, therefore, are not considered as significant. Using antisera raised against fusion protein of two putative ORFs (ORFla of C1, ORFlb of C2), a 14 kDa phosphoprotein is detected in MDV (oncogenic and attenuated) lytically infected cells, and MDV transformed cell lines (Hong and Coussens, 1994). Since the protein is encoded by an ORF which spans a 2.3-kb intron, including the 132 bp repeat, the detection of this 14 kDa protein in attenuated strains suggests that removing the 132-bp repeat by splicing does not affect protein expression. However, subtle changes in amino acid content coincident with attenuation and expansion of the 132-bp repeat region cannot be ruled out. Interestingly, three proteins (ppl4, pp38, and meq) expressed in MDV-transformed cell lines are all located in the repeat region of MDV. These data support the posible association of this region with MDV tumorigenic potential. Among them, only meq is a partial homolog of jun/fos oncogene. Both pp14 and pp38 are MDV unique, serotype-1 specific antigens. Therefore, whether MDV encodes a specific oncogene, or pp38 and pp14 play indirect roles in tumorigenicity, such as stimulation of other cellular factors, will be important issues for understanding MDV oncogenicity. Furthermore, these proteins are detected in MDV transformed cell lines, whether they are expressed in MDV tumors has not yet been tested. The mechanism by which latent infection changes to neoplastic proliferation needs to be investigated further. To understand MDV pathogenesis and tumorigenicity, we are interested in studying gene expression and regulation, especially genes in repeat regions of the MDV genome. The subject of this dissertation is focused on: 1) identification and localization 46 of an IE gene in the IRL region, 2) identification of the gene product encoded by this IE gene and characterization of the properties of the protein. Charter 11 Identification of an Immediate-early Gene in the Marek’s disease Virus Long Internal Repeat Region Which encodes a unique 14 kDa polypeptide Yu Hong and Paul M. Coussens Molecular Virology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824 J.Virol. 68:3593-3503, (1994) 47 ABSTRACT Marek’s Disease Virus (MDV) is an oncogenic avian herpesvirus whose genomic Structure is similar to herpes simplex virus and varicella-zoster virus. Repeat regions of the MDV genome have been intensively investigated due to a potential relationship to MDV oncogenicity and abundant expression of immediate-early transcripts. In this report, a 1.6 kb immediate-early transcript was localized to the BamHI-I2 region by Northern hybridization analysis. With cDNA cloning and sequencing, two cDNAs of 1.4 kb (C1) and 1.35 kb (C2) were identified. Both cDNAs are derived from spliced mRNAs spanning the BamHI-H and 12 fragments. C1 and C2 use the same splice acceptors and 3’ ends, but differ at their 5’ ends and utilize different splice donors. The upstream promoter-enhancer region of Cl cDNA has been defined as a bidirectional regulatory region shared by the MDV pp38 gene. Sequencing analysis shows two small open reading frames (ORF) within each cDNA (ORFla and ORF2 in C1, ORFlb and ORF2 in C2). Potential ORFs of the sequence have no significant homology with any known protein in the Swiss-Protein data base. DNA fragments encoding ORF 1a and ORFlb were cloned into pGEX-3X vectors to produce GST-fusion proteins and induce antisera. In Western blot analysis of MDV infected cell lysates, a 14 kDa polypeptide was identified by antisera against both ORF 1a and ORFlb. This 14 kDa protein is expressed in cells which are lytically infected with MDV strains GA, Mdll passage 16 (oncogenic) and Mdll passage 83 (attenuated), as well as in the MDV latently infected and transformed MSB-l cell line. 48 INTRODUCTION Marek’s disease virus (MDV) is a highly cell-associated avian herpesvirus which induces T-cell lymphomas and peripheral nerve demyelination (Marek’s disease) in chickens (Calnek, 1985; Churchill and Biggs, 1967). Although MDV has been effectively controlled by vaccination with an antigenically related but apathogenic herpesvirus of turkeys (HVT), the mechanism of MDV tumorigenicity is still unknown. Recent efforts in this regard have been focused on identification of genes which may be responsible for, or related to malignant transformation by MDV. MDV was classified as a gamma-herpesvirus, based on its biological characterization (Roizman and Sears, 1991). However, the overall genome structure of MDV is more similar to human alpha-herpesviruses (e.g., varicella-zoster virus and herpes simplex virus), consisting of unique long (UL) and unique short (Us) regions, each bounded by a set of inverted repeats (T RL, IRL, IRs and TR.) (Cebrian et al., 1982; Fukuchi et al.,1984). As with other herpesviruses, MDV gene expression is coordinately regulated and sequentially ordered in a cascade fashion (Maray et al., 1988; Nazerian and Lee, 1976; Schat et al., 1989). Three major kinetic classes of genes are expressed as immediate early (IE), early (E), and late (L) genes. IE genes are expressed immediately upon infection and do not require de novo viral protein synthesis. Characterization of RNA transcripts isolated from MDV infected cells treated with cycloheximide (CHX) indicates that transcripts from IE genes are clustered in repeat regions similar to the locations of other herpesvirus IE genes (Roizman and Sears, 1991; Schat et al., 1989). In addition, MDV IE transcripts can be detected not only in lytically infected cells but also in transformed cell lines in which MDV infection is considered latent. To understand 49 50 MDV gene expression and regulation during MDV tumor induction, we are interested in examining IE transcripts in MDV repeat regions and investigation of related IE gene products. Repeat regions of the MDV genome have been intensively investigated due to speculation that transcripts within this region may be important in MDV induced tumorigenicity. Several investigators have demonstrated that serial in vitro passage of virulent MDV in primary chicken embryo fibroblast cells results in loss of MDV tumorigenicity. This attenuation was found to correlate with amplification of a specific 132 bp repeat sequence found within the MDV TRL and IRL (BamHI-H and D, respectively) (Bradley et al. 1989, a; b; Chen and Velicer, 1991; Fukuchi et al., 1985; Maotani et al., 1986; Silva and Witter, 1985). Bradley et al. reported that a 1.8 kb gene family is transcribed rightwardly from the expanded region of the BamHI-H fragment. ' According to Bradley et al. these transcripts are expressed only in oncogenic MDV, but absent or truncated in attenuated MDV. Antisense inhibition of proliferation of a MDV- derived lymphoblastoid cell line using an oligonucleotide complementary to the putative splice donor sequence of the 1.8 kb gene family supports the hypothesis of an association between this gene family and the tumorigenic potential of MDV (Kawamura et al., 