f mam» ...._ Lil-c ,_A -> new-..“ THESlS NIVERSITY LIBRARIES ' IIIIIIIIIIIlIIIIIIIIIIIIIIIIIII||II|II IZIIIIIIII . 3 1293 01564 99 LIBRARY Michigan State University This is to certify that the dissertation entitled Identification of a Marek's disease virus gene homologous to ICP27 of herpes simplex virus type 1, and investigation of MDV ICP27 gene regulatory functions presented by Delin Ren has been accepted towards fulfillment of the requirements for Ph . D. degree inAnimal Sc ience I6 Major professor Date 3:524 ' H g lqjé MSU is an Affirmatiw Action/Equal Opportunity Institution 0-12771 PLACE N RETURN BOX to remove We checkout horn your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Ie An Afflnneflve Action/Equal Opportunlty InetItqun Wane-9.1 IDENTIFICATION OF A MAREK’S DISEASE VIRUS GENE HOMOLOGOUS TO ICP27 OF HERPES SIMPLEX VIRUS TYPE 1, AND INVESTIGATION OF MDV ICP27 GENE REGULATORY FUNCTIONS By Delin Ren A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1 996 ABSTRACT IDENTIFICATION OF A MAREK’S DISEASE VIRUS GENE HOMOLOGOUS TO ICP27 OF HERPES SIMPLEX VIRUS TYPE 1, AND INVESTIGATION OF MDV ICP27 GENE REGULATORY FUNCTIONS By Delin Ren Marek’s disease virus (MDV) gene expression, like other Ot-herpesviruses, is regulated in a cascade fashion with gene expression classified into immediate-early, early, and late groups. Compared to other a-herpesviruses, such as herpes simplex virus type 1 (HSV-l), MDV immediate-early genes have not been as extensive studied. HSV-l ICP27, an immediate-early protein, acts as a multifunctional factor and is highly conserved in the (1-, [3-, and y-herpesvirus subfamilies. In these Studies, we identified an MDV immediate-early protein, MDV ICP27, which is a homolog to HSV-l ICP27. The MDV ICP27 gene is located within the EcoRI-B fragment of the MDV genome and encodes 473 amino acids. The predicted amino acid sequence of MDV ICP27 suggests this protein shares structural domains highly conserved to HSV-l ICP27. Detection of MDV ICP27 gene transcripts in MDV infected cells treated with cycloheximide suggested that MDV ICP27 gene is transcribed as an immediate-early gene. Bacterial trpE- and GST-fusion proteins of MDV ICP27 were expressed for production of polyclonal antisera. Two specific polypeptides of 55 kDa and 52 kDa were detected in MDV serotype 1 infected cells using anti GST—ICP27 antiserum. MDV ICP27 is a phosphoprotein and is predominantly located in the nuclei of CEF cells infected with serotype-1 MDV or with a fowlpox virus recombinant expressing MDV ICP27. Transient expression assays indicated that MDV ICP27 possesses both intrinsic trans- activation and trans-repression activities. MDV ICP27 is able to transactivate an MDV pp14/pp38 bi-directional promoter. In contrast, MDV ICP27 strongly represses the MDV thymidine kinase (TK) early gene promoter. In addition, a heterologous RSV-LTR is also stimulated by MDV ICP27. By deletion mutant analysis, we further demonstrated that amino acids 207 to 378 are critical for MDV ICP27-mediated transrepression activity. Substitution of 3’ RNA processing signals in the RSV-LTR constructs does not obviously affect MDV ICP27-mediated transactivation activity. In contrast, the MDV TK promoter shows a negative response to MDV ICP27 when the SV40 small T 3’splicing site and the early poly(A) signal in the TK promoter construct are replaced with the MDV ICP27 gene poly(A) signals. These results suggest that MDV ICP27- mediated transactivation and transrepression activities may be mediated through different transcriptional mechanisms. In contrast to HSV-l ICP27, MDV IVCP27 does not display any co-operative activity with MDV ICP4. Copyright by Delin Ren 1996 To my wife, Chongyang Huang, for her love, support, and patience, and to my great kids, Nan-Nan Ren and Charley Huang Ren. ACKNOWLEDGMENTS A special thanks to my advisor, Dr. Paul M. Coussens, for his guidance, encouragement, and financial support. I wish to express my sincere thanks to Dr. Lucy F. Lee, for her guidance, friendship, and financial support. I also wish to thank members of my guidance committee, Dr. Robert F. Silva, Dr. Susan E. Conrad, and Dr., Melvin T. Yokoyama for their valuable time and helpful suggestion. I especially thank all the people I have worked with in Dr. Coussens’s lab and Dr. Lee’s lab for their friendship and helpful suggestion. Finally, I wish to express my deepest appreciation to my parents, my parent-in - laws, my brother, and my sisters, for their love, support and continue encouragement. vi TABLE OF CONTENTS List of tables ......................................................................................... xi List of figures ....................................................................................... xii List of abbreviations ............................................................................. xiv Chapter I. Literature review .................................................................................. 1 Part I. Marek’s disease (MD) and Marek’s disease virus (MDV) .................. 2 1. History ......................................................................... 2 2. Biology of MDV .............................................................. 3 A. Virion structure ........................................................... 3 B. Serotypes of MDV ...................................................... 3 C. MDV isolation and cultivation ........................................ 4 3. PathologyofMDV4 A. MDVinfection .......................................................... 4 B. Pathogenesis ofMDV 5 4. Molecular biology of MDV ................................................ 7 A. Genome structure and physical map of MDV ........................ 7 vii B. Genome sequences and gene identification 8 C. MDV gene expression ..................................................... 9 a. MDV IE gene expression ............................................. 9 b. MDV early gene expression ....................................... 11 c. MDV late gene expression ......................................... 13 Part II. Herpesvirus immediate-early (IE) gene products and their functions 1. 2. ............................................................................................................... 16 Activation of HSV-1 IE genes by VP16 ................................. 16 ICP27 gene families ......................................................... 17 A. HSV-l ICP27 ............................................................ 17 a. Basic protein properties of HSV-1 ICP27 ......................... 17 b. Regulatory function of HSV-1 ICP27 ............................. 19 B. Varicella-zoster virus (VZV) ORF 4 .................................. 22 C. ICP27 homolog in B-herpesvirus ..................................... 23 D. ICP27 homolog in y-herpesvirus ...................................... 24 Other HSV-l IE genes ...................................................... 24 A. HSV-l ICP4 ........................................................... 24 B. HSV-l ICPO ............................................................ 27 C. HSV-l ICP22 and ICP47 .............................................. 30 The B—herpesvirus IE genes and their functions ......................... 30 The y-herpesvirus IE genes and their functions .......................... 32 viii Chapter 11. Identification and characterization of Marek’s disease virus genes homologous to ICP27 and glycoprotein K of herpes simplex virus-1 ..................................... 35 Abstract ...................................................................................... 36 Introduction ................................................................................ 37 Materials and methods .................................................................... 40 Results ....................................................................................... 44 Discussion .................................................................................. 49 Chapter III. A Marek’s disease virus immediate-early protein, MDV ICP27, possesses both positive and negative regulatory activities in vitro .......................................... 65 Abstract ...................................................................................... 66 Introduction ................................................................................. 68 Materials and methods .................................................................... 73 Results ....................................................................................... 80 Discussion ................................................................................... 88 Chapter IV. Discussion ........................................................................................ 111 1. Summary of results and conclusions .............................................. 112 2. Future research directions .......................................................... 119 Appendix .......................................................................................... 122 List of references ................................................................................ 127 LIST OF TABLE Chapter 111 Table 3.1 Effects of MDV ICP27 and MDV ICP4 on target promoters .. . . . . . .. 105 xi LIST OF FIGURES Chapter I. Figure 1.1 BamHI map of MDV serotype 1 DNA and location of genes on MDV genome .............................................................................................. 15 Figure 1.2 Schematic structure of predicted polypeptides of MDV ICP27 and HSV—l ICP27 ............................................................................................................................. 22 Chapter H. Figure 2.1 Diagram of MDV-GA structure and location of MDV gK and MDV ICP27 ........................................................................................................ 54 Figure 2.2 Nucleotide sequence of a 3.2 kb fragment of MDV GA form EcoRI-B ........................................................................................................ 56 Figure 2.3 Alignment of the C-terminal amino acid sequences of three herpesvirus ICP27 homologs. ................................................................................... 58 Figure 2.4 Northern blot analysis MDV ICP27 and gK .................................... 60 Figure 2.5 The trpE-ICP27 fusion protein expressed in E. coli ............................. 62 Figure 2.6 Immunoprecipitation analysis of MDV ICP27 translation products in vitro and detection of MDV ICP27 in MDV-infected cells .......................................... 64 xii Chapter 111. Figure 3.1 Transcript maps of MDV ICP27 and MDV ICP4, and Structures of MDV ICP27 and ICP4 transient expression constructs .............................................. 95 Figure 3.2 Subcellular localization of MDV ICP27 by indirect immunofluorescence staining .............................................................................................. 97 Figure 3.3 Characterization ofMDV ICP27 polypeptides 99, 100 Figure 3.4 Schematic structures of reporter constructs and effects of M-ICP27CMV on target promoters ................................................................................... 102 Figure 3.5 Dose-dependent feature of MDV ICP27-mediated transactivation and transrepression activities 104 Figure 3.6 Effects of 3’RNA processing signals on MDV ICP27-mediated regulatory activities ............................................................................................ l 07 Figure 3.7 Functional dessection of MDV ICP27 polypeptides ..................... 109, 110 Appendix Figure A.1 Expression of GST—ICP27 fusion proteins in E. coli .......................... 124 Figure A.2 Specificity analysis of MDV ICP27 antisera ................................... 126 xiii AGP BMLFI BRLFI BZLFI CAT cDNA CEF C/EBP CHX CIP CMV HCMV CsCl DEF DR EBV EBNA-l EHV LIST OF ABBREVIATIONS Agar gel precipitation (EBV) BamI-II M leftward reading frame 1 (EBV) BamHI R leftward reading frame 1 (EBV) BamHI Z leftward reading frame 1 base pairs chloramphenicol transferase complementary DNA chicken embryo fibroblast CCAAT/enhancer binding protein cycloheximide calf intestinal phosphotase cytomegaloviurs human cytomegaloviurs cesium chloride duck embryo fibroblast direct repeat early Epstein-Barr virus EBV nuclear antigen-1 equine herpes virus xiv EPA FFE FITC FPV GST HCF HCMV HIV HSV—l HVT ICPO ICP4 ICP22 ICP27 ICP47 IE IFA IgG (SV40) early polyadenylation signal feather follicle epithelium fluorescein-S’isothiocyanate fowlpox virus glycoprotein B glycoprotein C glycoprotein D glycoprotein K glutathione-S-transferase host cell factor human cytomegalovirus human immunodeficiency virus herpes simplex virus type 1 herpesvirus of turkeys (HSV) infected cell protein No.1 (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 indirect immunofluorescence assay immunoglobulin G XV IRL IRS kb IUdR LAT LTR MD MDV mMDV meq Mta NC NLS NuLS Oct-l ORF ORF4 OTF-2 PAA PAGE internal repeat long internal repeat short kilobase kilodalton 5’-iodo-2’deoxyuridine late latency-associated transcripts long terminal repeat Marek’s disease Marek’s disease virus mild Marek’s disease virus Marek’s EcoRI Q (EBV) M transactivator nitrocellulose membrane nuclear localization signal nucleolar localization signal octamer binding factor-1 open reading frame (V ZV) open reading frame 4 octamer binding factor-2 phosphonoacetic acid polyacrylamide gel electrophoresis xvi PCR PI Rta SDS TBP TFIIB TFIID TK TRL TRS IS Us VP 1 6 VMDV vaDV VZV Zta polymerase chain reaction post inoculation restriction enzyme (EBV) R transactivator sodium dodecyl sulfate TATA-binding protein transcription factor IIB transcription factor IID thymidine kinase terminal repeat long terminal repeat short temperature-sensitive unique long unique short (HSV) Virion protein No.16 virulent Marek’s disease virus very virulent Marek’s disease virus varicella-zoster virus (EBV) Z transactivator xvii Chapter I Literature Review Part I. Marek's disease and Marek’s disease virus 1. History Marek's disease virus (MDV) is a cell-associated avian herpesvirus that causes Marek’s disease (MD) in chickens, resulting in T-cell lymphoma and mononuclear nerve demyelination (Churchill and Biggs, 1967; Nazerian et al., 1968; reviewed by Calnek, 1985). MD was first reported by Joseph Marek, a Hungarian veterinarian in 1907 (Payne, 1985), but it was not until 1967, that a herpesvirus was identified as the etiological agent. At the same time, MDV was successfully propagated in tissue culture (Churchill and Biggs, 1967; Solomon et al., 1968; Nazerian et al., 1968). Prior to use of vaccines, MD constituted a serious economic threat to the worldwide poultry industry because of heavy annual losses, due to death and condemnation. MD has been effectively controlled by vaccination since early 1970’s with attenuated MDV serotype 1 derivatives or with an antigenically-related but apathogenic herpesvirus of turkeys (HVT). Thus, economic losses due to MD are no longer as serious as prior to vaccine development (reviewed by Witter, 1985). Vaccination, however, is not completed effective against infection by certain strains of very virulent MDV (vaDV). Thus, development of a new generation vaccines using molecular biology approaches has been promoted (Yanagida et al., 1992; Nazerian et al., 1992). MDV is one of several related herpesviruses that can induce neoplastic diseases in their natural hosts, other such related herpesvirus is Epstein-Barr virus (EBV). MD vaccines provide the first example of controlling a natural neoplastic disease by vaccination. MD, therefore, provides an excellent animal model for understanding the oncogenicity of other herpesviruses, such as EBV, and may significantly contribute to comparative medical science. 2. Biology of Marek’s disease virus A. Virion structure MDV virions consist of 162 hollow-centered capsomeres with icosahedral symmetry (Schat, 1985). In MDV-infected tissue culture, most virions are hexagonal naked particles or nucleocapsids, 85-100 nm in diameter, and are usually found in nuclei but occasionally in the cytoplasm of infected cells. Enveloped MDV particles, 150-160 nm in diameter, are rarely seen and are principally associated only with nuclear membranes (Nazerian et al., 1971). Large numbers of cytoplasmic enveloped virions (273-400 in diameter) are only observed in feather follicle epithelium (F FE) of infected chickens. B. Serotypes of MDV Based on agar gel precipitation (AGP) and immunofluorescence assay (IF A), MDV has been classified into three serotypes (Bulow and Biggs, 1975). This serotype classification has been confirmed through use of monoclonal antibodies (Lee et al., 1983). Oncogenic MDV is only associated with serotype 1. However, there is a wide variation in pathologic potential within strains of serotype 1. Therefore, MDV serotype 1 is further classified as mild MDV (mMDV), virulent MDV (VMDV), and very virulent MDV (vaDV), according to pathogenesis and oncogenicity. Serotype 2 MDVs are naturally occurring non-oncogenic viruses. An apathogenic herpesvirus of turkey (HVT) is classified as serotype 3. Serial passage in vitro of vaDV or VMDV will lead to attenuation with concurrent loss of oncogenicity and pathogenesis. HVT, Serotype 2, and attenuated serotype 1 MDVs are widely used as vaccines against to MDV (Witter, 1985). C. MDV isolation and cultivation MDV can be isolated from chickens 1 or 2 days post-inoculation or 5 days after contact exposure and throughout the life span of infected chickens. Because MDV is highly cell-associated, intact viable cells are usually used for inoculation. Primary isolates of MDV can be propagated on chicken embryo fibroblast (CEF), duck embryo fibroblast (DEF), and chicken embryo kidney (CEK) cells. DEF and kidney cell cultures are commonly used for MDV serotype 1 infection, whereas CEF cells are suitable for propagating serotype 2 and serotype 3 (Calnek and. Witter, 1991). Recently, a chicken embryo fibroblast cell line, OU2, was used for MDV infection. MDV OU2 cell lines infected with MDV serotype 1 strain MDll were established (Abujoub and Coussens, 1995). MDV OU2 cell lines are similar to certain lymphoblastoid cell lines, and are capable of transferring MDV infection to primary CEF monolayer cultures. However, MDV OU2 cell lines are also capable of Supporting a cytolytic infection of MDV (Abujoub and Coussens, 1995). 