1991). Recently, Peng et al. (1992) further developed a cDNA library from this BamHI-H gene family and identified four cDNA clones. While two cDNAs of 1.9 and 2.2 kb, were reported as nonspliced transcripts, two other cDNAs, 1.5 and 1.9 kb were recognized as single spliced transcripts spanning the BamHI-H and BamHI-l2 fragments of MDV DNA. Protein products associated with these cDNAs or other transcripts from the BamHI-H gene family have not been identified. Other transcripts that are initiated or terminated 51 within or near the 132 bp repeat region have also been described (Chen and Velicer, 1991). Recently, a 38 kDa phosphoprotein expressed both in MDV lytically infected cells and transformed lymphoblastoid tumor cell lines was localized to the BamHI-H region and is transcribed in a leftward direction (Chen at al., 1992; Cui et al., 1991). A basic- leucine zipper gene, designated meq, (identified as a homolog of fos/jun oncogene family), has been mapped to the rightward region of BamHI-I2 fragment, within the MDV IRL. By using antiserum against a synthetic peptide deduced from the meq DNA sequence, a 40 kDa protein was detected in MDV transformed lymphoblastoid cell lines but not in cells lytically infected with MDV strain GA (Jones et al., 1992). In this study, we report analysis of an IE gene localized within the MDV IRL. By cDNA cloning and sequencing, we have determined that distinct transcripts are derived from this same gene by altered splicing patterns. In all cases examined, the 132 bp repeat region is not present in mature mRNA species. Computer sequence analysis revealed only small open reading frames within our cDNA transcripts, consistent with previous report (Peng et al., 1992). Using antisera raised against fusion proteins, we demonstrate that a 14 kDa protein encoded by two small ORFs in these transcripts is expressed in cells lytically infected with both oncogenic and attenuated MDV, as well as in cells latently infected and transformed by MDV. Our results have important implications regarding the role of proteins encoded within the MDV IRL in MDV-induced tumorigenicity. MATERIALS AND METHODS Cell culture and virus. Duck embryo fibroblast (DEF) cells and chicken embryo fibroblast (CEF) cells were prepared, maintained, and infected with MDV as described previously by Glaubiger et al. (1983). Cell-associated MDV strain GA passage 7, Mdll passage 16 , designated as a low passage (LP) and passage 83, designated as a high passage (HP) used in this study were obtained from the Avian Disease and Oncology Laboratory (ADOL), US. Department of Agriculture, East Lansing, ML, and have been described (Wilson et al., 1994). To obtain immediate early RNA transcripts, 100 ug/ml cycloheximide (CHX) was added at the time of infection. RNA was extracted 16 to 24 hours post infection/CHX treatment. Early RNA was obtained by treating cells with 100 ug/ml phosphonoacetic acid (PAA) at the time of infection. Fresh medium containing 100 ug/ml PAA was added to infected and control cultures after 24 hours. RNA was extracted after an additional 24 hours incubation in PAA. The MSB-l cell line (a producer, expression cell line) (Akiyama and Kato, 1974) was used as a representative of MDV-induced lymphoblastoid cell line and was cultured in Leibovitz L15-McCoy 5A medium supplemented with 10% fetal calf serum at 41°C, in a humidified atmosphere containing 5% CO2 . RNA isolation and Northern blot analysis. Total cellular RNA was isolated from uninfected and MDV-infected DEF cells using the guanidinium-phenol:chloroform method (Chomczynski and Sacchi, 1987). Polyadenylated [poly(A)”] mRNAs were purified from total RNA using the polyATract mRNA kit (Promega, Madison, WI) according to the manufacturer’s specifications. 52 53 For Northern blot analysis, 0.5 ug poly(A)" mRNA was loaded per well, electrophoresed through 1.2% formaldehyde/agarose gels and transferred to Hybond-N nylon membranes (Amersham corp., Arlington Heights, IL) essentially as described (Sambrook et al., 1989). or-nP-labeled probes were generated using a random primed labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) as recommended by the manufacturer. Hybridization was performed in high stringency conditions (50% formamide, 3X SSC, 5% dextran sulfate, 50 mM phosphate buffer, 5X denhardt’s, 0.1% SDS and 100 ug/ml salmon sperm DNA, at 42° C for 12-16 hs ). Transcription sizes were determined by comparison with a 0.24 to 9.5 kb RNA ladder (Bethesda Research Labs, Bethesda, MD) run on the same gel. RNA blots were stripped of probe DNA and rehybridized with a chicken B—actin gene probe (Cleveland et al., 1980). Intensity of RNA bands on autoradiographies were analyzed on a FB910 Densitometer using Zeineh 1-D Videophoresis 11 software (FisherBiotech). cDNA library construction, screening and Southern blot analysis. To enrich for cDNAs derived from IE genes, poly(A)" mRNAs isolated from MDV infected DEF cells, treated with cycloheximide, were utilized. Construction of a cDNA library was performed as described by Hu et al. (Hu et al., 1992). Briefly, plasmid pBluscript II KS+ was digested with the restriction enzyme EcoRV to generate blunt ends. Oligo (dT) tails were added to the blunt ends with T4 terminal transferase. After removal of the oligo(dT) tail at one end by digesting with SmaI, poly(A)" mRNA was annealed to the Oligo (dT) tail of the remaining end and cDNA was synthesized directly along the vector-primer. Following digestion of RNA and second strand cDNA synthesis, blunt 54 ends were generated by treating with T4 polymerase and religated to generate recombinant cDNA clones. cDNA clones were transformed into E.coli DHlOB cells (Bethesda Research Labs, Bethesda, MD) and the library was screened by in situ hybridization as described (Sambrook, et al., 1989). Positive colonies were isolated and expanded for analysis of plasmid DNA by Southern hybridization. Primer extension. Primer extension studies were performed essentially as described previously (Ausubel et al., 1991). Three oligonucleotide primers were designed based on cDNA sequences described in the text. Oligonucleotide primer 1 is: 5’-AGGAAATATATCGGG- GTACGGCCGT-3’; oligonucleotide primer 2a is: 5’-ATGGAAAGTGGGTCCGCAGTC- AATG-3’; and primer 2b is 5’-GTCAATGCATCCGGGGTCGTTCCCA-3’. The primers were 5’- end labeled with [732p] ATP and annealed to 30 ug total RNA isolated from uninfected and MDV-infected DEF cells for 90 minutes at 65°C. After cooling at room temperature for 30 minutes, reverse transcription was carried out at 42°C for one hour. Reactions were terminated by phenol-chloroforrn extraction and ethanol precipitation. Precipitated nucleic acids were resuspended and analyzed on 9% polyacrylamide/7 M urea sequencing gels. Viral genomic DNA and cDNA clone sequencing. Viral genomic DNA sequencing was initially performed on a 2.3 kb BamHI-XbaI subfragment of the BamHI-I2 fragment from an MDV strain GA BamHI library (kindly provided by M.Nonoyama). This subfragment was subcloned