3. Pathology and immunity of MDV A. MDV infection MDV infections consist of productive infection (also known as lytic infection), latent infection, and transformation. Productive infection can be further divided into fully productive and semi-productive infection. Fully productive infection, resulting in development of large numbers of enveloped and fully infectious virions, is only observed in feather follicle epithelium (FFE) of infected chickens. Semi-productive infection (or restrictive infection) mostly occurs in all other tissues and in cell culture (Calnek et al., 1970). In semi-productive infection, most virions are not enveloped. During semi- productive infection, virions are not released in an infectious form, instead of virus spreads from cell to cell. Both latent infection and transformation are not productive and only a few genes are expressed in latent and/or transformation infection. MD is horizontally transmitted by direct or indirect contact with infected birds. The infection period, clinical signs, and gross lesions of MDV can be induced experimentally in chickens. Chicks inoculated at 1 day of age, start secreting virus at 2 wk post- inoculation (PI). Clinical signs and gross lesions appear about 3-4 wk PI. The major clinical signs and gross lesions of MD can be described as either classical or acute MD. Classical MD predominantly affects peripheral nerves, but spinal roots or root ganglia are sometimes involved. Asymmetric progressive paresis, characterized by drooping wings is commonly observed in both natural or experimental diseases. A particular characteristic of infected birds is one leg stretched forward and the other backward (Biggs, 1967; Calnek and Witter, 1991). Lymphoid tumors can be seen in a variety of organs but frequently, only one or a few organs are grossly affected in classical MD. In contrast, multiple lymphoid tumors are observed more frequently in acute MD, affected tissues including the gonads, lungs, kidneys, liver, heart, spleen, bursa and skin. B. Pathogenesis of MDV MDV gains entry via the respiratory tract where it is most likely internalized by phagocytes. B cells appear to be the primary target cells, although some T cells may also be involved (Shek et al., 1983; Calnek, 1985). Primary degenerative changes are the major features in this early stage of lytic infection. At about 6-7 days PI, infection switches to a latent infection coincident with the development of immune responses. T cell-mediated immune responses play a central role in this switch, resulting in T cell activation (Payne, 1985; Calnek, 1985). CD4+ T helper cells are the principal targets at this stage, although a few B cells may still be involved (Schat et al., 1991). Latent infection is persistent and can last for the lifetime of the birds. Following latent infection, susceptible birds (but not genetically resistant birds) can develop a second lytic infection. This phase of infection usually is coincident with permanent immunosuppression. Lymphoproliferation and development of T cell tumors are commonly seen at this stage (Buscaglia et al., 1988). Massive lymphomas can be observed in visceral organs, skin, muscle, and neural tissues. Lymphoproliferative changes are only observed in birds infected with virulent serotype 1 MDV. The composition of MDV-induced lymphomas is complex, consisting of a mixture of neoplastic, inflammatory, and immune cells. Both T and B cells are present, but T cells predominate (Hudson and Payne, 1973). Neoplastic cells contain 5-15 copies of viral DNA and can be grown as continuous lymphoblastoid cell lines in vitro (Ross, 1985). It is believed that MDV transformed cell lines are latently infected and provide a useful system for studying both MDV latency and transformation. MDV transformed cell lines can be distinguished as either producer or non-producer cell lines. Producer cell lines are those cells can be rescued after in vitro co-cultivation or following inoculation into susceptible chickens. In non-producer cell lines, Viral antigens are not detectable and virus cannot be rescued by co-cultivation (Schat et al., 1989; Calnek and Witter, 1991). Both humoral and cell-mediated immunity are involved in MDV infection. However, cell-mediated immune responses are critical for immunity. Functional T cells are required for both genetic resistance and vaccinal immunity (Sharma et al., 1975). 4. Molecular biology of MDV A. Genome structure and physical map of MDV The genome of MDV is a linear double-stranded DNA molecule with a density of 1.705 g/cm3 in CsCl, and a base composition of 46% guanine plus cytosine. The molecular weight of MDV DNA is 108-120 x 106 daltons, equivalent to 180 kb (Lee et al., 1971; Cebrian et al., 1982; Fukuchi et al., 1984; Hirai et al., 1979). The structure of MDV DNA consists of unique long and unique short regions (UL, Us), flanked by inverted repeat regions (IRL/TRL, IRS/TRS, respectively) (Figure 1.1) (Cebrian et al., 1982; Fukuchi et al., 1984). MDV was originally classified as a y- herpesvirus because its lymphotropism is similar to that of Epstein-Barr Virus (EBV), a prototype of the y-herpesvirus family (Roizman et al., 1981). However, the genome structure and gene arrangement of MDV are more similar to that of (ac-herpesviruses, such as herpes simplex virus type 1 (HSV-l), and varicella-zoster virus (VZV) (Buckmaster et al., 1988; Roizrnan et al., 1992; Karlin et al., 1994). Furthermore, MDV genes, particularly those identified in UL and Us regions, display significant homology to those of HSV-1 and VZV (Zelnik et al., 1993; Ren et al., 1994; Brunovskis and Velicer, 1995; Lee et al., 1995). In addition to the inverted repeats, several direct repeats (DR) have been identified in MDV genomes (Hirai, 1988). These DR sequences consist of more than 100 bp repeats and are mostly located within the internal or terminal repeat regions (Hirai, 1988). DR] is a tandem direct repeat of 132 bp repeat units located within TRL and IRL Serial in vitro passage of virulent MDV in primary CEF cells results in a loss of MDV oncogenicity. The loss of oncogenicity has been found to correlated with an expansion of the 132 bp DRl (Fukuchi et al., 1985; Maotani et al., 1986). Recently, a 1.8 kb transcript family and several gene products have been identified within this region (Bradley et al., 1989; Cui et al., 1991; Chen and Velicer, 1992; Hong and Coussens, 1994; Peng et al., 1994). The development of a BamHI restriction enzyme (RE) map and associated clones was central to studies of MDV (Figure 1.1) (Fukuchi et al., 1985). The MDV BamHI map has been the basis for most gene identification and localization studies. RE maps of all three serotypes of MDV have been constructed and are also useful for comparative studies between MDV serotype genomes (Igarashi et al., 1987; Ono et al., 1992). B. Genome sequences and gene identification MDV sequencing and gene identification started in the late 1980’s. The first gene, encoding A antigen (homolog to herpes simplex virus gC), was identified by Isfort et al. in 1987, and sequenced by Coussens and Velicer in 1988. Using random sequence analysis, 35 MDV and 24 HVT genes have been compared to both y- and a- herpesviruses (Buckmaster et al., 1988). The results indicated that MDV and HVT bear greater genetic similarity to a- than to y- herpesviruses. In the following few years, MDV DNA sequencing and gene identification has progressed due to advances in molecular biological techniques. The unique short regions of MDV and HVT have been completely sequenced (Brunovskis and Velicer, 1995; Zelnik et al., 1993). Although the UL regions of MDV and HVT have not yet been completely sequenced, many oc- herpesvirus gene homologs have been identified (reviewed by Velicer and Brunovskis, 1992; Lee etal., 1995). These results have provided the basis for the reclassification of MDV to the (at-herpesvirus family (Roizman et al., 1992). C. MDV gene expression Like other herpesviruses, MDV gene expression is temporally regulated in a cascade fashion (Maray et al., 1988; Schat et al., 1989). Generally, herpesvirus genes have been divided into three kinetic families: immediate-early gene (IE or or), early (E or [3), and late (L or 7) genes, based on the requirement for viral protein synthesis and/or viral DNA replication (Honess and Roizrnan, 1974). a. MDV IE gene expression IE genes are expressed immediately upon infection with no requirement for de novo protein synthesis. Compared with other herpesviruses, studies on gene expression and regulation of MDV are far behind. This is mostly due to the highly cell-associated 10 properties of MDV. Cycloheximide, a metabolic inhibitor, can block protein synthesis and accumulate immediate-early transcripts in virus-infected cells. By cycloheximide treatment, numerous IE transcripts have been detected in MDV lytically infected cells and lymphoblastoid cell lines (Maray et al.,1988; Schat et al., 1989). However, all these reports are based only on Northern hybridization analysis, without exact gene and gene product identification. Recently, several MDV IE genes have been reported and three MDV IE genes are homologous to HSV-l ICP4, ICP27 and ICP22 (Anderson et al., 1992; Ren et al., 1994; Hong and Coussens, 1994; Brunovskis and Velicer, 1995). Anderson et al. (1992) reported an ICP4 gene homolog that is the major transcriptional activator in HSV-l infection (Roizman and Sears, 1995). MDV ICP4 is located in BamHI-A fragment within the MDV inverted repeats. MDV ICP4 is 4,245-nucleotide long and encodes 1415 amino acids. Based on sequence analysis, MDV ICP4 predicted polypeptide sequence can be divided into five regions in which region 2 and region 4 have higher amino acid conservation than others. A very highly conserved serine—rich domain is also found in region 1 (Anderson et al., 1992). Several potential cis-acting elements are observed in the MDV ICP4 control region or within the MDV ICP4 coding region, including an ICP4 consensus biding site, Oct-1 site, and TAATGARAT motif that is a consensus element recognized by VP16, a Virion transactivator (Anderson et al., 1992; Roizman and Sears, 1995) Another essential tat-herpesvirus IE gene, ICP27, has been mapped to the EcoRI-B fragment of MDV DNA (Ren et al., 1994). MDV ICP27 is a 473-amino-acid polypeptide 11 and shows 26% amino acid identity with HSV-l ICP27. Interestingly, MDV ICP27, by comparison to other a-herpesvirus homologs, shares a higher amino acid conservation with a cysteine-rich domain and a potential zinc-binding motif within the C-terminal region (Figure 1.2A) (Ren et al., 1994). A basic and arginine-rich domain has also been found between amino acids 150 to 200. Using MDV ICP27 specific antiserum, two polypeptides with molecular weights 55 and 52 kDa can be detected in MDV serotype 1 infected cells (Ren et al., 1996). Functions of both MDV ICP4 and MDV ICP27 are less well understood and will be discussed in more detail in Part II. Recently, a 1.6 kb IE transcript has been mapped to the MDV BamHI-I2 region ( Hong and Coussens, 1994). By cDNA cloning and sequence analysis, two cDNAs (C1 and C2) have been identified as spliced transcripts from this 1.6 kb transcript. C1 (1.4 kb) and C2 (1.35 kb) share identical splice acceptors and 3’ ends, but differ in their 5’ end and in the splicing donor site (Hong and Coussens,1994). Both C1 and C2 encode an identical highly phosphorylated protein, ppl4, which is predominantly found in the cytoplasmic. Interestingly, MDV pp14 can be detected not only in MDV serotype 1 infected cells, but also in an MDV transformed lymphoma cell line, MSB-l (Hong et al., 1995). However, function of MDV pp14 is not clear. MDV ICP22 homolog is mapped to MDV Us region, but no more information is available at this time (Brunovskis and Velicer, 1995). b. MDV early gene expression Early genes are expressed afier onset of immediate-early gene expression. Early gene 12 expression is regulated by IE gene products (Roizman and Sears, 1995). Most early gene products are involved in nucleotide precursor metabolism and viral DNA synthesis. Therefore, in the presence of drugs that block viral DNA synthesis, such as phosphonoacetic acid (PAA), early gene expression is enhanced rather than reduced. Thymidine kinase (TK) is a typical early gene and has been extensively studied in the oc- herpesvirus family (Roizman and Sears, 1995). A TK gene homolog has been reported in MDV (Scott et al., 1989). Other (Jr-herpesvirus early gene homologs such as DNA polymerase and ribonucleotide reductase have also been identified (Sui et al., 1995; Lee et al., 1995). Importantly, an MDV unique phosphoprotein, termed pp38, has been reported by several labs (Silva and Lee, 1984; Cui eta1.,1991; Chen and Velicer 1992). Expression of MDV pp38 is relatively insensitive to PAA, indicating that pp38 may belong to the early gene family (Chen and Velicer, 1992). Initially, pp38 expression was thought to be limited to serotype 1 MDV specific and was abundantly expressed in an MDV transformed lymphoid cell line, MSB-l. These observations promoted several labs to investigate the relationship between pp38 and MDV oncogenicity (Cui et al., 1991; Chen and Velicer 1992). More recently, however, pp38 homologs have also been found in both MDV serotype 2 and HVT (Ono, et al., 1994; Smith et al., 1995). Interestingly, both pp38 and pp14 genes share a common control region that has been defined as a bi- directional promoter (Chen and Velicer, 1991; Cui et al., 1991; Hong and Coussens, 1994). There are several cis-acting elements including two TATA boxes, two Spl sites, two CAAT sites and one octamer motif within this control region (Cui et al., 1991). 13 c. MDV late gene expression Late (or y) genes comprise the largest kinetic class of genes in most herpesviruses. Most late gene products are structural proteins for Virion capsid, tegument, attachment, cell fusion, and envelope. A key feature of late gene transcription is the requirement for viral DNA replication. Based on dependence for viral DNA replication, late genes can be further divided into yl and y2. Transcription of yl genes occurs prior to initiation of viral DNA synthesis and does not depend stringently on viral DNA replication. As viral DNA synthesis begins, yl genes are expressed abundantly and y2 gene expression begins. Protein synthesis of yl and y2 continues throughout the remaining replication cycle (Wagner, 1991; Roizman and Sears, 1995). Glycoproteins are a major component of this kinetic class and have been extensively studied. Most of the glycoprotein gene homologs have been identified in MDV, including gB, gC, gD, gE, gH, gI, gK, and gL (Ross et al., 1989; Isfort et al., 1987; Ren et al., 1994, Yoshida et al., 1994; Brunovskis and Velicer 1995). Two of these glycoproteins, gB and gC, have been extensively studied in MDV because of their unique biological properties. The MDV B-antigen, a major immunogen for inducing neutralizing antibodies, is a complex of three glycoproteins consisting of gp100, gp60, and gp49. MDV B-antigen has been confirmed as a HSV—l gB homolog (Ross et al., 1989; Chen and Velicer, 1992; Yanagida et al., 1992). Expression of MDV gB gene by fowlpox Virus and baculovirus recombinants followed by inoculation into genetic susceptible birds suggest that MDV gB is a valuable candidate for recombinant vaccine (Yanagida et al., 1992; Nazerian et al., 1992; Niikura et al., 1992). MDV gC is another interesting glycoprotein and was originally termed l4 MDV A-antigen (Isfort et al., 1987; Coussens et al., 1988). MDV gC is a secreted protein and can be readily detected in the supernatant of infected cell cultures. However, expression of MDV gC is significantly reduced in attenuated MDV strains (Bulow and Biggs 1975). Compared with low-passage MDV, no DNA sequence alterations have been observed within the gC coding regions or promoter regions in attenuated MDV. These results indicate that reduced expression of MDV gC may be due to alteration of viral or cellular proteins that regulate gC promoter activity in attenuated MDV (Wilson et aL,l994) 15 .35» >92 «o .5382 Ea >92 .8 0.3883 9:89.. 05 AB ”<75 _ 2528 >92 we 9E 3.88385 5858.. Esem 2:. A5 a; 95E."— 225 age 3 3 Al A>...v 3.383 Al l.v AI All lv Al lvlv EB EB .82 56— M» or; Um m» E. :l s Ii. m JEWE— D My:— sfi_ D _ ”E. Jo in in J5 JB :5 9.5 in i x .ITTITIE :T x n u; u n “T u a T-.. c < Noam m .6527/ m an: m mar. n O o. a 3.. o o mama .o «a o. o o PART II. Herpesvirus immediate-early gene products and their functions As described above, herpesvirus genes are expressed in a temporal regulation pattern during the lytic infection cycles. Much of our knowledge regarding gene expression regulation in herpesvirus has been derived from a prototype a-herpesvirus, HSV-l. Thus, in this section, review will be mostly focused on HSV-l IE genes, however the comparative aspects in those of 13- and y- herpesviruses will also be discussed in some detail. 1. Activation of HSV-1 IE genes by VP16 HSV-l contains five IE genes, ICPO, ICP4, ICP22, ICP27, and ICP47. Two of these IE genes, ICP4 and ICP27, are essential for viral replication and viral gene expression during lytic infection. Although HSV-l IE gene expression does not require de novo protein synthesis, both viral proteins and cellular factors are involved in the IE gene activation. A Virion tegument protein, named VP16 (V mw65, a-TIF), plays a central role in IE gene transactivation (reviewed by Roizman and Sears, 1995). HSV—l VP16 contains 490 amino acids, has a molecular weight of 54 kDa, and is synthesized during the late phase of lytic infection. During Virion assembly, VP16 is incorporated into the tegument between the capsid and Virion envelope. VP16 is subsequently released during infection and specifically trans-induces the IE gene transcription. Apparently, transactivation of IE genes requires at least one cis-acting sequence element, TAATGARAT (R = purine), within the IE promoter region (Mackem and Roizman, l6 17 1982; Gaffney et al., 1985). Further studies has indicated that VP16 has no substantial affinity for double-stranded DNA (Marsden et al., 1987), but functions by forming a multi-component complex on TAATGARAT sites, together with the cellular POU domain protein Oct-1 and host cell factor (HCF) (also called CFF or C1 factor) (Triezenberg et al., 1988; Katan et al., 1990; Kristie and Sap, 1995). 2. ICP27 gene families A. HSV-l ICP27 a. Basic protein properties of HSV-1 ICP27 HSV-l ICP27 is located in the unique long region of the HSV-l genome between coordinates 0.745 and 0.761. The open reading frame is 1536 nt long and encodes 512 amino acids. HSV-l ICP27 is localized to the nuclei of infected cells and is phosphorylated, resulting in different forms of proteins being detected on SDS-PAGE. It appears that HSV-l ICP27 contains both stable phosphate groups and phosphate groups that cycle on and off during infection. It is speculated that the different phosphorylated forms of ICP27 may specify its different regulatory activities, however, this aspect has not been thoroughly investigated (Ackerman et al., 1984; Wilcox et al., 1980; Sandri- Goldin, 1991). An assessment of the predicted amino acid sequence of HSV-1 ICP27 reveals that the primary structure can be divided into two halves with a hydrophilic amino terminal half and a relatively hydrophobic carboxyl terminal half (Sandri-Goldin, 1991). The first 64 amino acids from the N-terminus consist of 38% acidic residues and a serine-rich segment. The second striking feature of the N-terminal region is a highly 18 basic and arginine-rich domain between amino acid residues 110 and 175. This basic and arginine-rich domain contributes to nuclear localization and will be discussed in some detail below. The carboxyl terminal half of ICP27 plays the major functional role in HSV-l ICP27-mediated transactivation or transrepression. A potential “zinc-finger” motif has been found within the last 60 amino acid residues of C-terrninal region (Sandri-Goldin, 1991). Interestingly, this zinc finger motif is highly conserved in most ICP27 homologs including those of [3- and y- herpesviruses (Peara et al., 1994; Zhao et al., 1992; Winkkler et al., 1994; Wong and Levine, 1986). Studies with ICP27 temperature-sensitive (ts) and deletion mutants indicate that HSV-l ICP27 is essential for viral replication, especially for viral DNA synthesis and early and late gene expression (Sacks et al., 1985; McMahan et al., 1990). HSV-l ICP27 is localized to the cell nuclei in both viral infected cells and cells transfected with ICP27 expression plasmids (Ackermann et al., 1984). HSV—l ICP27 possesses a strong nuclear localization signal (NLS), between amino acid residues 110- 137, which bears similarity to the bipartite NLSs found in Xenopus laevis nucleoplasmin and other nuclear proteins. The sequence of HSV-1 ICP27 between residues 110 and 152 can function as a nuleolar localization signal (NuLS) (Mears et al., 1995; Robbins et al., 1991). The sequence of NuLS includes ICP27’s strong NLS and 15 contiguous residues consisting entirely of arginine and glycine. This arginine and glycine rich element is very similar to an RGG box, a putative RNA-binding motif found in a number of cellular proteins involved in nuclear RNA processing (Kiledjian and Greyfuss, 1992). Interestingly, Hibbard and Sandri-Goldin (1995) further demonstrated that two arginine— l9 rich regions located within the NuLS are required but not efficient for wild-type nuclear localization of ICP27. More importantly, when the NLS and R-rich regions are substituted by the heterologous NSL (SV40) or by the RNA binding domain of HIV-1 Tat individually, ICP27 mutants are defective in modulating late gene expression. Therefore, arginine-rich regions may be required for efficient nuclear localization and for the regulatory activity of ICP27 involved in co-operative activation of viral late gene expression (Hibbard and Sandri-Goldin, 1995). b. Regulatory functions of HSV-1 ICP27 Studies from ts mutants with lesions within ICP27 and from deletion mutants have shown that HSV-l ICP27 acts as a transactivator and a transrepressor (McCarthy et al., 1989; Sacks et al., 1985). The immediate-early genes (ICP4 or ICPO) and early genes are over-expressed in ts mutant infected cells, while late genes are poorly expressed. Phenotype analysis of these mutants indicates that ICP27 is required for the switch from early to late gene expression. Evidence from transient experiments also indicated that ICP27 acts either as a repressor or as an activator depending on the target genes examined (Hardwieke et al., 1989; Rice et al., 1990; Su and Knipe, 1989). In these transient expression assays, ICP27 appears to have little or no affect on expression of a wide range of target promoter constructs. However, when a plasmid encoding ICP27 is combined with effector plasmids encoding ICP4 or ICPO, both positive and negative effects can be observed (Hardwieke et al., 1989; Su and Knipe, 1989; Rice et al., 1989). With regard to the distinct regulatory activities of ICP27, various approaches have been employed to map functional domains within HSV—l ICP27. Rice et al. (1989) 20 reported that mutated polypeptides that possessed either 406 or 504 amino acids of ICP27 failed to activate gene expression but retained full transrepression activity. A polypeptide containing the amino-terminal 263 amino acids retained partial transactivation ability, but it was unable to transrepress target gene expression. Therefore, HSV-l ICP27 possesses two genetically separable activities which can modulate gene expression in transfected cells. One activity positively affects gene expression, while the other inhibits gene expression (Rice et al., 1989). In contrast, using in frame insertion or deletion strategies, Hardwick et al. reported that mutants with insertions between position 262 and 406 lost their transactivation activity but retained transrepression function, whereas those with insertions between position 434 and 504 lost both activities (Hardwick et al., 1989). Thus, they concluded that the C-terminal half of ICP27 is required for the transactivation activity of ICP27 but only the C-terminal 78 amino acids are critical for transrepression activity (Hardwick et al., 1989; Sandri-Goldin, 1991). Smith et al. (1991) sought to determine whether any of the ICP27 insertion mutants would display a dominant phenotype, in other words, to determine whether these mutants would interfere with wild- type ICP27 activities. By both transient and stable expression analysis, they found that mutations in the activation regions of ICP27 between residues 262 and 406 were dominant to wild-type ICP27 and specifically interfered with its transactivation function. In contrast, those mutants defective in repressor function, residues 434 to 505, cannot compete with wild-type ICP27 (Figure 1.2B) (Smith et al., 1991). Regulatory activity of HSV-1 ICP27 is independent of target promoter sequences but depends on the presence of different mRNA processing signals (Sandri-Goldin and Mendoza, 1992). The ICP27 activation function correlates with different 21 polyadenylation sites, whereas repressor function correlates with the presence of introns either 5’ or 3’ to the target gene-coding sequences. This hypothesis is supported by the following observations: First, HSV—l ICPO and ICP4-mediated transactivation of TK promoter can be repressed 12-fold by HSV-l ICP27, when the TK reporter plasmid contains an SV40 early polyadenylation signal (EPA) and a small T intron. When the SV40 EPA and small T intron are replaced with a synthetic poly A hexanucleotide (AATAAA), HSV-l ICP27 is able to stimulate (but not repress) the TK promoter. Splicing studies have also suggested that ICP27 directly inhibits splicing by sequestering snRNPs (Sandri-Goldin and Mendoza, 1992). Second, ICP27 affects the accumulation of CAT poly(A) mRNA in transfection assays with CAT expression plasmids containing different 3’RNA processing signals. ICP27 determines which polyadenylation signal is used where more than one signal is present. Generally, late gene polyadenylation signals were used more efficiently than those of early gene (McLauchla et al., 1989; Sandri- Goldin and Mendoza, 1992). HSV—l ICP27 also affects host mRNA accumulation. In infection with viral mutants defective in ICP27, the accumulated levels of three spliced host mRNAs are much higher than those seen with wild-type HSV-l. Thus, it appears that HSV—lICP27 contributes to the decrease in cellular mRN A levels during infection by inhibiting splicing (Hardwick and Sandri-Goldin, 1994). More recent studies support the hypothesis that ICP27 is involved in stabilizing mRNA and binding to 3’ ends (Brown et al., 1995). However, it is not clear whether binding involves specific poly(A) signals or the long AU-rich instability-associated motifs presenting in these transcripts. The RNA binding motifs within HSV-l ICP27 polypeptide remain to be identified. There are two potential domains that may be related to RNA binding. The repetitive 22 RGGRRGRRRGRGRGG motif between amino acid residues 138 and 152 is very similar to a well-defmed RNA-binding motif, the RGG box (Mears et al., 1995; Kiledjian and Dreyfuss, 1992). In addition, a putative zinc-finger motif which is highly conserved in most ICP27 homolog families could also function as a RNA-binding motif (Brown et al., 1995; Sandri-Goldin, 1991). The polypeptide structure and functional domains of HSV-1 ICP27 are summarized in Figure 2.2B. Highly hydrophilic region Relatively hydrophobic region I n I I Acidic domain Basic (40%) & (30%) R'fiCh (24%) Cys-rich & zinc-finger l 40 150-200 360 473 MDV ICP27 NH2 Acidic domain Basic (32%) & Cys-rich & zinc-finger (36%) R-rich (27%) ”O '7' 400 512 HSV-IICP27 NH2 . " _.,,.,_,.,..,J -CO0H / l \ 262 505 ACT INLS.NuLS.&RGGbox] ,3, $05 REP 262 407 DOM Figure]. 2 Schematic structures of the predicted polypeptides of MDV ICP27 (A), and HSV-l ICP27 (B). ACT, activation region; REP, repression region; DOM, dominant region; NLS, nuclear localization signal; NuLS, nucleolar localization signal B. Varicella-zoster virus (V ZV) ORF4 Varicella-zoster virus (V ZV) open reading frame 4 (ORF4) encodes a 452-amino acid polypeptide which is the VZV homolog to HSV-l ICP27 (Inchaupe and Ostrove, 1989; 23 Perera et al., 1994). The protein properties and functions of ORF 4 can be summarized as below: (1) ORF4 protein shares an overall amino acid identity of 27% with HSV-l ICP27, while certain regions, especially in the C-terminal portion, can be as high as 45%. A cys-rich domain and a putative zinc-finger motif are also conserved in the C-terminal region. (2) ORF4 protein acts as a transcriptional activator that can effectively activate certain VZV gene promoters such as ORF62 (ICP4 homolog), TK gene promoter, and a heterologous HIV-LTR (Inchaupe and Ostrove, 1989; Defechereux et al., 1993). (3) ORF4 has little or no effect on late gene promoters. (4) ORF4-mediated induction of gene expression occurs primarily at the level of transcription since 3’RNA-processing signals are not dominant determinants for the ORF4-mediated transactivation activity (Perera et al., 1994). (5) Oligonucleotide-directed site-specific mutagenesis indicates that of 10 cysteine residues in the ORF4 polypeptide, only C-421 and C-426 are essential for transactivation function. Both C-421 and C-426 are located within the putative zinc- finger motif conserved in numerous IVP27 homologs. Perera et al. (1994) further suggests that protein-protein interactions may be essential for ORF 4 inducibility and that amino acids C-421, C-426, and H-417 may play a critical role in these interactions. C. ICP27 homolog in B-herpesvirus The UL69 Open reading frame of human cytomegalovirus (HCMV) is homologous to the immediate-early protein ICP27 of HSV-1. Unlike HSV-l ICP27 which is transcribed as an immediate-early gene, HCMV UL69 belongs to the early gene family and is detected approximately 7 hours infection infection (Winkler et al., 1994). Protein of 24 HCMV UL69 localizes within intranuclear inclusions that are characteristic for HCMV infection. Cotransfection assays have shown that HCMV UL69 is able to transactivate an HCMV early promoter, UL112, as well as several heterologous promoters, whereas HCMV late promoters could not be activated by UL69 (Winkler et al., 1994). In addition, UL69 protein cannot substitute for HSV-l ICP27 in the context of HSV-1 infection, suggesting functional differences between the two proteins (Winkler et al., 1 994). D. ICP27 homolog in y-herpesvirus ICP27 gene homolog is not only conserved in a- and B- herpesvirus, but also in y- herpesviruses. These include BMLFl of EBV and the lE-52 gene of herpesvirus saimiri (Kenney et al., 1989; Nicholas et al., 1988). They will be described in some detail in a later section. 3. Other HSV-l IE genes A. HSV—l ICP4 As a major transcriptional activator, ICP4 is one of the most extensively studied immediate-early proteins in HSV—l (Reviewed by Roizman and Sears, 1995). ICP4 is encoded by the IE175 gene located within the short unique repeats. Therefore, there are two copies of ICP4 in each HSV—l genome. ICP4 is a highly phosphorylated protein and predominantly localizes in nucleus of infected cells. ICP4 is 1,298 amino acids in length with a predicted protein molecular weight of 133 kDa. However, there are at least three 25 modified polypeptides in denaturing SDS-PAGE with molecular weights of 160 kDa, 163 kDa, and 170 kDa (Pereira et al., 1977). Although all oc-proteins are phosphorylated in HSV-l, ICP4 appears to be the only poly(ADP) ribosylated a-protein (Preston and Notarianni, 1983). A more recent report indicates that ICP4 is also guanylated and adenylated (Blaho and Roizman, 1991). Through studies involving viral mutant analysis, transient expression assays, and biochemical analysis, it has been established that ICP4 is essential for viral growth and for viral gene expression (Dixon and Schaffer, 1980; DeLuca, and Schaffer, 1985; Roizman and Sears, 1995). The regulatory functions of ICP4 can be summarized as two major activities: (i) transactivation of early and late gene expression; (ii) transrepression of expression of ICP4 gene promoter and possibly other immediate-early gene promoters. The most striking feature of ICP4 is its DNA binding preference. Faber and Wilcox (1986) initially identified a strong binding site with a consensus sequences of ATCGTCNNNNYCGRC where R=purine, Y=pyrimidine, and N=any base. Subsequent studies have reported numerous ICP4 binding sites that did not correspond to this consensus sequence (Michael et al., 1988). Thus, HSV-l ICP4 can bind to both consensus and non-consensus sites (Michael et al., 1988). Although it is clear that ICP4 is essential for induction of early gene and some late gene expression, exhaustive studies have failed to reveal any evidence for the existence of ICP4-specifc induction sequences in target promoters (Coen et al.,1986). It has been suggested that the binding of ICP4 to specific sites is not required for ICP4-mediated transactivation (Gu and DeLuca, 1994). However, specific ICP4-binding sites are required for ICP4-mediated repression activity 26 (Gu and DeLuca, 1994; Smith et al., 1995; Roizman and Sears 1995). Studies have shown that ICP4 can transactivate a minimal promoters in which the only recognizable cis-element is a TATA box (Imbalzano and DeLuca, 1991). This finding suggests that ICP4 operates through the basal transcriptional machinery acting on the TATA box. This hypothesis was supported by a subsequent study that was reported by Smith et a1 (1993). By gel retardation and footprinting assays, they found that ICP4 forms a tripartite complex with TFIIB and either TATA—binding protein (TBP) or TFIID. Formation of this complex was not result of the simple tripartite occupancy of DNA but the consequence of protein—protein interactions (Smith et al., 1993). Therefore, it was speculated that formation of the TPB-TFIIB-ICP4-DNA complex was involved in the mechanism of ICP4 function, particularly including its repression activity. A more recent report has provided a strong correlation between tripartite complex formation and repression activity of ICP4 (Kudus et al., 1995). Both tripartite-complex formation and transcriptional repression are efficient when the ICP4-binding site is downstream of the TATA box, with a short distance (less than 40 bp) and in a proper orientation (Kudus et al. 1995). In contrast, when the TATA box and the ICP4-binding site was separated by more than 50 bp, both tripartite-complex formation and repression were concomitantly reduced. This observation strongly suggests that ICP4-mediated repression activity is strikingly related to the degree of tripartite-complex formation on the ICP4 promoter. In addition, when the ICP4-binding site was in its natural orientation, threefold greater tripartite-complex formation was observed. It also suggested that ICP4 predominantly represses transcription in a direction-dependent manner and not simply by blocking transcription (Kudus et al, 1995). 