into pUC18 and used to 55 generate overlapping deletions using EonII and S1 nucleases. At the same time, Sau3A and Tan libraries of the 2.3 kb BamHI-Xbal fragment were constructed in M13 vectors. Nucleotide sequences of both strands were obtained by the dideoxy-chain termination method (Sanger et al., 1977) using the Sequenase enzyme (United States Biochemical Corp., Cleveland, OH). Both forward and reverse 17-mer universal primers were used to sequence the ends of subcloned DNA. For acquiring the junction sequence of BamHI- I2 and BamHI-H, a HindIH-Xbal subfragment was isolated from the EcoRI-F fragment of a MDV strain GA EcoRI library (kindly provided by R. F. Silva, ADOL, East lansing, MI). This subfragment was cloned into pUC18 and sequenced in both directions as described above. For cDN A clone sequencing, the dideoxy-chain termination method was conducted as described for viral genomic DNA clones. T7 forward and KS reverse primers were used for the end sequences of each cDNA. Computer analysis of DNA sequence. Nucleotide sequences were analyzed using the Genepro program (Riverside Scientific Enterprises, Seattle,WA) and MacVector 3.5 (International Biotechnologies, Inc., New Haven, CT). Amino acid sequences of the putative polypeptide encoded by open reading frames (ORF) were searched against the protein sequence data deposited within Swiss-Protein data bases using the Genetics Computer Group program FASTA from the University of Wisconsin (Devereux and Smithies, 1984). GenBank accession number. The nucleotide sequences of the C1 and C2 cDNA reported in this paper have 56 been given GenBank accession numbers L26394 and L26395, respectively. Expression of GST fusion proteins and antibody production. The vector system used to express C1 and C2 ORFS in E .coli is plasmid pGEX-3X (Pharmacia, Alameda, CA) which contains a glutathione S-transferase (GST) gene under the control of an isopropylthiogalactopyranoside (IPTG)-inducible tac promoter. DNA fragments containing ORF 1a of C1 cDNA and ORFlb of C2 cDNA were ligated into the 3’ end of the GST ORF as in-frame insertions. The respective GST fusion proteins were purified using glutathione-sepharose 4B (pharmacia, Alameda, CA) as recommended by the manufacturer. Purified fusion proteins were applied to a 0.1% sodium dodecyl sulfate (SDS)-10% polyacrylamide gel. Positions of the fusion proteins were identified by Coomassie brilliant blue staining. Sizes of fusion proteins were estimated by comparison with protein MW standards run on the same gel. New Zealand White rabbits were initially immunized with 400-500 ug of purified fusion proteins in Titer-Max adjuvant (Cthx Co., Norcross, GA). The rabbits were boosted with the same amount of protein in Titer-Max every four weeks and bled ten days after final boost. The immune sera were preabsorbed with GST carrier protein to remove cross-reacting antibody. Antibody titers were determined by Enzyme-Linked Immunosorbent Assay (ELISA) as described (Sjogren and Jeansson, 1990). Western immunoblot analysis. Cultured cells were lysed with triple-detergent lysis buffer (Sambrook et al., 1989) and separated on 12.5% or 15% polyacrylamide gels containing 0.1% SDS. Proteins were 57 electrophoretically transferred to nitrocellulose filters. Immune detections were performed using an Amersham ECLTM western blot kit according to the manufacturer’s specifications. The filters were blocked with 5% nonfat milk and probed with anti-ORF 1a and ORF 1b sera at 1:100 dilution. Donkey anti-rabbit immunoglobulin conjugated with horseradish peroxidase was used as second antibody. Proteins were detected by luminescence reagent and exposed to X-ray film. Protein sizes were estimated with reference to prestained protein MW standards (Bio-RAD, Richmond, CA) run on each gel. RESULTS Detection of an immediate early transcript within BamHI-I2 region. Previous studies have Shown that MDV IE genes are mainly located in repeat regions, but there have been conflicting reports on the sizes and numbers of transcripts from these regions (Bradley et al., 1989, a; b; Chen and Velicer, 1991; Maray et al., 1988; Peng et al., 1992; Schat et al., 1989). To avoid problems associated with unprocessed precursor transcripts and introns, poly(A)” mRNAs isolated from uninfected, MDV-infected DEF cells, and infected DEF cells treated with CHX or PAA were purified for Northern blot analysis. Initial studies revealed that the BamHI-I2 fragment hybridized to a 1.6 kb transcript. This 1.6 kb BamHI-I2 transcript was abundantly expressed in cycloheximide treated cells (enhanced 3.5 fold over untreated MDV infected cells normalized relative to a chicken actin probe), and is identified in PAA treated cells after longer exposure (data not shown), suggesting it is expressed with immediate-early kinetics (Figure 1A). To verify location of this 1.6 kb IE transcript, various subfragments of the 5.1 kb BamHI-I2 fragment were utilized as probes in Northern hybridization. These studies confirmed localization of the 1.6 kb transcript to the leftward BamHI-Xbal region of BamHI-Iz, juxtaposed to the right region of the BamHI-H fragment (Figure 18). Thus, this 1.6 kb transcript may be related or identical to the 1.8 kb transcripts which hybridize to the BamHI-H fragment (Bradley et al., 1989 b), and is also consistent with the 1.7 kb transcript of BamHI-I2 identified by Peng et al. (1992). Smaller RNA species (1.0 kb, 0.5 kb and 0.4 kb) expressed with immediate-early kinetics were also observed in Northem hybridizations along with the 1.6 kb transcript (Figure 1A, and 18). As described later 58 59 in this report, smaller RNA species are most likely derived from undegraded intron RNAS Viral genomic DNA sequence of the BamHI-Xbal region from a MDV BamHI-I2 fragment. Data from Northern blot hybridization indicated that an immediate-early transcript was encoded within the leftward BamHI-Xbal region of the BamHI-I2 fragment. This region of MDV DNA was subcloned and completely sequenced as described in Materials and Methods. Despite abundant transcription in infected cells, no large open reading frame (ORF) was found in either direction of this region, though several smaller (<100 amino acids) ORFs were identified (data not shown). To confirm the accuracy of sequence data and acquire the junction sequence between the BamHI-H and BamHI-I2 fragments, a HindIII-Xbal subclone derived from the EcoRI-F fragment was also sequenced. As before, no continuous large open reading frames were observed (data not shown). Two possibilities were considered to explain a lack of ORFS within an abundantly transcribed region: (i) transcripts from the BamHI-I2 region may function similar to latency associated transcripts (LATS) in HSV-1 infected neurons (Rock et al., 1987; Spivack and Fraser, 1987), or (ii) RN As encoded within this region are extensively spliced. Data from Peng et al. (1992), Bradley et al. (1989 a; b), and Chen and Velicer ( 1991), suggested the later case was more likely. However, a protein product from this region had not been identified by any of these groups. Isolation of cDNA and cDNA sequence analysis. 