27 B. HSV-l ICPO HSV-l ICO (or Vmw 110) is encoded by a spliced IE gene that lies in TRL and IRL, therefore like ICP4, there are two copies in the viral genome (Preston et al., 1978). DNA sequence and SI nuclease mapping analysis showed that theHSV-l ICPO gene contains three exons separated by two introns. Like other IE gene products, ICPO is predicted to be 775 amino acids with a molecular weight of 80 kDa, however, in denaturing SDS- PAGE gel it has a molecular weight of 110 kDa. ICPO is highly phosphorylated and predominantly located in nuclei of infected cells (Wilcox et al., 1980; Perry et al., 1986). Primary structure of ICPO exhibits some striking features that, at least partially, reflect the protein activities. The first 71 amino acid residues of the [CFO N-terminus are highly acidic, adjust to two zinc-finger motifs that have been described as functional DNA- binding domains in several transcriptional factors (Berg, 1986; Evans and Hollenberg, 1988). This potential zinc-finger region is critical for the effects of ICPO both in vitro and in vivo (Evertt et al., 1991). By site-directed mutagenesis, it has been found that substitution of Cys or His residues in the zinc-finger domain abolishes ICPO-mediated transactivation (Moriuch et al. 1992). Two proline-rich regions are present in exon 3, but the function of these proline-rich domains is unclear. ICPO may dimerize or oligomerize, based on biochemical properties of the purified intact ICPO protein (Chen, et al., 1992; Everett et al., 1991). Transient expression assays indicate that ICPO has unusually powerful and promiscuous activities. The list of promoters that can be transactivated by ICPO includes promoters from all three kinetic classes of HSV-1 genes, heterologous promoters such as 28 HIV-LTR and SV40 early promoter, and host gene promoters (reviewed by Everett et al., 1991). ICPO response elements have not been clearly identified, partially due to its unusually broad range of induction on target promoters. Promiscuous activity of ICPO suggests that ICPO may nonspecifically bind to target promoters and interact with some transcription factors, in which ICPO functions as a bridge molecule (Chen et al., 1992; Everett, 1991). However, the mechanism of how ICPO mediates gene activation is not clear. In some cases, ICPO shows synergistic activity with ICP4. By both in vitro and in vivo assays, ICPO exhibits much stronger transactivation activity in synergy with ICP4 than in absence of ICP4. By a serial in-fi'ame insertion mutant analysis, the carboxyl region (between amino acids 633 and 723) and a Cys-rich domain in the N-terminus were identified as essential for ICPO synergy with ICP4 (Everett et al., 1991; Everett, 1988) ICPO localizes in a very unusual punctate pattern consisting of several dozen phase- dense granules in DNA transfected cells. In contrast, ICPO normally localizes in a much smaller micro-punctate pattern at early times in virus infected cells (Chen et al., 1991; Everett et al., 1988; Giufo et al., 1994). More recently, a short basic amino acid motif VRPRKRR mapped to amino acid sequences 500-506 has been identified as a functional motif for ICPO nuclear localization (Mullen et al. 1994). A similar motif with a sequence of GRKRKSP has also been identified in HSV-l ICP4 protein between amino acids 726-732 (Mullen et al., 1994). By analysis of deletion mutants and analysis of ICPO/ICP4 hybrid protein, it has been suggested that this nuclear localization motif does not contribute to ICPO unusual punctate pattern. In contrast, amino acid residues from 105 to 244 are critical for conferring the punctate localization feature (Mullen et al., 29 1994; Giufo et al., 1994). ICP4 and ICP27 can inhibit ICPO nuclear localization both in transiently transfected cells and in virally infected cells (Zhu et al., 1994). These negative effects may contribute to the functional cooperation among these three or- proteins. Based on mutation studies in viva, it appears that ICPO is not essential for lytic infection in cell culture (Chen and Silverstein 1992; Everett 1991). Under high multiplicity infections, ICPO function is dispensable for viral DNA replication, viral polypeptide synthesis, and Virion formation (Sacks and Schaffer, 1987; Everett 1991). Importantly, ICPO has also been suggested to play a role in the efficient establishment and reactivation of latency, where viral gene expression is limited to transcription of the latency-associated transcript (LAT) and no infectious virus can be detected (Leib et al., 1989) . However, viruses with mutations in both copies of ICPO do not reactivate from latency as efficiently as wild type viruses. The role of ICPO in establishment of latency may increase the efficiency of virus replication in primary infections and in ganglionic neurons, the site of latent infection (Leib et al., 1989). In reactivation from latency, ICPO may boost viral gene expression at the onset of latency (Cai and Schaffer, 1992; Leib et al., 1989). Therefore, an alternative pathway, independent of VP16-Oct-1, may exist for activating IE gene expression, particularly in the early stage of reactivation from latency where VP16 is absent. ICPO has been suggested to play a back-up role for VP16 in IE gene expression (Elsshiekh et al., 1991). However, the molecular mechanism by which ICPO genes are regulated to exert reactivation of latency is not clear. 30 C. HSV-l ICP22 and ICP47 HSV-l ICP22 and ICP47 have been less studied than other HSV-l IE genes. HSV—l ICP22 is encoded by the U81 gene, located in the unique short region. ICP22 is a dispensable gene since deletion of ICP22 had no effects on DNA synthesis and Virus infection. However, in some cell cultures, such as RAT-l cell lines and human embryonic lung cells, plating efficiency of virus with ICP22-mutant was reduced and the yield of virus was dependent on the multiplicity of infection (Sears et al., 1985; Roizman and Sears, 1995). It has been reported that HSV-l infection results in a rapid alteration of phosphorylation on the large subunit of cellular RNA polymerase II. This modification generates a novel form of the large subunit, designated IIi (Rice et al., 1994). Further studies suggest that 22/n99, an HSV-l mutant containing a nonsense mutation in ICP22, is significantly deficient in IIi induction. In 22/n99 infected Cells, late gene transcription is less efficient, and antisense transcription through out the genome is diminished compared with that of wild type infection (Rice et al., 1995). These results suggest that HSV-l ICP22 may be necessary for virus-induced aberrant phosphorylation of RNAP II and for normal patterns of viral gene transcription in certain cell lines (Rice et al., 1995). HSV-l ICP47 is the only IE protein that does not Show obvious regulatory functions in HSV—l infection. Thus, the function of this gene is unclear. 4. The B-herpesvirus IE genes and their function Human cytomegalovirus (HCMV) is a prototype virus in the B-herpesvirus subfamily. HCMV is highly species specific and has the largest genome (229 kb) (Marcarski, 31 1995). As with (Jr-herpesviruses, gene expression of HCMV in infected cells occurs in a cascade fashion. At the immediate-early time, gene expression is restricted to four loci of the genome. The most abundantly expressed IE region, termed the major IE gene region, is located in the large unique (UL) component between 0.732 and 0.751 map units. Two transcripts from the major IE gene region, designated IE1 and IE2, have been extensively studied. A striking feature is that both IE1 and IE2 share a common regulatory region defined as an enhancer-containing promoter-regulatory region. This promoter-regulatory region is unusually strong and contains multiple sets of highly conserved repetitive elements. (Reviewed by Stinski et al., 1991). The IE1 gene, immediately down stream of the major IE promoter, encodes a spliced mRNA consisting of 4 exons, designated exons 1, 2, 3, and 4 respectively. The translation initiation codon of IE1 is located in exon 2. IE1 encodes a highly phosphorylated protein with a molecular mass ranging from 68 to 72 kDa. The largest polypeptide, designated 72kDa-E1, has been extensively investigated and will be discussed in more detail. The three different sized transcripts derived from IE2 region share a common 3' region, but differ in their 5’ regions, due to alternative splicing. Three polypeptides derived from these transcripts are termed 86-kDa IE2, 54-kDa IE2, and 28-kDa IE2, based on their molecular weights. Functions of these IE proteins are dramatically different, although they share some common domains. 72- kDa IE1 and 86-kDa IE2 are the best characterized proteins. Briefly, 86-kDa IE2 independently transactivates Viral early and weak late gene (y, gene) promoters, as well as heterologous early promoters from adenovirus, while it negatively regulates the major IE promoter. In contrast, 72-kDa IE1 positively regulates both IE1 and IE2 gene 32 expression in HCMV infected cells, but has no significant effect on early and late gene expression (Hermiston et al., 1987; Malon et al., 1990; Stinski et al., 1991). Furthermore, 86-kDa IE2-mediated transactivation is augmented inithe presence of 72- kDa IE1. However, both IE1 and IE2 gene proteins fail to stimulate true late (72) gene expression. This suggests that additional regulatory proteins encoded by HCMV may play a role (Depto and Stenberg, 1992; Puchtler and Stamminger, 1991), but these factors remain to be identified. 5. The y-herpesvirus IE genes and their functions Epstein-Barr virus (EBV) is a prototype of y-herpesviruses that can infect human B lymphocytes and induce cellular proliferation or transformation. Latently infected lymphocytes persist for life and can be established as continuous lymphoblastoid cell lines. EBV transcription has been analyzed for the most part in lymphoblast cell lines. Most of the infected lymphocytes are latently infected and only limited number of viral transcripts can be detected. It is difficult to study gene regulation in EBV since EBV lacks a lytic tissue culture system. B cells latently infected with EBV can be induced to express lytic cycle genes by treatment with variety of agents including phorbol esters, butyrate, and anti-immunoglobulin antibiotics (Hamper et al., 1974; Raab-Traub and Gilligan, 1991; Hayward and Hardwick, 1991). There are three immediate-early genes in cells lyticly infected with EBV, including the Z transactivator (Zta), the M transactivator (Mta), and the R transactivator (Rta). Zta, also termed BZLFl, is a major transactivator for induction of EBV lytic gene 33 expression. Zta gene has been mapped within the BamHI-Z fragment. The Zta proteins are encoded by two spliced mRNAs transcribed from two different promoters. A 0.9 kb mRNA is initiated from an unusual TATA box (TTTAAA) and encodes Zta protein, whereas a 2.8 kb bicistronic mRNA is transcribed from an upstream promoter of BRLFl and encodes both Zta and Rta proteins. Zta is a highly phosphorylated protein and is localized to the nucleus (Daibata et al., 1992). By cotransfection assays, several EBV promoters have been identified as Zta responsive promoters in which Zta can recognize and bind to a highly conserved seven-hp sequence, ZRE (Zta response elements). The interesting thing is that Zta not only binds to homologous promoters but also is capable of binding to a sequence TGACTCTA, the AP-l recognition site for c-jun/fos transcriptional activation. Exon 2 of Zta has a high homology to a basic DNA binding domain conserved in c-jun/fos family (Lieberman et al., 1990; Hayward and Hardwick, 1991). Expression of the Zta triggers disruption of latency in EBV-infected cells. Rta, also termed BRLFl, is encoded within a multi-spliced 2.8 kb mRNA that is bicistronic. This bicistronic transcript also contains the complete Zta coding sequences downstream of the Rta open reading frame. Rta is predominantly localized in the nuclei. Three EBV promoters have been identified as Rta responsive promoters, including the duplicate sequences DSL‘ DSR, and the Mta promoter. The Rta responsive elements have been mapped in all three promoters. These consensus elements function as enhancer elements and behave in a position and orientation independent manner (Kenney et al., 1989; Hayward and Hardwick, 1991). The heterologous promoter, HIV-1 LTR is also stimulated by Rta. However, Rta transactivation of the HIV-1 promoter does not require the HIV-1 enhancer. Thus, Rta may transactivate by at least two different mechanisms, 34 one mechanism involving certain enhancer elements and another mechanism being enhancer independent (Quinlivan et al., 1990). Mta, also known as BMLFl, is the product of the BSLFZ/ BMLFl open reading frames that consist of two alternatively spliced mRNAs. Mta consists of three major polypeptides with molecular weights of 45, 50, and 60 kDa. The 50-and 60-kDa Mta polypeptides are phosphorylated. Mta is homologous to HSV-l ICP27. Thus, Mta is the only tit-herpesvirus IE gene homolog that has been identified in EBV. In cotransfection assays, Mta can transactivate CAT reporter constructs derived from both homologous and heterologous promoters. This Mat-induced activation of promoters is reporter-gene dependent and functions at the post-transcriptional level (Kenney e al., 1989). However, the mechanism of Mta-mediated transactivation is not clear. As described above, ICP2 7 gene families play some critical roles for herpesvirus growth and for viral gene expression. The specific aims of this project are I ) to identify the ICP27 homolog in MDV and firrther characterize the gene product; 2) and, to investigate and evaluate the regulatory fimctions of the MDV ICP27 homolog. We will focus on in vitro studies of the transcriptional regulatory mechanism of the MDV ICP27 homolog. Chapter 11 Identification and Characterization of Marek's Disease Virus Genes Homologous to ICP27 and Glycoprotein K of Herpes Simplex Virus-l Delin Ren], Lucy F. Leez, and Paul M. Coussens" lMolecular Virology Laboratory, Department of Animal Science, Michigan State University, East Lansing, Michigan 48824; and 2USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, 3606 East Mount Hope Road, East Lansing, Michigan 48823 Virology 204:242-250 (1994) Sequence data from this article have been deposited with EMBV/Gene Bank Data Libraries under Accession No. U10040 *To whom reprint requests should be addressed. 35 ABSTRACT We have identified two Marek's disease virus (MDV) genes within the EcoRI-B fragment of MDV-GA genomic DNA. EcoRI-B is 11.3 kb long and maps within the long unique (U L) region of MDV genome. A 3.2 kb fragment of EcoRI-B has been sequenced and contains two open reading frames, ORF53 and ORF54. ORF53 (MDV gK), a homolog to HSV-l glycoprotein K (gK), is 1,062 nucleotides (nt) long and encodes 354 amino acids (39.5 kDa). ORF54, designated MDV ICP27, based on significant similarity to HSV-l ICP27, is 1,419 nucleotides long and encodes 473 amino acids (54.5kDa). In Northern blot hybridization, two overlapping transcripts (2.9kb and 1.6kb) were detected in MDV-infected DEF cells treated with cycloheximide, suggesting that both transcripts belong to the immediate-early gene family. Amino acid sequence analysis of MDV gK shows some common glycoprotein features, including a putative N-terminal signal sequence, four N-linked glycosylation sites, and four potential transmembrane domains. Comparison of the predicted amino acid sequence of MDV ICP27 with that of HSV-1 ICP27 and VZV ORF4 shows a high degree of conservation within the C-terminus. The C-terminal region of HSV-1 ICP27 has been demonstrated to be critical to its function. A conserved zinc finger metal-binding motif C(442)-X4-C(447)-X13-H(461)-C(467) was also found in the C-terrninus of MDV ICP27. Furthermore, MDV ICP27 upstream sequences contain four copies of consensus sequence elements similar to the tegument protein target sequence TAATGARAT. TrpE-ICP27 fusion protein was expressed in E. coli, and rabbit antisera were generated using purified fusion protein. A 55 kDa protein has been detected in both MDV GA and Mdll infected cells using immunoblot analysis. 36 INTRODUCTION Marek's disease virus (MDV) is a cell-associated herpesvirus that induces T-cell lymphomas and peripheral nerve demyelination in susceptible chickens (Calnek, 1985). MDV was originally classified as a gamma herpesvirus on the basis of its lymphotropism. However, MDV genomic structure closely resembles members of the alpha herpesvirus group (e.g. herpes simplex virus, and varicella-zoster virus) (Buckmaster et al., 1988; Roizman et al. , 1992). Herpesvirus genes are classified into three kinetic classes, immediate-early (IE), early (E) and late (L) genes, based on requirements for viral protein synthesis and DNA replication (Honess et al., 1974). To date, five immediate-early (IE) gene products have been identified and mapped in herpes simplex virus type-1 (HSV-l), including ICPO, ICP4, ICP22, ICP27, and ICP47 (Honess et al., 1974; Sacks et al., 1985). ICP4, as a major regulatory protein, plays an essential role throughout the viral replication cycle (DeLuca et al., 19850). ICPO has been shown as a potent transcriptional activator (Chen et al., 1992). HSV-l ICP27 is a 63 kDa phosphoprotein localized in the nucleus of infected cells (McCarthy et al., 1989). A series of temperature-sensitive (ts) mutants of ICP27 have been isolated and provided powerful tools to study gene function (Sacks et al., 1985). ICP27 is an essential protein which is required for virus replication and for modulation of early and late gene expression at transcriptional and post-transcriptional levels (McCarthy et al., 1989; Smith et al., 1991; Sandri-Goldin et al., 1992). Evidence from transfection studies has shown that ICP27, in the presence of ICP4 and ICPO, can 37 38 repress some early genes (e.g. thymidine kinase gene) and enhance expression of some late genes (e.g. VP5 and glycoprotein B) (Smith et al., 1992), but it has little or no trans-regulatory effect on target genes by itself (Sekulovich et al., 198 8). Genes encoding ICP27 homologues have been identified for other alpha hepesviruses, including varicella-zoster virus (V ZV) (Davison et al., 1986) and equine herpesvirus type 1 (EHV-l) (Zhao et al., 1992). Though similar in amino acid sequence (28% identity), VZV ORF4 has been shown to be functionally distinct from HSV-l ICP27 (Moriuchi et al., 1994; Perera et al., 1994). Unlike its HSV-l counterpart, VZV ORF4 efficiently activates heterologous promoters in the absence of other virus proteins (Defechereux et al. , 1993). To date, little is known about MDV immediate-early genes and the gene products they may encode. A gene encoding MDV ICP4, a homolog to HSV-l ICP4, has been identified and mapped within BamHI fragment A of the MDV genome (Anderson et al. , 1992). However, the function of MDV ICP4 remains to be determined. Recently, a 1.6 kb spliced immediate-early gene has been reported and mapped to BamHI-IZ fragment, within the MDV inverted repeat region (IRL). Protein products encoded by this gene appear to be MDV specific (Hong and Coussens, 1994). In this study, we report the DNA sequence of the two open reading frames within the MDV genome which encode proteins homologous to HSV-l glycoprotein K and ICP27. Two overlapping transcripts, corresponding to MDV gK and ICP27 genes, were identified in Northern blot analysis. Based on expression in the absence of protein synthesis, both transcripts may belong to the immediate-early gene family. MDV gK shows critical homology to HSV-l gK and has some common features of glycoproteins. 39 Because our laboratory is primarily concerned with understanding MDV gene regulation, further attention was focused on MDV ICP27. By comparing to other alpha herpesvirus counterparts, MDV ICP27 exhibits a significant conservation in its C-terminal region. Rabbit antiserum specific against MDV ICP27 was produced using trpE-ICP27 fusion protein in order to detect ICP27 gene products in MDV infected cells. MATERIALS AND METHODS Cells and viruses Duck embryo fibroblast (DEF) cells were prepared, maintained and infected with MDV according to previously described procedures (Glaubiger et al, 1983). Two low passage cell-associated MDV serotype 1 strains, GA (passageIO) and Mdll (passage12), were used for this study. DEF cultures were grown in Leibovitz-McCoy medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% calf serum at 37° C, in a humidified atmosphere containing 5% C02. Calf serum concentration was reduced to 1% following infection. DNA sequencing An EcoRI-B fragment (11.3kb) from the genomic library of MDV-GA was kindly provided by Dr. Robert Silva (USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory, East Lansing, MI). A series of overlapping subclones, representing the entire 11.3kb of EcoRI-B, were constructed in pUC18. DNA sequencing was performed on double stranded plasmids by dideoxy chain termination (Sanger et al. , 1977) using [a-3SS]ATP (NEN Research Products) and TAQuence DNA sequencing kits (United States Biochemical Corp., Cleveland, Ohio). DNA sequences were analyzed using MacVector 3.5 (International Biotechnologies), programs GAP and PILEUP of the University of Wisconsin Genetics Computer Group (GCG), GenBank release 80.0 (IntelicGenetics), and the Protein Identification Resource, Version 38.0 (National 40 41 Biomedical Research Foundation). Total cellular RNA isolation and northern blot analysis Total cellular RNA was isolated from mock-infected and MDV-infected DEF cells using the guanidinium-phenolzchloroform method as described by Chomczynski et al. ,(1987). Immediate-early (IE) and early RNAs were obtained by adding cycloheximide (CHX,100ug/ml) and phosphonoacetic acid (PAA, 100 ug/ml) at the time of infection, respectively. IE RNA was extracted 12 hours post-infection and CHX treatment. Early RNA was extracted 24 hours post-infection and PAA treatment. Total RNA (10ug) was loaded onto 1.2% agarose gels containing 6% formaldehyde and electrophoresed for 12 hours at 30V. RNA was transferred onto Hybond-N membrane (Amersham Corp., Heights, IL) as described by Sambrook et al., (1989). Two DNA fragments, ClaI-Clal (CC, Figure 2.1) and BamHI-Kpnl (BK, Figure 2.1) were used as probes and labeled using [a32P]dCTP (NEN Research Products). Northern blot hybridization was performed using standard procedures (Sambrook et al., 1989). Transcript size was determined by comparison to an RNA ladder marker (BRL, Gaithersburg, MD). Expression of trpE fusion protein The pATH vector systems, which encode a 37 kDa bacteria trpE ORF under control of an inducible trp promoter, were used to express trpE-MDV fusion proteins (Chen et a1, 1992). A 688 bp BamHI-CIaI fragment (BC), encoding MDV-ICP27 amino 42 acid residues 24 to 252, was cloned between BamHI and ClaI sites of pATHll (Figure 2.1). A 520 bp [{pnI-Bglll fragment (KBg) which represents the ICP27 ORF coding region between amino acids 205 and 378 was cloned into pUC18 to generate pUC18KBg. A second fusion protein was constructed by cloning an EcoRI and HindIII fragment from pUC18KBg into pATH3 (Figure 2.1). TrpE-ICP27 ORF fusion proteins were analyzed by 8% SDS-PAGE and partially purified as described previously (Chen et al., 1992). New Zealand white rabbits were injected with 200mg of fusion protein emulsified in Freund's complete adjuvant (Life Technologies, Inc., Gaithersburg, MD). Rabbits were boosted with the same amount of protein in Freund's incomplete adjuvant after 4 weeks interval and bled ten days following the last injection. Western blot analysis Mock infected and MDV infected DEF cell lysates were prepared with triple-detergent lysis buffer (Sambrook et al., 1989). Lysates were separated on 12% SDS-PAGE Minigels (Bio-Rad), and transferred to Nitrocellulose membrane (NC) (Scheicher & Schuel). NC membranes were blocked using 5% dry milk. Rabbit antiserum against fusion protein was used at a 1:100 dilution, and donkey anti-rabbit IgG conjugated with horseradish peroxidase was used as second antibody. Amersham ECLTM was used as substrate according to the manufacturer's specification. Protein translation in vitro and immunoprecipitation analysis The MDV-ICP27 gene was modified by the polymerase chain reaction (PCR) to create a BglII restriction site in front of the ATG codon. Primer sequences were 43 5'-CCCCAGATCTAAAAATGTCTGTAGATGCATTCT3' and 5'-CATGGTCATTCCA- CATCGAA3'. A 628 bp BglII-Kpnl fragment obtained from PCR products was ligated with a 1,077 bp KpnI-Xhol fragment which contains C-terminal coding regions of the ICP27 gene (Figure 2.1) and cloned between BamHI and XhoI sites of pBluescript KSII+/- vector (Figure 2.1, pBlue-ICP27). A 549 bp BamHI-Kpnl fragment of ICP27 was cloned into prokaryotic expression vector pRSET-A, directly downstream of a T7 promoter using histidine-tagged protein ATG as the translation start codon. The TNTTM T7 Coupled Reticulocyte Lysate System (Promega) was used for coupled in vitro transcription/translation. Translation products were labeled using [3SS]methionine (NEN Research Products). Immunoprecipitation analysis of labeled translation products was carried out as described previously (Silva and Lee, 1984). Rabbit antiserum against trpE-ICP27 fusion protein was used in this experiment. RESULTS DNA sequence analysis of MDV ICP27 and gK genes The MDV EcoRI-B fragment (11.3 kb), located in the long unique (UL) region, spans BamHI-H, K1, M, P3, N, and T fragments. EcoRI-B was chosen to initiate a search for immediate-early gene ICP27, based on structure similarity between HSV-l and MDV (Fukuchi et al., 1984; McGeoch et al., 1988). A partial restriction enzyme map of the MDV EcoRI-B fragment is shown in F ig.1. A 3.2 kb DNA fragment of EcoRI-B suspected to contain the ICP27 has been sequenced in both directions (Figure 2.2). Analysis of nucleotide sequences of this fragment revealed two open reading frames (ORFs) (Figure 2.1 and Figure 2.2). ORF53 is 1,062 nucleotides long and encodes 354 amino acids with a calculated molecular weight of 39.5 kDa. ORF54 is 1,419 nucleotides long and encodes 473 amino acid residues with a calculated molecular weight of 54.5 kDa. ORF54, designated MDV ICP27, has homology with HSV-l ICP27 and VZV ORF4 (Figure 2.3). ORF53, designated MDV gK, is homologous to HSV-l glycoprotein K (gK) (Data not shown). The collinear relationship of gK and ICP27 genes is consistent with their positions in other alpha herpesviruses (Davison et al., 1986; McGeoch et al. , 1988; Zhao et al. , 1992). Translational start codons (ATG) of each ORF have been assigned at nt 427 for MDV gK and nt 1,639 for MDV ICP27, based on comparison with HSV-l homologs and on similarity to Kozak's consensus sequence (Kozak et al., 1989). A number of potential transcriptional elements are present both upstream and downstream of gK and ICP27. Two TATA boxes are located 269 bp and 294 bp upstream from the putative ATG of 44 45 MDV gK. There are three potential TATA boxes located 24 bp, 28 bp, and 87 bp upstream of the putative ATG of MDV ICP27. Three poly(A) signals were found at positions 3,132, 3,142, 3,146. No potential poly(A) signals were identified within the ORF53-ORF54 junction region. Interestingly, four copies of sequence elements, which are very similar to the IE-specific element TAATGARAT responsive to presence of the Virion-associated a gene activator VP16 in HSV-l IE genes, have been found within the putative promoter region of MDV ICP27 (Figure 2.2). Analysis of MDV ICP27 and gK gene Transcripts Northern blot hybridization was performed to detect the gene transcripts of both MDV gK and ICP27. Two DNA fragments, which map within ORF53 and ORF54 were used as probes for detection of transcripts. Probe BK which maps within ORF54 (Figure 2.1), hybridized to two transcripts (1.6kb and 2.9kb) in both MDV GA infected DEF cells and CHX-treated cells (Figure 2.4 A, lane 2 and lane 3). No transcript was detected in mock-infected cells or GA infected cells treated with phosphonoacetate (PAA) (Figure 2.4 A, lane 1 and lane 4). 'Probe CC which maps within ORF53 (Figure 2.1), only hybridized to the 2.9 kb transcript in GA-infected cells and infected cells treated with CHX (Figure 2.4 B, lane 2 and lane 3). These data, taken together, suggest that MDV gK and MDV ICP27 transcripts overlap, sharing a common transcriptional terminus, most likely due to read through transcription of the MDV gK gene. MDV ICP27 is also transcribed independently of the MDV gK gene. This result is consistent with DNA sequence analysis in that there are multiple poly(A) signals in the region downstream of the ICP27 gene, but not between gK and ICP27 (Figure 2.2). Both 46 transcripts were abundantly expressed in untreated MDV infected cells and in MDV infected cells treated with CHX. Neither transcript was detected in MDV infected cells treated with PAA. Accumulation of transcripts in CHX treated cells but not in PAA treated cells is characteristic of MDV IE genes (Lee et al., 1974; Hong and Coussens, l 994). Comparative analysis of MDV ICP27 and gK predicted amino acid sequences Our laboratory is primarily concerned with investigating MDV gene regulation. Given the importance of HSV-1 ICP27 in early and late gene regulation in HSV-l infected cells, we opted to focus additional attention on MDV ICP27. To further define the ORF54 gene as a homolog of HSV-1 ICP27, the deduced amino acid sequence of ORF54 was compared with ICP27 homologs from other alpha herpesviruses. The average amino acid sequence identity and similarity between MDV ICP27 and HSV-l ICP27 are 25.8% and 42.5%, respectively; between MDV ICP27 and VZV ORF4 are 29.5% and 49.4%, respectively. A partial amino acid sequence is shown in Figure 2.3. Interestingly, significant amino acid conservation exists in the C-terminal region of all ICP27 homologues. We find 37.3% identity between MDV and HSV-l ICP27 and 32.7% identity between MDV ICP27 and VZV ORF 4 within the C-terminal region. In contrast, the N-tenninal half of MDV ICP27 has poor homology with both HSV-l and VZV, even though it shares a similar hydrophilic feature (data not shown). In addition, we note a conserved potential zinc finger metal-binding motif C(442)-X4-C(447)-X13- H(461)-C(467) within the MDV ICP27 C-tenninus (Figure 2.3). Based on computer analysis, MDV gK shows a significant similarity to other 47 alpha herpesvirus counterparts, including HSV-l glycoprotein K, and VZV ORFS. The average amino acid sequence identity and similarity between MDV gK and HSV-l gK are 26.6% and 49.7%; between MDV gK and VZV ORFS are 29.4% and 56.1%, respectively (Data not shown). Several common features existed between HSV-l gK and MDV gK; i) a possible N-terminal signal sequence (amino acids 1-30 in HSV-l gK and 1-29 in MDV gK); ii) several potential N-linked glycosylation sites present in N-terrninal regions (two N-glycosylation sites in HSV-l gK; four N-glycosylation sites in MDV gK); iii) the similar location of four potential hydrophobic transmembrane domains (Figure 2.2). Analysis of MDV ICP27 gene products Two DNA fragments, BC and KBg (Figure 2.1) were cloned into pATH bacteria expression vectors (Koener et al., 1991) to generate in frame trpE-ICP27 fusion proteins. Clone KBg, encoding amino acids 205 to 308 of MDV ICP27, produced a 57 kDa fusion protein in great abundance (Figure 2.5). On the contrary, clone BC, which encodes amino acids 24 to 252 of ICP27 ORF and has a significant hydrophilic characteristic, failed to express in trpE expression vectors (Figure 2.5). Antisera were produced by immunization of New Zealand white rabbits with trpE-KBg fusion proteins, as described in Materials and Methods. To demonstrate specificity of antiserum against trpE-KBg fusion protein, in vitro translation and immunoprecipitation studies of MDV ICP27 were performed as described in Materials and Methods. A trpE-KBg antiserum immunoprecipitated a 55 kDa polypeptide from pBlue-ICP27 primed in vitro transcription/translation products. As expected, no product was detected with a 48 pRSET-BK primed reaction (Figure 2.6A). These results indicate that trpE-BKg antisera can specifically detect a MDV ICP27 gene product. In HSV-l, ICP27 is essential for replication and growth of the virus (Sacks et al., 1985). To determine if MDV ICP27 is expressed in lytically infected cells, western blot analysis was performed using MDV-infected cell extracts and our trpE-KBg antisera. A 55 kDa protein was detected in both MDV GA and Mdll infected DEF cells, but not found in mock-infected DEF cells (Figure 2.6B). The size of this polypeptide is consistent with that predicted from translation of the MDV ICP27 ORF. DISCUSSION We have identified and sequenced two genes located within the EcoRI-B fragment of the MDV genome. These correspond to coding units for HSV-l UL53 and ICP27, based on gene arrangement and amino acid sequence similarity. MDV UL53 is similar to HSV-l UL53 encoding glycoprotein K (gK) (DebRoy et al., 1985). Several syncytial mutations of HSV-1 have been mapped to the UL53 gene (Pogue-Guile and Spear, 1987). The recent reports indicate that the natural HSV-l UL53 gene product is involved in cell fusion of HSV-1 infected cells (Hutchinson et al. , 1992; Ramaswamy and Holland, 1992). Although MDV gK shows some common glycoprotein features and critical similarity to HSV-l gK, such as N-terminal Signal sequence, N-linked glycosylation sites, and similar location of hydrophobic transmembrane domains, the precise function of MDV gK remains to be determined. HSV-l ICP27 was reported to act as a transactivator of late gene promoters, when expressed in combination with ICP4 and ICPO, but has little or no effect by itself (Evert et al., 1987; Smith et al., 1991). HSV-l ICP27 can also act as a transrepressor on several immediate-early and early gene promoters (Hardwieke et al., 1989, Rice et al., 1989). The positive and negative regulatory activities of HSV-1 ICP27 are separable (Rice et al. , 1989). Recent studies define the activator region as those sequences which lie between residues 260 and 434. The repressor region has been localized to the C-terminal 78 amino acids (Smith et al., 1991). In contrast, VZV ORF4 directly transactivates plasmids containing homologous or heterologous promoters and has no apparent transrepressing activities (Moriuchi et al., 1994). Interestingly, the C-terminal region of MDV ICP27, 49 50 HSV-l ICP27, and VZV ORF4 are strikingly similar (37.3% identity between MDV ICP27 and HSV-l ICP27; 32.7% identity between MDV ICP27 and VZV ORF4, see Figure 2.3). A putative zinc finger