60 In an attempt to distinguish between the possibilities described above, a cDNA library was constructed using poly(A)+ mRNA from DEF cells infected with MDV strain GA and treated with cycloheximide. In construction of this library, we considered both the reported sequence of BamHI-H and our own sequence of the MDV BamHI-I2 fragment. Both sequences contain many stretches of high AT content. Using oligo dT to prime cDNA synthesis in this area could lead to multiple starts within transcripts. For this reason, we chose a modified Okayama-Berg procedure (Hu et al., 1992) as described in Materials and Methods. This procedure is less likely to support priming within transcripts. The resulting cDNA library was screened with a 5.1 kb BamHI-I2 fragment by in situ hybridization. Three cDNA clones (C1 through C3) were isolated. Insert sizes for these clones were estimated to be 1.4 kb for Cl, 1.35 kb for C2, and 0.7 kb for C3 by Southern blot analysis. The three clones were sequenced on both strands, and compared to MDV genomic sequence data. C1 and C2 were identified as derived from different spliced RNAs and C3 was a partial cDN A clone. C1 and C2 transcripts both extend from BamHI- H into BamHI-I2 in a rightward direction (Figure 2). Cl and C2 start from different 5’ initiators, have different splice donor sites within the BamHI-H region, but share the same splice acceptor site and 3’ end within the BamHI-12 region (Figure 2). The complete nucleotide sequences of Cl and C2 are presented in Figure 3. The C1 cDNA clone has an insertion of 1295 base pairs before the poly(A) tail. The 5’ 38 bp of C1 are identical to nucleotides 701 to 738 of a previously published BamHI-H sequence (Bradley et al., 1989, b). Beginning with nucleotide 39, C1 sequence was aligned with the BamHI-I2 genomic sequence from nucleotide 224 to 1489. Sequence alignment revealed the presence of a 2.3 kb intron, which encompass the 132 bp repeat 61 region. A potential TATA sequence was found 42 bp upstream of the 5’ end of C1 and a putative polyadenylation signal sequence (ATTAAA) is located 17 bp upstream of the 3’ end of our C1 cDNA clone. This spliced transcript utilized a splice donor, GAAGGA, beginning at nucleotide 739 of BamHI-H sequence and a splice acceptor, ATCGTTGCAG, at nucleotides 214 to 223 of the BamHI-Xbal subfragment of BamHI- 12. The C2 cDNA contains a 1275 bp insert. The beginning 18 base pairs were identical to nucleotide 2071 to 2088 of the BamHI-H sequence (Bradley et al., 1989, b). The remaining sequence of C2 was aligned to the BamHI-I2 fragment, which is identical to C1 cDNA. C2 cDNA initiates 512 bp downstream of the 132 bp repeat region and ends at the-same 3’ site as C1 cDNA. The C2 transcript thus contains a 1.0 kb intron. This intron employs a splice donor, GTATGC, located at nucleotides 2088 to 2093 of the BamHI-H fragment, and the same splice acceptor used by the C1 transcript C2 cDNA therefore appears to be analogous to cDNA 3 identified by Peng et al. (1992). Despite significant attempts, we were unable to identify any cDNA clones which initiated within the 132 bp region of MDV BamHI-H. Transcriptional mapping of Cl and C2 cDNA. In order to determine the precise 5’ ends of our cDNA clones, primer extension experiments were conducted. Three oligonucleotide primers were designed, based on our cDNA sequence. The position and direction of these primers are schematically depicted in Figure 2. The first primer (P1), 5’ AGGAAATATATCGGGGTACGGCCGT 3’, was complementary to C1 cDNA sequence positions 14-38, which is just upstream of the 62 intron. The 42 bp extension observed in reactions with P1 and RNA isolated from MDV GA infected cells (data not shown) indicated the C1 transcript starts 13 base pairs downstream of a putative TATA sequence within BamHI-H. This location is consistent with the start site of the 1.8 kb gene family described by Bradley et al. (1989, b). An ATG translation start site was found 37 bp down stream of the putative TATA sequence. However, the optimal context surrounding AUG (A at -3 position, G at +4 position) was not found (Kozak, 1991). The TATA sequence, upstream of the C1 start site, belongs to a putative promoter-enhancer region containing a variety of potential transcription regulatory elements (Bradley et al., 1989, b; Cui et al., 1991). This promoter-enhancer region has been cloned into a chloramphenicol acetyl transferase (CAT) reporter plasmid in both orientations in our laboratory. Transient expression assays indicate that this region is, in fact, a bidirectional promoter activated by infection with MDV (Abujoub and Coussens, unpublished observations). The second primer (P2a) 5’ ATGGAAAGTGGGTCCGCAGTCAATG 3’ is complementary to C2 cDNA sequence position 29 to 53, which is 10 base pairs downstream of the splice acceptor. The third primer (P2b) 5’ GTCAATGCATCCGGGGTCGTTCCCA 3’ is complementary to C2 cDNA nucleotides 9 to 33 and spans the exon/intron junction. Primer extension bands of 135 bp, 99 bp, 77 bp and 54 bp in length were observed using P2a and P2b primers hybridized to RNAs isolated from MDV GA infected cells (data not shown). The 77 bp band matched the C1 initiation site. It is difficult to determine which extension product represents the actual initiation site of C2 RNA, as we can not exclude the presence of other spliced transcripts using the same splice acceptor as C1 and C2 or presence of unspliced transcript 63 precursors. To confirm the origin from which our cDNA clones were derived, Northern hybridization was conducted using the C1 cDNA insert as a probe. A 1.6 kb transcript was detected in poly(A)" mRNA isolated from MDV strain GA infected cells and enhanced in mRNA from infected cells treated with cycloheximide (Figure 4, lanes 2 and 3). No hybridization to uninfected cell mRNA was observed (Figure 4, lane 1). This result indicates that the cDNA was derived from the 1.6 kb immediate early RNA previously detected by a BamHI-I2 fragment probe. Consistent with sequencing and primer extension data, the C1 cDNA is 1.3 kb in length, without a poly(A) tail. Interestingly, minor transcripts of 1.0 kb, 0.5 kb and 0.4 kb observed in Northern hybridizations probed with the BamHI-Xbal subfragment of BamHI-I2 were not detected when'using the C1 cDNA insert as a probe (Figure 4). This result suggests that these smaller RNA species are most likely derived from undegraded intron RNAs. In support of this observation, unspliced RNA precursors (3.3 kb and 2.6 kb) as well as the 2.3 kb intron are readily detected in Northern blots of total RNA from MDV infected cells (data not shown). Analysis of potential open reading frames in MDV BamHI-I2 related cDNAs. Nucleotide sequences of C1 and C2 cDNA were analyzed using the Genepro, Macvector, and GCG programs. Translation of the cDNAs in all six reading frames identified two small potential open