metal-binding domain within the C-terminal repressor region of HSV-1 ICP27 is conserved in MDV ICP27. HSV-l ICP27 binds zinc in vitro (Smith et al., 1991) and the zinc finger motif was shown to be involved in binding DNA, RNA and protein-protein interactions (Breg et al., 1986). The importance of this element in ICP27 function is underscored by conservation in many alpha herpesviruses, such as VZV (ORF4) and EHV-1 (ORF3) (Perera et al., 1994; Zhao et al. , 1992). Based on structure similarities, we speculate that MDV ICP27 may have similar functions in MDV gene regulation. Four DNA elements were found within the MDV ICP27 putative transcriptional control region with striking similarity to the sequence TAATGARAT, an essential element of HSV-1 immediate-early gene promoters (Greves et al., 1990). A virion tegument protein, VP16, interacts with cellular factor Oct-1 (octamer-binding protein), and forms a protein-DNA complex with the target sequence TAATGARAT in HSV-l IE promoter regions ( Gelman et al., 1987; Moriuchi et al., 1993). Recently, the gene encoding MDV VP16 has been identified (Yanagida et al., 1992). Experiments to explore the possible activation of ICP27 expression by MDV VP16 are in progress. Two significant features of MDV ICP27 were demonstrated in this report: i) Two different size transcripts are highly expressed in MDV infected cells treated with cycloheximide. Herpesvirus immediate-early (IE) genes usually express immediately upon infection and do not require de novo protein synthesis. Thus IE transcripts are the only viral RNA transcribed when infected cells are treated with a protein synthesis 51 inhibitor, such as cycloheximide. Our results suggest that both transcripts observed in northern blots with ICP27 probes belong to the immediate-early family; ii) Two transcripts were detected using probe BC (which maps within the ICP27 gene ORF). However, only the larger transcript (2.9kb) was detected using the probe CC (which maps within the gK ORF). Read-through transcription of the ORF53-54 gene region is likely due to absence of polyA signals between MDV gK and ICP27. The transcriptional pattern of MDV ICP27 and gK is similar to that of VZV and EHV-1 (Inchauspe et al., 1989, Zhao et al., 1992); but different from that of HSV-1 which contains two independent polyA signals (DebRoy et al., 1985). It will be of considerable interest to learn whether MDV gK is translated as an immediate-early glycoprotein or if gK translation is repressed until later in infection. HSV-l ICP27 is a phosphorylated protein located predominately in the infected cell nuclei, where it can be detected with monoclonal antibodies (Ackermann et al., 1984). HSV-l ICP27 is a multifunctional transcription/translation regulator. The importance of ICP27 is underscored by lethality of ICP27 deletions. It will also be of considerable interest to determine if MDV ICP27 displays the same multifunctional role as HSV-l ICP27. ACKNOWLEDGMENTS We thank N. Yanagida for critical technical assistance and computer analysis. This work was supported in part by a grant from Nippon Zeon awarded to Lucy F. Lee; by the Michigan Agricultural Experiment Station; by Research Excellence Fund State of Michigan; and by grant 92-8420-7430 awarded to P. M. Coussens under the Competitive 52 Research Grant program administered by US. Department of Agriculture. 53 Figure 2.1 Diagram of MDV-GA structure and location of MDV gK and MDV ICP27. Genes are indicated by arrows. DNA fragments or plasmid clones are indicated by unbroken lines. Clone names are shown below the appropriate paragon. Restriction enzymes used are: B, BamHI; Bg, Bglll; C, ClaI; E, EcoRI; K, Kpnl; Xh, XhoI. 54 Kb. -85.. 091.2,. 8+5... v5-52... 8 +2.20: 12% Away nnumo _ __ no on m m m— In m— miEoom 7.". .V\\\\\\\\\\\ .///////////A 7”] .Pmozw Q‘s 7///////////. Q20:— MDd-Z: \\\\\\\\\\\\\ mmh ”.592: mm. .3: 4m... 55 Figure 2.2 Nucleotide sequence of a 3,200 bp fragment of MDV GA from EcoRI-B. Predicted amino acid sequence of gK and ICP27 are indicated below the DNA sequence. Potentially important sequence motifs are indicated as follows: A, TATA box; *, polyA signal. The four potential IE-specific regulatory elements (TAATGARAT) are underlined. Glycoprotein signal sequence, N-linked glycosylation sites and potential transmembrane domains are shown and underlined. 56 TATATTTTTCCGGACTCGATTAICTTTATCAATAGCTCTCAGGGAAAGGGGGGTAGATATAAATGATTGTCCTGCCATTRCAACATTTGTAACAGAJCAC AITTTABATCATATTATAACATACGTATATGAGCATATACCAGATCACGCAATOGAATATCAAAAJCTTTCTGTCTCGTGTTGTCTTGTCAAATCGGATT ....... ....... GGATCCTCCTCCAGCTAATCCCCAATAAAACAASAGGATATCCTCACGGGTTTACAJCTGTCAGATTTAAGCATGCAAGAGCAAGGCGAGCGAGTGCACG TTCTTATTTGGCTCTGAACGTCCAJGCGCATGGTAGGTTCTGCGTATGTGTAATTCAACAGTGTTTTGCGGCCAACTGCGGAAATAATAAACTTCGCACA CTTTTCACGGTAGATATTCACTCGAAASGTCGATTAGAACATCAATAGCTCTCATCCGAATTTTATTTGCTATATCGATATATACTCTTTTATTCGTACT MDV 9R fl 3 I B I S I A L I Q I .L P A I 8 I T T V L L V V TTATCTA1CGACCCTTTCCCAAAATGGATCTGGATGTATCTATGCTACCTTGGTCCACAGTAGCCTCTATGACGCCAAAAACTTCACCTCGGAACAATAT ]L_jL_JL_J[ L 8 Q I C 8 G C I T A. T L V D 8 8 L T D A. R I P T I R Q I II-glycoe N—glycoe AATTCTACCTTGATATATACAGCACTGGGGAATAAATTGCCTCTGGATGGTGGATTTGACGATTTCAGCGACGTATGTCGTACATATCTAGTCAACTTAA R 8 T L I T T A L G R R L P L D G C P D D P 8 D V C R T I L V R L Il—glyooe N-glycoe CCTCTA TTTCCGGACTCCCTTCACACGTTTCTACCAAGCCCAAGATTCGAICGCTAGTAGCAACCCGCAATTGTCTGACGTATCTTTGGAGGATACATAT T 8 I 8 G L A 8 H V 8 T R P R I R 8 V V C T R R C V T T L I R I H I TCCTCTCTGGGGTTGTATACAATATTTTATGTCATTCGTGAATGGAGACCCATGTTTGGTGTAGTAAGATTTGAGGATGATGCTATA Q 8 L i 3 a L Q L I I I I I I I R R R R R R P G V V R P 3 D D A I hydrophobic donnin 1 ATACCAAAAATT TGATATCTAGTCTTTTACTCAAAACAACCTACACTAAAAJGTCTAGATTTATGTGCGAGAIAA S T A R T T R R T A. A. R V I S 8 V L L R T T T T R R S R P R C R I IEAIQIATAAAAATGCTTTGAGTAGGACTTTTAAAGATGATCCCATATCATTTTTGTTTCATCACCCTATCGCAGCAGTTCTTATTATTACTGAGGGTTI R I T R l’ A L 8 R T P R D D P I 8 P L P R R P I A Hydrophobic domain 2 AEIEEEAITABGGCCTCACTGTCTTTGTCTRGCGACACTATCCATCTATTTTCTACCASCTCAAAAAGTTCTCTCGAAATGGTTTTTATCCATAACAGGC gang5991.;Laznsnxrvr’czxvnsxrtrns1x9 mamnuammummm I_L_I_G_I_I_I_LI_£_L_LL_L_L_A hydrofluobic domain 3 AAGATGAATGTGCCTTGGAAACTTCCCCATCTGGCGTACATCTTTTTTCCTCCAATTCTTGOGCCTCTTTAATATCTAACATATIAAIQAABETGCTGTA R D R C A L R T 8 P 8 C V R V P C 8 I C C A 8 L I 8 R I__L__I__fl__¥__fi__x hydrophobicdoaein4 TATATTGTTCATCATAATRTTCATTCTAACTATCGTAAGATATGAACGAACCCTTCAAATTGCATTCTTTGGGCCTCCCTATTTGCCTTAGTATACTTAA I V R T R R T L O I A L P C R. A T L P> P C P V D C A. A. R L G R T R 9 V R CTCA1CTCTTABATGATTTCCATTCCGOGGATATCACATAACCTACGGGGTTATATAGGTTCATATAGAGCTATAGGAGATTCTCTCCTCAAGTCTACCI ....... AfllfififlQIATTAICTATATCAAGATTAAACAAAAAAAAATCTCTGTAGASGCATTCTCTCGCGAGTCCCATGACATGATGAGTTTGTTGGACTATCATTT ““““““ MDV ICP27 R 8 V D A P S R I S D D H H 8 L L D T D P TCCTCCTCCGATCAAAATGCCGAAGTGACTGAAATGGAAACATCTCCAAAAACCGCTAATAACAAGAATGAAGTTTTATTCCCGCCACCC I I G 8 8 8 D R R A. I V T R R R T 8 A. R T A R R R R R V L P A P P TGTACGCAGGAACTTTTGACCGAAOGACCATCTOCTGATTCCAAAAATTCCCAAGGCCACGAIGACTCAAATTCAATATATGGCAAC C T Q I L L T R R P 8 P D 8 R R 8 Q C D D D 8 R 8 I T G R V I R D CTCAACACTCACCAAGTCGATATGCTACAAGGTGTCTTCACAATGCAATACCACCGAAACCTCTACGCTTRGCTAATTTGACAGTAGATTCTGCATGCAT A Q R 8 A. 8 R T A T R C L D R A I P R R R L R L A R L T V D S A C I AAACCGCCCCAOGGTACAGGCAATCGCAAACAATATCACAGACCTAATTTTCCGAJGTCACCCACTTCACAAGAAAAAATTCATCTACGA I Q T R R P R C T C R R R Q T R R R I P P R 8 P T 8 Q I R I R L R TCTCGGAGCCAAAAACACCAOOCCAGTCTAAATTICCACCGACCTCTCCAAGAAGGGCATCACCGAAGAAGATTCTACAGTC L I R R L C 8 R 8 I R O Q R 8 L I T D R R L Q R G R R R R R P T S AGAGACGTATTTATCATCAAAATCATIGTCACCATCGTACACACGATATACCGGTACCAITCGAAAAATATACAGTTTCCAGACAACATGATCTCCCTCT R R R I T D Q R R 8 R R R T R D I R V P L R R T R V S R Q R D L P V TICTTCAAAGAGAGAAACACCCTCTGGCCTCTATTTCAAATGAGTGTCATTTTCGCGTTTCGAGCAAAAATCGATGGGCT R R R L I R I L Q R R R R R L A. 8 I 8 R I C D P R V 8 8 R R R I A TTTTCAAflCAACGCGGAGAGTRCCTTAJCTCCTCCTCAGATAACATGGGAGTATTTATTGCAIGOGGGTCCAGAGCTACCAAACACGT A V L T P 8 8 R A. R 8 T L C 6 P Q I T I I T L L R A. G P R L R R T TCGAAATCAGACCTAGAATATCGCTACAAGCAAGTCCAGCACCAGAAGCCOTCTTCCCAGCTCAAAGTTTCATTGCCCCATTRGGGACTCCTCAAGAAAC P R I R P R I 8 L Q A 8 A A. R R A V L R G R 8 P I A A L G 8 A. R R T AAAACTRCAIGCTCTTTTAAAGTTRCGOCTAGTAAAICATCACCCCATTTTTAAGACCGCTGCTGCGGTTTTAGATAACCTCAGGCTG L 8 I L R L R A. V L R L R L V R R D P I P R T A G A V L D R L R L TAATCATGTCTAAATATCGAACAGAGAAACGCTCCATCGGGGATATCTTAABAAGATCTCCTCCTGAAGATATAAACGATTCCTTAA R L A P I I R C R T C T R R R 8 R C D R L R R 8 A. P R D I R D 8 L TTTTCTTATCGCOCATTCGTCGTCTGATCCATCBCACATCGGCCAGCAAATACAGTTRJRJGATAGACCCTRCAGGATGTRTCAIRGA T L C L I L L 8 R I R R V R R R T 8 C 8 R T 8 T R I D P R C C R I D CTATCTACCTCCAGAATBTATGACAAATATACTACOTTATGTAGATGCCCATACGAGCAGATGTTCTGATCCCGCATCTRACTTGTATATCAGCTGCACA T V P C I C N T R I L R T V D A R T R R C 8 D P A C R L T I S C T CTCATCCCTATTTATATCCATCGCAGGTATTTTTACTGCAATACTCTGTTTGGTATGTAAATAGTTATCTAAAAGACATCCTATATTTAGTATTCTACAC L R P I T I R G R T P T C R T L P G R> AATTTCTTCTCACGATATTACTAACTCCTCTAATAAAGTTAAATAAATAAACGTCTCAGATATCTCTTCTTRAAGTGTCGTTTTATTATCTATATAJCAC eeeeee eeeeeeeeee 100 200 300 400 500 25 600 58 700 91 800 125 900 150 1000 191 1100 225 1200 250 1300 291 1400 325 1500 354 1600 1700 21 1000 54 1900 87 2000 120 2100 154 2200 187 2300 221 2400 254 2500 287 2600 321 2700 354 2800 307 2900 421 3000 454 3100 473 3200 57 Figure 2.3 Alignment of three herpesvirus C-terminal ICP27 amino acid sequences. Amino acids identical to those found in MDV ICP27 are boxed. A C-X4-C-X13-H—X5-C conserved zinc finger metal-binding motif is boxed and shaded. 58 smsmscfl R ............... SK 14min SNAESTLCGP VDRISE G QVMHDP FGGQPFPAAN 3 GO GGPFDAE.TR Imosoosw AS ............................ GGCFPGI.KQ QI HA PE P F: _ CG RV 1 AH PSLYRT G IBALAS NT E LY sfiuLYRTFa. EAT sAD ..... KYG LKAR. .. LLNRDND TEI®MGDMQ AP SLBLCLIfi GS. SYM.I .GLCGLDELC RL IASFVFVI v AEI TLGV LoflrLPELLfl EQQRF c ITEYMFVMIA KFV DISC (3- t: NIQR C GLIE LAGVQE 270 308 243 320 358 293 365 405 343 413 454 393 460 501 441 473 512 452 59 Figure 2.4 Northern blot analysis of MDV ICP27 and gK. Total cellular RNA was isolated from infected or uninfected cells and IOug RNA was loaded per well as described in Materials and Methods. Panel A: DNA fragment BK (see Figure 2.1) was used as probe; Panel B: DNA fragment CC ( see Figure 2.1) was used as probe. Lane 1, mock-infected RNA; Lane 2, MDV GA infected—DEF RNA; Lane 3, MDV GA infected-DEF treated with cycloheximide; Lane 4, MDV GA infected DEF treated with PAA. 60 61 Figure 2.5 TrpE-ICP27 fusion protein expressed in E.coli. Two DNA fragments of the MDV ICP27 coding region were cloned into pATH vectors to express trpE-ICP27 fusion proteins (see Materials and Methods). Lanes 1, pATH-KBg (see Figure 2.1); Lanes 2, pATH-BC (see Figure 2.1); Lanes 3-4, pATH3 vector control; Lane 1, 2, 3 were induced using 1AA. TrpE-KBg fusion protein (57 kDa) and trpE protein (37 kDa) are indicated by the arrows. kDa 106.0— 80.0 — ‘ 49.5 — 32.5 — 27.5 —- 62 63 Figure 2.6 (A) Immunoprecipitation analysis of MDV ICP27 translation products in vitro. Lane 1, 3 and 5, pBlue-ICP27; Lane 2, 4 and 6, pRSET-BK; Lane 1 and 2, translation products were precipitated using rabbit antisera against trpE-KBg; Lane 3 and 4, normal rabbit serum; Lane 5 and 6, no serum control. (B) Detection of MDV ICP27 in MDV-infected cells. West blot analysis was performed as described in Materials and Methods. Mock-infected DEF cell lysate (lanel) is used as the negative control. MDV GA strain (lane 2) and Mdll strain (lane 3) infected DEF cell lysates were pre-absorbed using normal rabbit serum and precipitated by Protein A SepheroseR CL-4B (Pharmacia). MDV ICP27 was indicated by an arrow. Protein molecular weight was calculated by comparison to prestained protein standards (Bio Rad). 9mm HCIW'ACIIN n vs-Aaw u . :Iaa .- mfifl mdn m5? 06¢ l 0.6 r r «9. II mdr l mfifl l mdn l m6? I ode lad? an... Chapter III A Marek's Disease Virus (MDV) Immediate-early Protein, MDV ICP27, Has Both Positive and Negative Regulatory Activities in vitro DELIN REN', LUCY F. LEEZ, AND PAUL M. COUSSENS1 Department of A nimal Science, Molecular Virology Laboratory, Michigan State University, East Lansing, Michigan 488241; USDA/ARS Avian Disease and Oncology Laboratory, East Lansing, Michigan 488232 65 ABSTRACT We previously reported a Marek’s disease virus (MDV) immediate-early protein, MDV ICP27, which is a homolog to the HSV-l trans-regulatory protein, ICP27. Here, we report that MDV ICP27 is a phosphoprotein and dominantly localizes to nuclei of CEF cells infected with serotype-l MDV or with a fowlpox virus recombinant expressing MDV ICP27. In HSV-l infection, ICP27 acts as a multi-functional nuclear protein and contains separable positive and negative functional domains. Independently, HSV-l ICP27 has little or no effect on target promoters, but can enhance or repress target gene promoters in the presence of transcriptional activators ICP4 or ICPO. The regulatory activities of HSV-1 ICP27 are independent of target gene promoter sequences but, instead depend on presence of different mRNA processing signals. In this report, we present evidence that MDV ICP27 possesses both intrinsic trans-activation and trans-repression activities. MDV ICP27 can transactivates MDV ppl4 and pp38 bi-directional promoters, but strongly represses the MDV thymidine kinase (TK) early gene promoter. In addition, a heterologous RSV-LTR U3 promoter is also stimulated by MDV ICP27. We further demonstrate that the region from amino acids 207 to 378 are critical for MDV ICP27- mediated repression activity. The substitution of 3' RNA signals in RSV-LTR promoter construct does not obviously affect MDV ICP27-mediated transactivation activity. In contrast, MDV TK promoter shown a negative response to MDV ICP27 when the SV40 small T 3’splicing site and the early poly(A) signal were replaced with the MDV ICP27 gene poly(A) signals in the TK promoter construct. These results suggest that MDV ICP27-mediated transactivation and transrepression activities may be mediated through 66 67 different transcriptional mechanisms. In contrast to HSV-l ICP27, MDV IVCP27 dose not display the functional co-operative activity with MDV ICP4. No measurable activation or repression effects of MDV ICP27 on MDV immediate-early gene promoters (MDV ICP4 and ICP27) as well as a late gene promoter (gB) were observed. Thus, MDV ICP27 may play a minimal role in expression of these genes. INTRODUCTION Marek's disease virus (MDV) is a cell-associated avian herpesvirus that induces a T-cell lymphoma and nerve demyelination in susceptible chickens (Reviewed by Calnek, 1985). Originally classified as a y-herpesvirus on the basis of its lymphotropism. MDV has a genome structure and gene content with more similarity to members of the on- herpesvirus group (Buckmaster et al., 1988; Roizman et al., 1992; Karlin et al., 1994). Herpesvirus gene expression is typically regulated in a cascade fashion with genes classified into three classes, depending on expression kinetics. Immediate-early (IE, or a), early (E, or [3), and late (L, or 7) gene classification depends upon requirements for viral protein synthesis and/or viral DNA replication. In HSV-l, B and 7 genes are further subdivided into B, and [32, 71 and 72, respectively (Roizman and Sears, 1995). Much of our knowledge regarding gene regulation in a—herpesviruses has been derived from studies of the prototype HSV-l system. There are five IE gene products in HSV-l, including ICPO, ICP4, ICP22, ICP27, and ICP47. With the exception of ICP47, HSV-l IE gene proteins have demonstrable regulatory functions that affect subsequent IE, E, and L gene expression (Reviewed by Roizman and Sears, 1995). ICP4 and ICP27 play essential roles in virus infection (DeLuca et al., 1985; Sacks et al., 1985; Roizman and Sears 1995). ICP4, the major transcriptional regulatory protein, is the most extensively studied IE protein in HSV-l. The major function of ICP4 is to participate in HSV-l early and late gene transactivation and IE gene repression. ICP4 acts by binding to both consensus and non-consensus sites on DNA. The repression function of HSV-1 ICP4 is 68 69 dependent upon its DNA binding capability, in which ICP4 forms a tripartite complex with TBP and TFIIB on the consensus sequence of IE gene promoter regions (Smith et al., 1993). More recent studies demonstrate that both tripartite-complex formation and transcription repression were efficient when the ICP4-binding site was down stream of a TATA box, within a short distance and in proper orientation (Kuddus et al., 1995). I-ISV-l ICP27 is a 63kDa phosphorylated nuclear protein (Ackerman et al., 1984). Studies with ICP27 temperature-sensitive and null mutants have revealed that ICP27 is involved in the negative regulation of immediate-early and early genes, as well as host cellular genes, but positively regulates late gene expression (Sacks et al., 1985; McCarthy et al., 1989; Rice et al., 1990). The regulatory activities of HSV-1 ICP27 are independent of target gene promoter sequences but depend on the presence of different mRNA processing signals. Activation functions correlated with different polyadenylation sites, whereas repression function correlated with the presence of introns either 5' or 3' to target gene-coding sequences (Sandri-Goldin and Mendoza, 1992). Both positive and negative modulation domains have been mapped in the C-terminal half of HSV-1 ICP27 (Rice et al., 1989; Hardwicke et al., 1989). The N-terminal half of HSV-1 ICP27 protein is critical for nuclear localization (Mears et al., 1995; Hibbard and Sandri- Goldin, 1995). Interestingly, Rice et al. (1993) reported that the acidic amino-terminal region of HSV-1 ICP27 is also participates in its regulatory activities in both lytic infection and transient expression. Homologs of HSV-1 ICP27 have been identified not only in other a- herpesviruses, including Varicella-zoster virus (V ZV) ORF4 (Perera et al., 1994) and 7O equine herpesvirus-1 (EHV-1) gene 3 (Zhao et al., 1992; Zhao et al., 1995), but also in B- and y-herpesviruses, including the UL69 gene of human cytomegalovirus (HCMV) and BMLFI of Epstein-Barr virus (EBV) (Winkler et al., 1994; Kenney et al., 1989). All of the proteins encoded by these genes modulate gene expression. Studies of reporter-gene dependent activities of BMLFl indicate that BMLFI acts. in trans by a post- transcriptional mechanism (Kenney et al., 1989). In contrast, VZV ORF4-mediated induction of gene expression occurs primarily at the level of transcription (Perera et al., 1994). Despite their apparent functional differences, all ICP27 homologs share a high amino acid residue conservation and a cysteine-rich domain in their C-terminal region. Similar studies of MDV IE genes have been hampered by the highly cell- associated nature of MDV in vitro and low level of gene expression even in fully lytic infection. Characterization of RNA transcripts isolated from MDV infected cells treated with a metabolic inhibitor (e. g. cycloheximide) indicate that MDV IE gene transcripts are mainly clustered in repeat regions, similar to locations of other a-herpesvirus IE genes (Maray et al., 1988; Schat et al., 1989). Recently, several MDV IE genes were reported and three of them were identified as homologs to HSV-l ICP4, ICP22, and ICP27 (Anderson et al., 1992; Ren et al., 1994; Hong and Coussens, 1994; Brunovskis and Velicer, 1995). MDV ICP4 is highly homologous to HSV-l ICP4 and contains several similar functional domains as its counterpart in HSV-l (Anderson et al., 1992). MDV ICP4 is able to enhance expression of the MDV early pp38 gene promoter by transient expression assays using MDV transformed lymphoid cell lines (Pratt et al., 1994). MDV ICP4 is also able to transactivate a Rous sarcoma virus long terminal repeat 71 (RSV-LTR) U3 promoter (Banders and Coussens, 1994). The MDV ICP27 gene homolog has been mapped to the EcoRI-B fragment of MDV DNA (Ren et al., 1994). MDV ICP27 is a 473 amino acid polypeptide that has 26% amino acid identity with HSV-l ICP27. By comparison to other a-herpesvirus homologs, MDV ICP27 shares a high amino acid conservation and contains a cysteine-rich domain and a potential zinc- binding motif within it’s C-terminal region (Ren et al., 1994). Furthermore, a basic and arginine-rich domain has been found between amino acids 150 to 200 (Ren et al., 1994). Sequence and structure similarities suggested that MDV ICP27 may have important regulatory functions in MDV gene expression. In this report, we demonstrate that the MDV ICP27 gene product is a prominent nuclear protein and is subject to post-translation phosphorylation. To evaluate potential regulatory functions of MDV ICP27, we transiently expressed the MDV ICP27 gene in chicken embryo fibroblast (CEF). Six MDV gene promoters representing all three MDV kinetic classes, and a heterologous promoter (RSV-LTR) were selected as target promoters and transiently cotransfected with MDV ICP27 expression constructs into CEF cells. MDV ICP27 independently transactivated MDV pp14 and pp38 gene promoters, as well as RSV-LTR promoter, which contains MDV-mediated induction elements (Banders and Coussens, 1994; Sun and Coussens, manuscript in preparation). In contrast, MDV ICP27 repressed expression from the MDV TK promoter. MDV ICP27-mediated repression was mapped to amino acid residues 207 to 378. We also reported that the substitution of 3’RNA processing signals on target promoters display to controversy response to MDV ICP27. Surprisingly, two MDV IE gene promoters (ICP4 and ICP27) 72 and a late gene promoter (gB) were not responsive to MDV ICP27 gene transfection. Little or no cooperative effect between MDV ICP27 and MDV ICP4 was observed on any target promoter. MATERIALS AND METHODS Cells and viruses Preparation, propagation, and infection of CEF cell cultures with MDV and fowlpox virus (FPV) were as described previously (Solomon, 1975; Yanagida et al., 1992). MDV serotype 1 strain Mdll (passage 23 and passage 86) and GA (passage 14) were used for this study. A fowlpox virus recombinant expressing the MDV ICP27 gene (rFPV/ICP27) and a wild type fowlpox virus were kindly provided by Dr. N. Yanagida (USDA-ADOL, East Lansing, MI) for this study. Plasmid constructions A11 recombinant plasmids were generated by standard methods (Sambrook et a1 ., 1989). All enzymes used were from Bioehringer Mannheim (Indianapolis, IN), unless otherwise noted. To generate the MDV ICP27 expression plasmid M-ICP27CMV (Figure 3.1 B), a SpeI-Xhol fragment derived from plasmid pBlue-ICP27 (Ren et al., 1994) which contains the entire MDV ICP27 coding region from -10 bp upstream of the ATG codon to +274 bp downstream of a st0p codon was cloned into SpeI and XhoI sites of pBK/CMV vector (Stratagene, La Jolla, CA). To increase transcription efficiency in mammalian cells, a 200bp fragment of the prokaryotic Lac promoter region between the CMV promoter and the MDV ICP27 gene insert was removed and the plasmid was recircularized with T4 DNA ligase. To generate the MDV ICP4 expression plasmid M- ICP4CMV (Figure 3.1 B), a XhoI fragment containing 227 bp of upstream sequences and the entire coding region of MDV ICP4 was cloned into a XhoI site of pBK/CMV with 73 74 properorientation. The prokaryotic lac promoter region in M-ICP4 was removed as previously described. The three MDV ICP27 deletion mutant constructs, M-ICP27D1, M-ICP27D2, and M-ICP27D3, are derivatives of plasmid M-ICP27CMV (Figure 3.7 A). To generate M-ICP27D1 (a.a. 1-378), plasmid M-ICP27CMV was digested with BglII and XhoI, followed by end-filling with Klenow enzyme and ligation with T4 DNA ligase. For M-ICP27D2 (a.a. 1-207), a 1.1 kb fragment was excised from plasmid M- ICP27CMV with KpnI digestion and recircularized with T4 DNA ligase. To create M- ICP27D3 (a.a. 36-473), a 280 bp fragment including the lac promoter and 80 bp of MDV ICP27 coding sequences was removed from plasmid M-ICP27CMV by NheI and BamHI digestion followed by end-filling with Klenow enzyme and recircularization with T4 DNA ligase. Three reporter plasmids, pSph, pp38CAT, and pp14CAT, have been described (Banders and Coussens, 1994; Abujoub et al, 1996). Briefly, for pSph, a 137 bp fragment from the RSV-LTR U3 region containing MDV-mediated transactivation elements was fused with the chloramphenicol acetyltransferase (CAT) gene in plasmid pCAT-Basic (Promega, Madison, WI). For pp38CAT and pp14 CAT, a 700 bp fragment from the MDV pp38 gene and pp14 gene bi-directional promoter region has been placed upstream of the CAT gene in opposing orientations (Figure 3.4 A) (Abujoub and Coussens, 1996). To generate TKCAT, the oligonucleotides 5'-CACGCATGCTACAT- CTAATACCATGACC-3' and 5'-GGGTCTAGAGTTCAATGGGAGAGAA-3' were used as upstream and downstream primers respectively, to amplify the MDV TK gene promoter (-243, +1). The 250 bp fragment amplified by PCR using DNA isolated form MDV strain MDll infected CEF cells as template was digested with Sphl and XbaI, and 75 fused with the CAT gene in plasmid pCAT-Basic (Figure 3.4 A). In gBCAT, the oligonucleotides 5'-GGGAAGCTTGTATTTAAATGTGGCG-3' and 5'-GGGTCTAGA- GTGAGATGATCTTAATGTGC-3' were used as primers to amplify the MDV gB gene promoter (~361, +1) by PCR, followed by HindIII and Xbal digestion and cloning into the pCAT—Basic vector (Fig. 4 A). To create M-ICP27CAT, an Xbal-Bsml fragment corresponding to DNA sequences (-660, +17) of the MDV ICP27 gene promoter was blunt-ended with T4 DNA polymerase and cloned into pCAT-Basic in the proper orientation. M-ICP4CAT plasmid was generously provided by Dr. M. Boussaha (Boussaha et al., 1996). To create a panel of reporter plasmids with RSV-LTR U3 and TK promoter- driven CAT constructs differing only in their 3' RNA processing signals, a 250 bp PvuII fragment containing three poly (A) consensus elements followed by two GT-rich elements derived from the MDV ICP27 3’RNA region was cloned into the pBluescript KSII+/- vector in a SmaI site to generate p27PA. Oligonucleotide 5'-GCGGATAACA- AT'ITCAC-3', which is identical to the upstream sequences of the CAT gene and 31 bp from a HindIII site in the multiple cloning sites of the pCAT-Basic, was used as 5’primer. Oligonucleotide 5'-GGATCCGCTTATCACTTATTCA-3' carrying a BamHI site was used as 3’ primer to amplify the CAT open reading frame without the 3’ RNA processing signals, which includes a SV40 small T 3’ splicing site and the early poly A signal. The PCR amplified CAT gene fragment flanked with Xbal and BamHI was fused with 27PA. To create pCAT27PA (Figure 3.6 A), an Xbal-BamHI fragment from the pCAT-Basic, which contains the CAT gene and 3' RNA processing signals as described above, was replaced with anXbaI-Hindlll fragment containing the CAT gene followed by 76 a 27PA element. The pCAT27PA plasmid was used as parent plasmid to generate RSVCAT27PA, and TKCAT27PA, where the promoters are identical to those used in pSph and TKCAT, respectively, allowing evaluation of differences in 3'RNA processing signals only (Figure 3.6 A). Expression of GST fusion proteins and antiserum production A BamHI-Kpnl fragment from M-ICP27CMV corresponding to amino acids 23- 206 of MDV ICP27 was cloned into a pGEXZT vector (Pharmacia, Alameda, CA) to generate in frame glutathione S-transferase (GST) fusion proteins. Expression and purification of GST-ICP27 fiision proteins were performed according to manufacturer’s specification. Purified GST-ICP27 fusion proteins were used to generate rabbit polyclonal antiserum as described previously (Hong and Coussens, 1994). Indirect immunofluorescence assay CEF cells infected with MDV strain Mdll and with fowlpox virus recombinant expressing MDV ICP27 gene (rFPV/ICP27) were fixed with acetone/methanol (1:1). Uninfected CEF cells and cells infected with wild type fowlpox virus were used as negative control. Indirect immunofluorescence assay was performed as described by Harlow and Lane, (1988). Anti GST-ICP27 serum was used as the primary antibody at a dilution of 1:40. Goat anti-rabbit IgG conjugated with fluorescein-S'-isothiocyanate (FITC) (Sigma) was used as the second antibody at a dilution of 1:20. Cells were visualized using a Laser Scanning Confocal Microscope (Carl Zeiss, Inc.) with a 488 nm argon laser line and a 520/560 barrier filter. 77 Immunoprecipitation and protein dephosphorylation assay Mock-infected CEF cells, CEF cells infected with MDV serotype 1 strains MDll and GA, and CEF cells infected with ICP27FPVr were cultured in 100 mm plates and pre-incubated in phosphate-free Dulbecco Modified Eagle medium (Gibco-BRL, Gaithersburg, MD) for 1 hour. Cells were labeled in 500 uCi 32P orthophosphate per plate for 6 hours before harvesting (DuPont, Wilmington, DE). Cell lysates were immunoprecipitated with GST-ICP27 antiserum and protein A-agarose beads (Pharmecia, Alameda, CA) as described by Hallow and Lane (1988). Immunoprecipitates were washed, resuspended with 1X SDS-PAGE loading buffer, and analyzed by electrophoresis in a 12.5% SDS-PAGE minigel (Bio-Rad, Richmond, CA). For the protein phosphatase assay, samples were divided into two parts after final washing of immunoprecipitation reactions and one part was added with 20 IU of calf intestinal phosphatase (CIP) for 1-2 hours at 37°C. Dephosphorylation was terminated by washing with lysis buffer (50 mM Tris, 500 mM NaCl, 1%NP40). CIP treated and control samples were analyzed by electrophoresis in 12.5% SDS-PAGE minigels. Western blot analysis and Protein translation in vitro MDV infected CEF cells or uninfected CEF cells were lysed, cellular proteins were separated on a 12.5% SDS-PAGE minigel and electrophoretically transferred to nitrocellulose membranes. Immune blotting was performed as previously described (Ren et al., 1994). GST-ICP27 polyclonal antiserum was used at a 1:200 dilution and donkey 78 anti-rabbit IgG conjugated with horseradish peroxidase (Amersham, Life Science, England) was used as second antibody at a 1: 4000 dilution. Amersham ECL Western blot kit was used for detection essentially according to the manufacturer's specification. For protein translation in vitro, plasmid pBlueICP27, in which the MDV ICP27 gene was placed under the T7 promoter as described previously (Ren et al., 1994), was used as transcription template. The TNT/T 7 coupled reticulocyte lysate system (Promega, Madison, WI) was used for coupled in vitro transcription and translation as described previously (Ren et al., 1994). DNA transfection and CAT assay All DNA transfections for transient-expression assays were performed by electroporation with a Gene-Pulser Electroporater (Bio-Rad, Richmond, CA). Primary cultured CEF cells were removed from plates by exposure to 0.05% trypsin and washed twice with PBS. After final washing, cells were resuspended with HBS 2X buffer (50 mM HEPES, pH 7.1, 280 mM NaCl, 1.5 mM NazHPO4) at a density of 7.5x106 cells/ml. 800ul of cell suspension (6X106 cells) was mixed with defined quantities of plasmid DNA and incubated on ice for 15 to 20 minutes. Cell mixtures were transferred into an electroporation cuvette and electroporated with a single pulse at 350 mV and 960 uF with a capacitance extender. After pulsing, cells were incubated at room temperature for 10 minutes and then plated equally into three 60mm tissue culture plates in 3ml of Leibovitz-McCoy medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% calf serum (Gibco BRL, Life Technologies, NY). Cells were grown for 48 79 hours before harvesting. Total amount of plasmid DNA used for each individual transfection was adjusted to a consistent level by adding pBK/CMV. The amount of plasmid DNA used in each particular transfection experiment noted in the text and figure legends. A CMV-B-gal plasmid (0.3 ug, Kindly provided by Drs. R. F. Silva and J. D. Reilly, USDA-ADOL, East Lansing, MI) was included as internal control for transfection efficiency in each individual experiment. Cells were harvested 48 hours after transfection and disrupted by three cycles of freezing and thawing in 0.1M Tris-HCI (pH 7.8). Protein concentration of cell extracts was determined by Lowry Assay. CAT activity was assayed using the same amount of total protein for all samples in an individual experiment. CAT assays were performed using a simultaneous diffusion assay as described by Neumann et al., (1987). All transfections and CAT assays were repeated at least three times with three replica plates per individual experiment to control for variability. CAT activity was normalized by assessing the B-gal activity present in each cell extract as described previously (Sambrook etal,l989) RESULTS MDV ICP27 is a nuclear phosphoprotein. Protein subcellular localization is determined, at least partially, by the function of the target protein. To determine if MDV ICP27 is transported to infected cell nuclei, as most other herpesvirus IE proteins are, we performed indirect immunofluorescence assays. As expected, MDV ICP27 specific staining was predominantly localized to the nuclei in both CEF cells infected with MDV serotype 1 strain Mdll (Figure 3.2 A) and with rFPV/ICP27 (Fig.2 B). In wild type MDV infected cells, ICP27 displayed nearly exclusive nuclear localization with diffuse staining well-distributed within the nuclei (Figure 3.2 B). rFPV/ICP27 infected CEF cells were chosen as positive control because they offer abundant expression of MDV ICP27 that was readily detected by MDV ICP27 specific antiserum. Second, other MDV gene products expressed in PFV recombinants exhibited the same subcellular localization as in the wild type MDV infected cells (Yanagida et al., 1992; Yoshida et al., 1994). As shown in Figure 3.2 B, ICP27 specific staining occurred with a very strong nuclear localization in rFPV/ICP27 infected cells. However, the distribution pattern was distinguished from wild type MDV infected cells in that specific staining was granulated with defined high density regions (Figure 3.2 B). As expected, ICP27 specific staining signals were not detected in either mock infected CEF cells or CEF cells infected with wild type FPV (Figure 3.2 C and D). We previously reported that a 55 kDa polypeptide was detected by anti trpE-ICP27 specific antiserum in MDV serotype-1 infected DEF cells (Ren et al., 1994). Another smaller polypeptide (52 kDa) was also detected in MDV serotype 1 infected CEF cells as well as 80 81 in rFPV/ICP27 infected cells when GST-ICP27 antiserum was used in immunoprecipitation or Western blot analysis (Figure 3.2 A). Failure to detect the smaller polypeptide with trpE-ICP27 antiserum may be due to the relative poor title of this antibody, particularly since the fragment used for production of this polypeptide was derived from the C-terminal region of MDV ICP27, a region of poor predicted antigenicity. In contrast, the fragment used for GST fusion antibody expression was from the N-terminal half of MDV ICP27, a region with very high antigenic features. The most likely explanation for presence of two distinct ICP27 species was post- translational modification of precursor polypeptide. Give the role of other ICP27 homologs in transcriptional regulation, and presence of 14 potential sites for phosphorylation of serine or threonine in the predicted MDV ICP27 amino acid sequence, it seemed most likely that the 52 kDa species