reading frames in both C1 and C2. In C1 cDNA, ORFla could encode 83 amino acid residues, resulting in a putative protein of 9,307 Da. ORFla has two potential casein kinase consensus phosphorylation sequences (amino acid 5 and 66), four potential histone kinase consensus phosphorylation sequences (amino acid 64 9, 42, 49 and 63), and three N-glycosylation sites (amino acid 3, 34 and 64). ORF2 could encode a 107 amino acid protein of 12,332 Da. ORF2 has four sites of histone kinase consensus phosphorylation sequence (position 7, 43, 44 and 71) and one potential site of N-glycosylation ( position 63). C2 cDNA could encode two ORFS of 76 (ORF 1b) amino acids and 107(ORF2) amino acids , respectively. The predicted size of an ORFlb encoded protein is 8,381 Da with one potential casein kinase phosphorylation consensus sequence (position 59), three potential histone kinase phosphorylation sequences (position 35, 42 and 56) and two potential N-glycosylation sites (position 27 and 57). ORF2 of C2 cDNA is the same as ORF2 in C1(Figure 5) and corresponds to ORF-F reported by Peng et al (1992). All potential ORFS were compared with protein sequence data deposited within the Swiss-Protein data base without finding highly significant homology. However, limited homologies were found with mouse zinc finger protein ZFP-27(mkr4) and myc proto-oncogene protein. ORFla has a 29% identity to mouse zinc finger protein ZFP-27 (mkr4) (21 of 78 amino acids overlapping) and ORFlb has a 28% identity to ZFP-27 (14 of 50 amino acids overlapping) (Chowdhury et al., 1988). The N-tenninus of ORF2 has a 30% homology with the myc proto-oncogene exon 3 (19 of 66 amino acids overlapping) (van Beneden et al., 1993) (data not Shown). A lack of continuous open reading frames within our cDNA sequences is consistent with results of Peng et al. (1992) and raises questions regarding the function of these abundantly expressed spliced transcripts. Identification of a 14 kDa Protein of ORFla and ORFlb gene products. Previous analyses of transcripts encoded by the BamHI-H and I2 regions of MDV DNA have failed to determine if any protein product is produced by these extensively 65 spliced RNAS. Given the potential importance of this region in viral oncogenicity, identification of a protein product associated with BamHI-H and I2 transcripts would be of considerable interest. To generate adequate amounts of protein for antibody production, DNA fragments which contain ORFla (83 amino acids) of C1 and ORFlb (76 amino acids) of C2 were cloned into the 3’ end of the glutathione S-transferase (GST) gene of pGEX-3X vectors (in frame insertions) and induced by IPT G to express fusion proteins in E.coli cells. Two fusion proteins GST-ORFla and GST-ORFlb were generated and purified by glutathione-affinity chromatography. Antibodies against these proteins were raised in rabbits as described in Materials and Methods. The immune antisera were preabsorbed with purified vector protein (GST) to remove cross-reacting antibodies. After the second boost, antisera titer reached a 1:1000 dilution as determined by ELISA test (data not shown). Western blot analysis was conducted to identify putative protein products of the C1 and C2 clones. Both ORF 1a and ORF 1b antisera detected a 14 kDa protein in CEF infected with MDV strain GA , but not in control CEF cells (Figure 6 A, B, lane 1 and 2). As described previously, ORFla spans a 2.3 kb intron which includes the 132 bp repeat region amplified in the genome of attenuated MDV. To determine whether ORFla and ORFlb gene products, identified in this study, incur any structural changes when the 132 bp region expands in attenuated MDV, we employed Md11p16 as a representative of low passage (oncogenic) virus, and Md11p83 as a high passage (attenuated) virus. Both fusion protein antisera detected a 14 kDa protein in lysates of cells infected with low and high passage Mdl 1(Figure 6 A, B, Lane 3 and 4). Since these gene products originate from the repeat region, which is speculated to be associated with MDV tumorigenicity, 66 we further explored the gene products using the MDV lymphoblastoid cell line MSB-l. A 14 kDa protein was readily detected by both ORFla and ORFlb antisera in the MSB-l tumor cell line without IUdR induction (Figure 6 A, B , lane 5). These results suggest that ORFla and ORFlb indeed encode polypeptides in MDV infected cells. Furthermore, both oncogenic and attenuated strains of MDV produce similar amounts of this polypeptide. DISCUSSION Reports on size, number, and direction of transcripts derived from MDV repeat segments (IRL and TR), especially around the 132 bp expansion region, have been well documented (Bradley et al., 1989, a; b; Chen and Velicer, 1991; Maray et al., 1988; Schat et al., 1989). Many reports offer confusing and often conflicting data regarding initiation, splicing, and termination. Transcripts derived from the BamHI-H fragment may comprise a 1.8 kb gene family, including three transcripts of 1.8, 3.0, and 3.8 kb as reported by Bradley et al. (1989, a; b). Four groups of transcripts are postulated to initiate or terminate within or near the 132 bp repeats (Chen and Velicer, 1991). Three transcripts of 4.1, 3.0 and 1.9 kb in BamHI-H and six transcripts from 1.8 to 5.9 kb in BarnHI-I2 were reported by Schat et al. (1989). Two transcripts (5 and 2.5 kb) in BamHI-H and similar sized transcripts in BamHI-I2 were reported by Maray et al. (1988). Among these transcripts, the 1.9 kb in BamHI-H and 1.8 kb in BamHI-I2 identified by Schat et al. (1989), as well as the 5 kb and 2.5 kb transcripts in BamHI-H reported by Maray et al. (1988) were cataloged as immediately-early gene transcripts. Here we report that a 1.6 kb major transcript which hybridized to BamHI-Iz, is highly expressed in MDV strain GA infected and CHX treated DEF cells. Accumulation of this 1.6 kb transcript in CHX treated cells suggests it is expressed with immediate-early gene kinetics. The decrease of this transcript in PAA treated cells is expected, because inhibition of viral replication by PAA can limit new viral infection and subsequently reduce accumulation of viral transcripts in infected cell cultures. Though CHX would also prevent virus spread, IE gene transcription is not limited by feedback inhibition as it would be in PAA treated or untreated cells. Complete nucleotide sequence analysis of 67 68 the 2.3 kb BamHI-Xbal fragment of MDV BamHI- I2 and its positioning relative to previously published MDV BamHI-H sequences (Bradley et al.,1989, b) revealed no large open reading frames in over 5 kb of continual viral genome. Several smaller ORFs (< 100 amino acids) which could comprise exons of a spliced gene were identified within the BamHI-I2 sequence. In support of this possibility, spliced IE mRNAs have been identified in other herpesviruses. ICPO of human simplex virus (HSV), IE1 and IE2 of cytomegalovirus (CMV), BZLF] and BRLFl of Epstein-Barr virus (EBV), and IE RNA 1 and IE RNA 2 of Bovine herpesvirus 4 (BHV-4) (Lau et al., 1992; Leib et al., 1989; Stinski et al., 1991; van Santen, 1993) all result from spliced transcripts. In CMV, exons 1, 2 and 3 of IE1 can be ligated onto the IE2 by alternate splicing to produce different mRNAs (Stinski et al., 1991). A variant form of splicing which omits the middle exon of BZLFl to produce a shortened protein is also described in EBV (Lau et al., 1992). It was therefore considered likely that the 1.6 kb transcript identified in our Northern blots was spliced. Given the confusing and conflicting reports regarding transcription in the IRL region of MDV, we analyzed the available sequence data for regions which may interfere with cDNA synthesis and S1 nuclease mapping. The sequence of BamHI-H from an internal EcoRI site to the right end BamHI site has been reported (Bradley et al., 1989, b). Within this sequence, multiple stretches of high AT content DNA exist. For example, nucleotides 1701 to 1900 contain two tracts , one with 19 A residues and another with 13 T residues. These sections could dramatically affect Sl mapping and act as sites for oligo dT initiation during cDNA synthesis. In our poly(A)+ mRNA Northern blots, smaller species of RNA (1.0, 0.5 and 0.4 kb) hybridize to the BamHI-XbaI fragment but are not 69 observed when a cDNA insert is used as probe. These results suggest that the small RNAs may be derived from undegraded or partially degraded intron RNA which can contaminate poly(A)*mRNA, perhaps due to specific AU rich sequences. In Northern blots of total infected-cell RNA, additional bands of 3.3 and 2.6 kb are visible when the RNA is probed with an intact I2 fragment Interestingly, these larger species correspond to the predicted sizes of unspliced RNAs from which our 1.6 kb transcript is likely derived. Given these facts, we chose to utilize an oligo dT tailed plasmid for initiation of cDNA synthesis. In this system, the poly (dT) primer extends from a double-stranded plasmid end. Thus, annealing to internal poly(A) tracts is thermodynamically less favored than annealing to terminal poly(A) tracts (e. g. internal annealing could only occur if the RNA bent to accommodate the complimentary plasmid strand). Poly(A)+ mRNA was also used to reduce the contribution of unspliced RNA precursors and spliced introns. In the analysis of over 3000 clones from this library, we detected no transcripts which initiated within the 132 bp repeat region. As this research was in process, Peng et al. (1992) reported a cDNA library constructed from MDV strain RBIB infected cells. Three classes of cDNA were detected from the long inverted repeat region and all were transcribed rightwardly. Among them, two cDNAs are derived from spliced mRNAs. The class II 1.9 kb cDNA (no.3 cDNA) has the same splice donor and acceptor sites as our C2 cDNA, but is 663 bp longer at the 5’ end and about 30 bp shorter at the 3’ end. The class I 1.5 kb cDNA (no. 4 cDNA) shares the same splice acceptor as the class II 1.9 kb cDNA, as well as our C1 and C2 cDNA, but uses a different splice donor site which is 250 bp downstream of our C1 cDNA splice donor. Thus, there are at least three cDNAs, which utilize different splice 70 donors but share a common splice acceptor site, present in the BamHI-H and I2 fragments of MDV. Two unspliced mRNAs (1.69 kb cDNA 1 and 2.2 kb cDNA 6) from the BamHI H and I2 regions were also reported by Peng et al. Previously, Iwata et a1. (1992) reported construction of a cDNA library derived from MDV strain Md5 infected cells. Twelve clones localized to the BamHI-H fragment, contained 1.1 to 1.8 kb insertions, and all were derived from unspliced mRNAs transcribed in a rightward direction. Iwata et al., therefore, concluded that the majority of mRNA from the BamHI-H region is composed of unspliced transcripts. Chen and Velicer (1991) also reported four groups of unspliced transcripts which initiated or terminated in the BamHI-H 132 repeat region in a bidirectional manner. On the basis of results presented in this report and extensive analysis of cDNA clones, we believe that many of these cDN A result from priming within transcripts and within relatively stable introns containing the 132 bp repeats. The 5’ end of Cl has been defined by a combination of primer extension and sequence analysis. The 5’ upstream promoter-enhancer has been characterized as a bidirectional regulatory region, shared with the pp38 gene. The structural arrangement of this bidirectional regulatory region shared by C1 and pp38 is similar to that of the HSV ICP4 and ICP22/47 genes (Preston et al., 1988; Wong and Schaffer, 1991). Whether C1 and pp38 gene expression is coordinately regulated, mutually exclusive, or their gene products are functionally synergistic will be an important issue in extending our knowledge of MDV gene regulation. Protein similarity searches of putative C1 and C2 encoded polypeptides against the Swiss-protein data base revealed only limited homology with zinc-finger proteins and the myc proto-oncogene. However, these similarities were not in conservative or 71 functional regions. The regions of similarity between ORFla, ORFlb, and the mouse zinc-finger protein ZFP-27 is not within the zinc-finger motif region (Chowdhury et al., 1988). Similarly, ORF2 has a limited homology with exon 3 of the myc proto-oncogene, a region outside the conservative "myc-box" (van Beneden et al., 1986). Therefore, we do not believe these similarities have significance regarding potential gene functions. We did not find similarity between ORF 1a or 1b and the mouse TLM oncogene as reported by Peng (1992), because the region in question was not present in our cDNA sequence due to altered Splicing. We also did not find any similarity between ORF-F, (corresponding to our ORF2), and the fer/fps family of kinase-related transforming proteins as reported by Peng et al. (1992). It is therefore likely that any protein product encoded by these cDNAs are MDV specific. Using predicted amino acid sequences deduced from our cDNA nucleotide sequence, fusion proteins and specific antibodies have been prepared. A 14 kDa protein was detected in Western blots of MDV infected cell lysates by antisera against both ORF 1a and ORF 1b. It is possible that these two ORFS encode proteins similar in size which differ slightly at their amino-terminal end. ORFla is predicted to encode 83 amino acids. An additional 2 amino acids would be added if 5’ sequence determined by primer extension is included. ORF lb could encode only 76 amino acids and is 18 amino acids shorter at the amino-tenninal end than ORF-D described by Peng et al. (1992). If we assume the 5’ end of ORF-D is the same 5’ end for our ORF 1b, ORF 1b could encode 93 amino acids. Thus, the calculated sizes of polypeptides encoded by ORF 1a and 1b would be proximately 9.6 kDa and 10.3 kDa, respectively. The predicted sizes of polypeptides encoded by ORFla and 1b, therefore, are smaller than the polypeptides identified by 72 ORF la and lb fusion protein antisera in SDS-PAGE gels. According to computer data analysis, ORF 1a and lb both could be heavily phosphorylated (six sites for ORF 1a and four sites for ORFlb) and glycosylated (three sites for ORFla and two sites for ORF 1b). Thus, post-translation modification, such as phosphorylation or glycosylation, may be an important factor contributing to the discrepancy between predicted and apparent protein sizes. Alternatively, ORF 1a and 1b may be exons encoding only part of a larger polypeptide. We believe this latter possibility is remote, since our primer extension data indicate the Cl and C2 transcripts end very near the 5’ ends contained in our cDNA clones and we did not detect any larger polypeptide in Western blots. Our data suggests that the 132 bp repeat region is removed by splicing and does not affect the protein encoded by transcripts C1 and C2 from this region of MDV DNA. These results also suggest that expansion of the 132 repeat may not be as critical in MDV attenuation as was previously thought. However, our data cannot rule out the possibility of subtle changes in amino acid content coincident with attenuation and expansion of the 132 bp repeat region. Expression of the 14 kDa C1/C2 gene products in an MDV induced lymphoma cell line indicates these proteins may play at least some role in maintenance of the transformed state or latent infection. Detection of the C1/C2 14 kDa proteins in MDV induced lymphoblastoid cells represents the third such MDV antigen identified in latently infected transformed cells after the identification of pp38 (Chen et al., 1992; Cui et al., 1991) and meq (Jones et al., 1992). All of the proteins identified in MSB-l cells are encoded within viral repeat regions. Whether these proteins have coordinated functions in MDV gene regulation and cell transformation remains to be investigated. The 14 kDa 73 proteins identified in this study are derived from an IE gene. Investigation of whether these proteins can activate or inhibit expression from other MDV genes and whether they execute different roles mediated by altered splicing patterns, will provide important clues regarding the nature of MDV induced tumorigenicity and pathogenicity. Acknowledgment This project was supported by the Michigan Agriculture Experiment Station, Research Excellence Fund, State of Michigan, and Grants 90-34116-5329 and 92-8420- 7430 awarded to P. M. Coussens under the Competitive Research Grants Program administered by the U. S. Department of Agriculture. We thank Melinda Wilson and Amin Abujoub for reviewing the manuscript and their helpful discussions. We also thank R. Southwick for his expertise with computers and assistance with graphics preparations. 74 Figure 1. Northern blot hybridization to identify immediate-early gene transcripts in MDV BamHI-12. A). MDV strain GA, passage 7, was propagated in duck embryo fibroblasts (DEF). To obtain immediate-early and early RNA transcripts, cycloheximide (CHX) or phosphonoacetic acid (PAA) was added at the time of infection and was present during the entire culture process. Poly(A)+ mRNAs were isolated and electrophoresed in 1.2% formaldehyde/agarose gels, transferred to Hybond-nylon membranes and hybridized with the 5.1 kb MDV BamHI-I2 fragment. Lane 1 is uninfected DEF, lane 2 is DEF infected with MDV GAp7, lane 3 is DEF infected with GAp7 and treated with CHX, lane 4 is DEF infected with GAp7 and treated with PAA. Sizes of various transcripts were determined by comparison to a 0.24 to 9.5 kb RNA ladder. B). The same membrane used in panel A was stripped and reprobed with the 2.3 kb BamHI-Xbal subfragment of MDV BamHI-12. C). Schematic representation of MDV genomic structure with its BamHI sites (Fukuchi etal., 1984). The enlarged portion is a restriction map of the BamHI-I2 fragment with the heavy line representing the 2.3 kb BamHI-Xbal subfragment probe. 75 MDV GA p7/CHx MDV GAp7/PAA X 5 it a. < ‘3 Q E MDV GAp7/PAA E '5 < < o 0 > > a o E 2 LI. LL I“ I.” D D 1.6 Kb .— 0.5 Kb . *— o.4 Kb (22 Q, It, I3 P2 9, s s r If, Q2 02 BamHI Li l2 0 I 5 C F Fri . E \ it 1 IR 3 \\\\NngH 12I|LI A P, hum ‘ : j. I r v A 1' x .1 11111 I - I; II II . . . l r I {771117 Y .7 It -- MDV TR U 8mm ’- L R 1. IR 5 Us TR 5 12 (5.1 kb) BamH] Xbel BamHI 2.3 kb 76 Figure 2. Schematic representation of the location, structure and primer extension analysis of Cl and C2 cDNAs. Partial restriction map of the MDV BamHI-H and I2 fragments is presented with a putative TATA sequence for the C1 transcript in a horizontal box. The dark line represents the region from which cDNAs C1 and C2 are derived. Grey shaded boxes represent the 132 bp repeat region. Location of C1 and C2 cDNAs is shown with approximate positions of their respective introns. Arrow heads indicate the direction of transcription for C1 and C2. Oligonucleotides used for primer extensions were designed based on cDNA sequence. P1 primer was complementary to C1 cDNA position 14 to 39, just upstream of the intron. Two primers, P 2A and P 28 were designed for the C2 transcript. P 2A is complementary to C2 cDNA sequence position 29 to 53, 10 base pairs downstream of the splice acceptor. P 2B is complementary to nucleotide 11 to 35 of C2 clone and crosses over to the spliced intron. Primers P1, P 2A and P 2B are depicted with small arrows. Dashed lines indicate sequences extended by primer extension analysis. 77 .NoSwE m .m ..... N <75U NU mg I. <8 I .. .m .m Figure 4. 82 Figure 5. Schematic representation of the positions of ORFS in C1 and C2 cDNA clones. Nucleotide sequences of C1 and C2 cDN A were analyzed using the Genepro and GCG programs. A partial restriction map of the BamHI-H and I2 fragments is shown with the putative TATA sequence upstream of C1 clone highlighted. Grey shaded boxes (DR) represent the 132 bp direct repeat region. ORFla of Cl clone, which is interrupted by a 2.3 kb intron, encodes a putative 83 amino acid polypeptide while ORFlb of C2 (which crosses over a 1.0 kb intron) encodes a putative 76 amino acid polypeptide. ORF2, which is completely located in exon 2, encodes 107 amino acid residues. 83 .m Squ .m .m Ego <20U NO [ n=m-0 .m aimU D m— mmo _<<<._. 100 amino acids) ORFS lead to cDNA isolation. The genomic sequence was used for alignment and analysis of C1 and C2 cDNAs which have been detailed in chapter 11. Fusion protein cloning and identification. As described in chapter 11, DNA fragments of ORF 1a and ORF 1b were cloned into pGEX-3X for expression of fusion proteins. An illustration of the vector and data from identification of fusion protein clones are presented in Figure 3. To confirm specificity of antiserum to ORF la encoded protein, GST-ORF 1a fusion protein was cleaved by Factor Xa to remove vector GST proteins. Cleaved ORFla protein was then detected by anti-ORAla serum (Figure 4). Effect of phosphorylation and glycosylation on ppl4 migration. Immunoprecipitation data indicated p14 is a phosphorylated protein (ppl4). Phosphorylation may be one factor contributing to slow migration of ppl4. To further investigate how post-translational modifications might affect the apparent size of pp14, two treatments were conducted: 1) MDV-infected cell lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate. 2) MDV-infected cells were treated with tunicamycin (TM), an inhibitor of N-linked glycosylation. TM and CIP treated cell lysates were compared with untreated cell lysates to identify any size discrepancy of pp14 118 119 by western blot analysis (Figure 5). Double-stained immunofluorescence on MDV infected cells. As detailed in chapter 111, monoclonal anti-MDV/gB antibody was employed as a positive control to confirm that immune signals detected by anti-ORFla serum are truly in MDV-infected cells. Data from double staining immunofluorescence which shows signals detected by both antibodies (anti-ORFla and anti-gB) are primarily present in the same cells (Figure 6). 120 Figure 1. Location of BamHI-Xbal subfragment in MDV genome and computer analysis of the viral genomic DNA sequence ORFS. A). MDV genome structure and location of the region sequenced. Dark lines represent sequenced fragments from the MDV BamHI library and EcoRI library. Sequence was determined for both strands. B). Translation of the viral DNA sequence in all six reading frames by Genepro program. Upper vertical bars indicate methionine or start codons. Lower vertical bars represent stop codons. puter r and m the Is. BI. gram. .1 stop 121 Figure l. A. V 32.... U. IE} If, / \, EcoR r zoom / , , I‘m” Hindi" XIII B. 1 IL'll III II IIII EHLuL—l II I ll 2 IIH‘HI ll lllullll II—III LI'--‘III‘-I IU-J-I Illll II 3 I I'll IIIIIIH III lIII I II-II |-“-I l1 ‘1 r-‘-‘Ir"I-“II II-‘II lH-JH‘I llll'" '3 I'JIIII—E‘IIIII'J'I'JIJ'"I I-"'-'r' I IIIHIII‘II II II I-"--IIII'III‘-I IIIIl-JII Illlllll ‘2 lllll I‘I-i—‘II Hi I I I II IIr‘IIIIIlI lllll IMF“ II‘III_-'II 122 Figure 2. Nucleotide sequence of the BamHI-XbaI region in MDV BamHI-I2 fragment. 1 TTTCACTCTC 61 AGAATAATCA 121 CCAATTTTCG 181 TCTATGCCTC 241 TGTATATGAA 301 ACCTACCATC 361 CGGACCCACT 421 GGTGATCGCA 481 TGCCOTAOTA 541 GGAATTCGAC 601 TGCACATTAT 661 TCTTACAATA 721 TACAGATCAC 781 GGATAATTGG 841 AAAGTGTGTA 901 AGGCCGGTGT 961 TGTTGTAGGG 1021 TGGCCGTGTT 1081 TGTGACGTCA 1141 AGTATCTCGC 1201 CCTACCTCAC 1261 TCCCOCATTC 1321 ATTATCGATA 1381 GTACACGACA 1441 GACGCTTCTG 1501 GGGACGTTCA 1561 ATCCTGTAAA 1621 ATTTTATCTT 1681 GCAAATGTAC 1741 TATATGTTCT CCCTAAAAAA CTGCTTCGAA CAGAGGTAAA AGGAAGAACA GCAGGACTGA CGTGTTGATT TCCATCTCGA TCAGAAGTTT GAAACGAGAG CCTCTCGAAT CAAACGTACG CGTCTGTATA TGTTTATTGA CATCTCAATT TGCGATTCGC ATGTAGAGAG TTCGAGAGGG GTAGCATACA TGTTTAGGTT CCCATTTGTA GCTTCTTCGT CGATATAGTT ATACGGAAGC ACACGGAAAT TAAACGATTG TTGTCTTTGT TCGGTCGAGC TCGTATACTC AGTAACCGCC GTGCGCTTCT AAAAAGGAGG CGGAGCTCGA TATCCATGCG TATCTACTTT AAAAAAACTA ACAGCTGTGA AACGGATACT TTGTGAGAGG CTGTGAGGTT CCAGTATAAA CATTAGTAAC TACGCACGAA AGTTCAACGG TCTCGAGGCT TTACCCTTCC TCTACATCTT GTGAGACCTA AGCAGTACAC TGAGCATGTA TCATTCGGCG TCGCGATCGC TGCAGCCAAT AAAGGGGATA CCTGTCTCAA CGGAAGTAGC TTTTCTAATT ATTAAAAGGT CCCTTCGCGT GTAAATTAAC CAACTAGCTG TTTGGTTACC TTCATCATCC TGATCTATTT GTTGGATTTT TTAAGTTCTA TATCGTTGCA GCGACAACTA GCTCGCTCCT CTGGCAGAGA TAGTAGCTAG TGCAGAAAGA CATATAAGTC TATGAAATTT TTTTTTTTTT CCAACTTTCT CGATCTCCTT AACATCAGTC ATGGCGAAAG AGAAAAATGG GTGGGAAATA AGAAGTGTCT GCTTGGTCCG ACTTCCTTGT AGTCTTTGTT GGTCGCCGAG ATTTTGAATG TACGGATATT ACGGTACGAT TTGCGGTGTC GCGGCATCTA CAGGTAGTGC GGGATTATGC GTGGTGGCAA GTGAGTGAGA CTAACAAAGT GACCCCGGAT GGAACGGAAC TAATTTCCAA TTCCAGCAAG GCGGATAATG CGCTGCGTAT TGTAAGAATG GAGTATACCT GCACATTGAC CTGTCGGTCG CGGATCACAT GCATGCATGT TTTGCCGTCC AACTGTTAAC TAGGTAATAG GGAGACGCGC CGGAATCGGG TTACATAGAA TGCGGAAATA ACGAACGCGG TATGTATATT GGTTCACTGT CAAGGTTAAA GGTTGCTTCA CAATTTTTGT CCCCCCAGAA E15111}. TTTGGGGGAT TGTGGACTTT ATTAAGAACG GTGCCGGTGT GCATTGACTG ATCAAGTTTT CTTTGTAACA AGAAAGAAGT AGTCGCTGTT AGTTATGTAT TAATGCTTCG AGAATTAGCA ATCTACCGGA TGGTATATTG GGAGCGGAGA GGAACACGAT GCCTGTTCGG TCTAAAAAGA AAAAATCATA AAAGAAGGTC ATCGGAGCCG TGTATGACCG GAACGGATAT ACGTGTAGCG TTTCAGCCTC ATGCGTGTTC AAAAGTTACT TGCGTCTCGA CTGTTTGTCG 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 TTTGCCACTA TTCGAGAGCG ATTTTTGTTG GGCAGCTCCT CCTTTTATTA TTGCTTACTC AGGTGCCTTT ATCATCTCAT CCGCTCTTCC GCCCTGTAAA AACTCAATGA TTCGGCCGGG TTGTTATTCT CAGGATTTAT TAAGTTTTTT ATAGCTGAGT GAAGTGTCTG TACCCCTCGC TAATTAGCAG 123 TTTCCGGACC TTTGATCCTC ATGGTTCCAA ACGGAATTGT TTAAGTCGCT GCCCGGAATT GGGCCCTGAT CTCAGCATAA AACCAGCGAA AGTAGCGGCG GAATTATGAA TGGCGCGAAA TTATAACGTC GTTCCCATGA TTAGCTTAAG AGACAGATGC ATATGCGATC TTATACTTTA TTTTACCGCC CCCGAACACT TTCCAACTAA (Figure 2. continued) GGGGAGGGGG ACCGTGCCGA TGGGGTAGTT ATTGGGTATT GAAATTTTTG TGAAGTCACC CCTTTCTTAA GGTGTGACGA GAGGCCTTTA CTGTCGATTT TGTGTTATAT CTGCGTTTTT TTTTTTTTGC GAAGGTATTT ATAACCGATA TTATAGGTGC GTGCTGATTT TTACTCAGAA ATGTCGAGAG XbaI GTCATCTAGA 124 Figure 3. Fusion protein expression vector and fusion protein clone identification. A). Plasmid pGEX-3X with multiple cloning sites labeled. ORF 1a was cloned in SmaI sites; while ORF 1b was cloned into BamHI and SmaI sites. B). Cells expressing fusion protein clones were identified by SDS-PAGE stained with Coomassie brilliant blue. T means total cell lysates; F means glutathione-sepharose purified fusion proteins. Lane 1 and 2 are GST-ORFla fusion proteins. Lane 3 and 4 are GST-ORF 1b fusion proteins. Lane 5 and 6 (G) are GST proteins. Lane 7 (M) is prestained protein marker. lion. lSlOfl 1c. T me 1 sins. A. 125 BamHI Smat EcoRI (~920) Figure 3. 126 x \‘2’ .9 .5! 1 ll. 0.” 3 9 Ii; 5 I~ 0) ‘0 0) 37—» , 27“» a: 10+ -"’ 1 2 3 Figure 4. Western blot showing antibody specificity to ORFla protein. GST—ORF 1a fusion proteins were bound to glutathione sepharose 4B and cleaved by Factor Xa. The cleaved ORFla protein was analyzed by western blot using anti-ORF 1a serum. Lane 1 is uncutted GST-ORFla fusion protein, lane 2 is Factor Xa cleaved ORFla protein, lane 3 is GST protein. Protein sizes were estimated by comparison with prestain protein marker. 127 86:8- '1! ,...-- rflfi—PP“ ”1234‘ Figure 5. Western blot analysis of post-translation modification. Lane 1 is cell lysate from CEF, lane 2 is cell lysates from CEF infected with MDV strain GA, Lane 3 is cell lysate from MDV infected cells which treated with TM, while lane 4 is cell lysate from MDV infected CEF and digested with CIP. 138 Figure 6. Double-stained immunofluorescence of MDV infected CEF cells. Anti- ORFla with secondary antibody conjugated with PE and monoclonal anti-MDV/gB antibody with secondary antibody conjugated with FITC were utilized to conduct double staining. A). Fluorescent labels were identified by 5130/30 nm band pass filter to show anti—gB-FITC stain. B). 580/30 nm band pass filter was used to detect anti-ORFla-PE stain. LIST OF REFERENCES REFERENCES Abujoub, A., and P. M. 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Adenovirus vector expressing functional herpes simplex virus ICPO. J. Virol. 62.:4544-4553. ‘ifi‘fi‘ HICHIGRN STRTE UNIV. LIBRRRIES llllllllllllllHIMIllllIlllllllllllllllWill 31293010441909