represented a precursor polypeptide and 55 kDa species resulted from phosphorylation of this precursor. To determine if MDV ICP27 is indeed phosphorylated, CEF cells infected with rFPV/ICP27 were labeled with 32P and immunoprecipitated with anti GST-ICP27 antiserum as described in Materials and Methods. SDS-PAGE analysis of immunoprecipitation samples revealed that only the 55 kDa polypeptide was detectable in 32P labeled rFPF/ICP27 infected cell extracts. A similar phosphoprotein was not detected in mock-infected or wild type FPV infected cells (Figure 3.3 B). Significant reduction in intensity of 32P labeled ICP27 bands in CIP treated samples suggested that the 55 kDa form of MDV ICP27 was sensitive to phosphatase (Figure 3.3 C). MDV ICP27 can selectively transactivate or transrepress different target 82 promoters. To evaluate if MDV ICP27 is a transcriptional activator, we undertook transient expression / reporter construct experiments. Although this method cannot fully mimic the complex regulatory interactions that occur in MDV infected cells, it provides an appropriate strategy for studying the regulatory properties of a given protein. We constructed an MDV ICP27 gene expression plasmid, M-ICP27CMV, in which the coding region of MDV ICP27 gene was placed under the strong constitutive promoter of human cytomegalovirus immediate-early promoter (CMV). Expression of MDV ICP27 from this construct in transfected CEF cells was verified by Western blot analysis (Figure 3.7 C). Seven reporter constructs where various target promoters were fused with the CAT gene were co-transfected with M-ICP27CMV plasmid as described in Materials and Methods. Basal CAT activity of reporter promoters in the absence of effector plasmid (M-ICP27CMV) was arbitrarily set as 1.0. Fold induction of CAT activity following cotransfection of reporter plasmid with M-ICP27CMV was calculated relative to basal CAT activity. As summarized in Figure 3.4 B, four reporter constructs were responsive to MDV-ICP27, including pp14CAT, pp38CAT, TKCAT, and pSph. Three reporter constructs, M-ICP4CAT, M-ICP27CAT, and gBCAT, exhibited no obvious responsiveness to MDV ICP27. MDV pp14 and pp38 genes are controlled by a bi-directional promoter region (Chen and Velicer, 1991; Cui et al., 1991; Hong and Coussens, 1994 ). Co-transfection studies indicated that MDV ICP27 can significantly transactivate both pp14 and pp38 promoters. CAT activities of in extracts of cells co-transfected with pp38CAT and pp14CAT were increased 12.5 fold and 3 fold, respectively, in the presence of M- 83 ICP27CMV. In contrast, the activity of another MDV early promoter, the TK gene promoter, was strongly depressed (12.5-fold) by co-transfection with M-ICP27CMV (Figure 3.4 B). Interestingly, MDV ICP27 can also stimulate a heterologous promoter construct, pSph, which was derived from RSV-LTR U3 sequences. The relative CAT activity of pSph was increased approximating 2.0 fold when co-transfected with M- ICP27CMV (Figure 3.4 B). By comparison to other responsive promoters, activation of pSph by MDV ICP27 appears rather weak, however, pSph exhibits a very high level of basal CAT activity. Therefore, the reduced level of induction may be partially due to enhancement beyond the liner range of our CAT assay. To define more clearly whether MDV ICP27CMV can directly affect target promoters and if concentration of M-ICP27 plasmid was critical to reporter promoter activation, increasing amounts of M-ICP27CMV plasmid were co-transfected with a consistent amount of various reporter plasmids. As shown in Figure 3.5A, when 2 ug of pp14CAT was co-transfected with 0.25 ug of effector plasmid, CAT activity increased only 1.1 fold. Increasing effector plasmid concentration up to 2.0 ug per plate, a peak of CAT activity 4-fold above basal expression levels was observed. A dose-dependent pattern was also observed in MDV ICP27-mediated transrepression activity with the TK reporter plasmid, TKCAT, with a maximum repression of 33-fold at 1.5 ug effector plasmid per plate (Figure 3.5 B). MDV ICP27 dose not co-operate with MDV ICP4. In HSV-l, it is clear that ICP27 has little or no intrinsic effect on target gene expression. Instead, HSV-l ICP27 is able to affect both positive and negative regulatory 84 activities when in the presence of two additional HSV-l transcriptional activators, ICP4 or ICPO (Sandri-Goldin et al., 1992). In contrast, ICP27 homologs in VZV (ORF4) and EHV (1 UL3) independently transactivate certain target promoters (Inchauspe, et al., 1989; Zhao, et al., 1995). To determine if MDV ICP27 could co-operate with MDV ICP4 in promoter activation or repression, we constructed a MDV ICP4 expression plasmid, M-ICP4CMV (Figure 3.1 B), and employed transient co-transfection assays. Three MDV ICP27 responsive promoter constructs, pp14CAT, pp38CAT and pSph, were also stimulated by MDV ICP4 alone (Table 3.1). This finding is consistent with previously published reports ( Pratt et al, 1994; Banders and Coussens, 1994). Addition of MDV ICP27 had no demonstrable effect on promoter activation by MDV ICP4 with any of the all three target promoter constructs (Table 3.1). Thus, MDV ICP27 and MDV ICP4 are both capable of independently activating homologous and heterologous gene promoters but exhibit no co-operativity This pattern of independent activation without co-operation supports the observation that MDV ICP27 and ICP4 are functionally more similar to VZV ORF4 and ORF62, respectively, than to their respective HSV-l counterparts. Effects of 3’RNA processing signals on MDV ICP27-mediated trans regulatory activities. Our preliminary in vitro studies indicated that MDV ICP27 selectively and independently modulates certain MDV gene promoters as well as a heterologous (RSV- LTR) promoter. To determine if 3' RNA-processing signals affect MDV ICP27- mediated gene-modulation as has been described for HSV-l ICP27, we created a panel 85 of reporter constructs, RSVCAT27PA and TKCAT27PA (Figure 3.6 A) which are derived from pSph and TKCAT, respectively. Each plasmid differs from it’s parent only in the 3' RNA-processing signals. As summarized in Figure 3.7 B, pSph (SV40 poly A signal) and RSVCAT27PA (ICP27 poly A signal) respond similarly to MDV ICP27 expressed by co-transfection with M-ICP27CMV. This result suggests that different 3'RNA processing signals may not affect MDV ICP27-mediated transactivation activity of the RSV-LTR promoter. However, the basal activity of RSVCAT27PA activity was reduced 2-3 fold relative to pSph (Data not shown), presumably as a result of the reduced stability of an unspliced transcript. Interestingly, CAT activity in cells transfected with TKCAT decreased 10-12 fold in the presence of MDV ICP27, but no measurable fold change (increase or decrease) was observed when TKCAT27PA was co-transfected with M-ICP27CMV. We also noted that the basal promoter activity of TKCAT27PA was 2 to 3-fold less than the parental TKCAT construct (data not show). However, no obvious repression activity was observed even a over range of concentration of TKCAT37PA was cotransfected with M-ICP27CMV. These results indicated that the abolishment transrepression of MDV ICP27 on the TK promoter was not due to the reduced basal promoter activity, but due to the 3'RNA processing signal substitution of TK promoter construct. Functional dissection of MDV ICP27. Our studies imply that MDV ICP27 may possess complicated regulatory activities since it not only transactivates homologous (e. g. pp14 and pp38) and heterologous (RSV-LTR) gene promoters, but also strongly transrepresses the MDV 86 TK promoter. To further map and gain an insight into potential MDV ICP27 functional domains, we created three MDV ICP27 deletion mutants, as illustrated in Figure 3.7A and described in Materials and Methods. In M-ICP27D1, a 95 amino acid segment containing the cysteine-rich domain and a potential zinc-finger motif was deleted from the C-terminus of MDV ICP27. In M-ICP27D2, the entire C-terminal half of MDV ICP27 was deleted. In M-ICP27D3, only 35 amino acids from the N-terminus were deleted. To map the MDV ICP27 domain responsible for promoter repression, 2.0 ug of TKCAT plasmid was transfected alone or in combination with 1.5 ug of MDV ICP27 mutant expression plasmids. Altered MDV ICP27 polypeptides expressed from M- ICP27D1 and M-ICP27D3 repressed MDV TK promoter activity to the same extent as wild type MDV ICP27 (Figure 3.7B). In contract, M-ICP27D2 caused little or no repression on MDV TK promoter activity. These results suggest that amino acids from 207 to 378 contain functional domains involved in negative modulation of the MDV TK promoter. All three MDV ICP27 mutants lost the ability to transactivate RSV-LTR and pp14 gene promoters. To verify that differences between MDV ICP27 mutants were not due to altered protein expression or transfection efficiency, CEF cells were transfected with 3.0 ug of each plasmid DNA by electroporation and cell extracts were obtained after 48 h. Immunoblot assay was performed using GST-ICP27 antiserum as described in Materials and Methods. As shown in Figure 3.7C, comparable specific polypeptides were detected in cell extracts from M-ICP27CMV and the three mutants. Furthermore, all polypeptides exhibited a molecular size consistent with their respective deletion. As expected, no MDV ICP27 specific polypeptide was detected in cell extracts transfected with pBK/CMV plasmid. Immunoblot results indicate that the loss of regulatory 87 activities in mutated M-ICP27CMV constructs were not due simply to mutation-induced polypeptide instability. DISCUSSION We previously reported that a MDV immediate-early protein, MDV ICP27, has significant amino acid identity and protein structure similarity to HSV-l ICP27 and VZV ORF4 protein (Ren et al, 1994). In the present study, we demonstrated that MDV ICP27, like it’s HSV-l homolog, is a phosphorylated nuclear protein. Predominant nuclear localization of MDV ICP27 in MDV serotype 1 infected cells and in CEF cells infected with rFPV/ICP27 suggest that MDV ICP27 may contain signal peptides which direct the protein to the cell nucleus during MDV infection. Recently, a strong nuclear localization signal (NLS) and a nucleolar localization signal (NuLS) have been identified and mapped within the N-terminal half of HSV-1 ICP27 (amino acid residues 110 to 137 and 110 to 153, respectively) (Mears et al., 1995). NLS of HSV-1 ICP27 bears similarity to the bipartite NLSs found in Xenopus laevis nucleoplasmin (Robbins et al., 1991), whereas the NuLS includes NLS as well as 15 contiguous residues which consist entirely of arginine and glycine residues (the RGG box ), a putative mRNA binding motif. (Mears et al., 1995). Although neither the bipartite NLS elements nor an RGG box has been observed in MDV ICP27, MDV ICP27 contains a region rich in basic amino acids (40% basic amino acid residues and 24% arginines) from amino acid residues 151 to 200. Despite absence of a universal consensus sequence for NLSs, most are short sequences with a high basic amino acid content (Robbins et al., 1991). Conservation of basic and arginine rich in MDV ICP27 suggests that these sequences have some potential relationship with its predominant nuclear localization of MDV ICP27 property. However, more defined mutagenesis studies will be required to verify 88 89 this possibility. MDV ICP27 can significantly and independently tansactivate MDV pp14 and pp38 gene promoters in transient transfection experiments. It is notable that pp14 and pp38 genes are located within MDV genomic repeat regions (IRL and TRL), which have been intensively investigated due to a potential relationship to MDV oncogenicity and abundant expression of immediate-early transcripts (Maray et al., 1988; Schat et al., 1989). MDV pp14 and pp38 genes share a common control region which has been defined as a true bi-directional promoter and both gene products can be detected in MDV oncogenic serotype 1 transformed lymphoblastic cell lines (Cui et al., 1991; Chen and Velicer, 1992; Hong and Coussens 1994). It is also noteworthy that pp38 and pp14 genes are classified as belonging to different kinetic classes. MDV ppl4 is classified as an immediate-early gene whereas pp38 is considered as an early gene (Hong and Coussens, 1994; Chen and Velicer, 1992). There are several cis-acting elements including two TATA boxes, two Spl sites, two CAAT sits and one Oct-1 site within the pp38/ pp14 control region (Cui et al., 1991). Pratt et al. (1994) demonstrated that MDV ICP4, can enhance pp38 and pp14 gene expression in MDV transformed lymphoblastic cell lines. This observation was further supported by our in vitro studies using M-ICP4CMV expression plasmid. Our studies combined with previous reports lead us to conclude that MDV pp14 and pp38 gene promoters are responsive to MDV ICP27 and MDVICP4. Furthermore, MDV ICP27 in vitro translation products did not specifically retard any DNA fragments derived from the pp38 and pp14 promoter region as determined by mobility-shift assay (Data not shown). This observation suggests that MDV ICP27- mediated transactivation may not directly involve DNA-protein interactions. However, it 90 is equally possible that modification present on ICP27 (e.g. phosphorylation) are required for direct DNA binding. Ongoing studies with mutant ppl4 and pp38 promoters constructs will further define which cis-acting elements, if any, within these promoters, are critical for MDV ICP27- or MDV ICP4- mediated transactivation (Abujoub and Coussens, 1996). There are considerable differences in ICP27 homologs with respect to modulating TK gene promoters. For example, HSV-l ICP27 by itself has no or little effect on HSV- l TK promoter activity, but represses HSV-l ICP4- or ICPO-mediated transactivation of the HSV-l TK promoter (McCarthy et al., 1989; Rice and Knipe , 1990). In addition, HSV-l ICP27 mediated transrepression of the HSV-ITK promoter occurs via a post- transcriptional regulatory mechanism which depends on the 3' RNA processing signals (Sandri-Goldin et al., 1992). In contrast, VZV ORF4 can independently transactivate both homologous TK promoter as well as HSV-l TK promoter (Inchauspe and Oatrove, 1989). Like HSV-l ICP27, MDV ICP27 also possesses both positive and negative regulatory activities. It not only positively modulates certain MDV gene promoters, but also strongly represses the MDV TK gene promoter. Interestingly, the 3’RNA processing signals dose not affect MDV ICP27-mediated promoter transactivation. In contrast, MDV ICP27 failed to transrepress the TK promoter, when the SV40 small T splicing site and the early gene poly(A) were substituted with MDV ICP27 poly(A) signals (Figure 3.6A). However, it is not clear if MDV ICP27 is involved in mRNA processing. These results suggest that MDV ICP27-mediated transactivation and transrepression may be via the different transcription machinery. This apparent fimctional conflict prompted us to further analyze target promoter sequences. The MDV 91 TK promoter sequence displays some features different from those found in the HSV-l TK promoter. C/EBP (CCAAT box), Spl (two sites), OTF-l (octamer motif) cis-acting elements in HSV-l TK promoter region are not found in the MDV TK promoter. Whether these promoter differences lead to functional results such as MDV ICP27- mediated transrepression of the TK promoter is, at present, not clear. Our studies clearly demonstrate that MDV ICP27 can either positively or negatively regulate certain MDV gene promoters. Our further experiments using deletion mutants indicated that the amino acid segment from 207 to 378 is critical for MDV ICP27-mediated repression on MDV TK promoter. However, we failed to map a specific region which involves MDV ICP27-mediated transactivation activity. Lacking transactivating activity of all three deletion mutants of MDV ICP27 suggested that MDV ICP27-mediated positive regulatory is not a single region, but seems a multiple region related activities. Although evidence obtained from these deletion mutants implies that MDV ICP27-mediated both positive and negative regulatory functions possess the genetically separable activities, we still do not know if these two sets of regulatory activities are entirely independent. Two MDV IE gene promoter constructs (M-ICP4CAT and M-ICP27CAT) and one MDV late gene promoter construct (gBCAT) revealed no measurable response to M-ICP27CMV co-transfection (Figure 3.4 B). However, M-ICP4CAT and M- ICP27CAT display very low basal CAT activities in transient transfection assays (Data not show). As a result, these reporter constructs may be not sensitive in detecting repression activity if MDV ICP27 has negative effects on MDV IE gene expression. In HSV-l infection, ICP27 is required for efficient late gene expression. However, VZV 92 ORF4 protein has little or no effect on VZV late gene expression (Sandri-Goldin et al., 1992; Perera et al., 1994). Failure of MDV ICP27 to augment expression from gB promoter/reporter construct imply that MDV ICP27 may play the minimal role for expression of MDV late genes. Co-infection of susceptible birds with MDV and ALV leads to enhanced incidence of avian leukosis virus-induced lymphoid leukosis (Bacon et al., 1989). In vitro, MDV gene products or cellular proteins induced by MDV infection efficiently transactivate Rous Sarcoma virus long terminal repeat (RSV-LTR) promoters (Tieber et al., 1990). MDV-responsive elements within RSV-LTR promoters were localized to a 28-bp segment from -109 to -l37 within RSV-LTR promoter. Furthermore, we also demonstrated that MDV ICP4 was, at least, partially responsible for MDV-mediated transactivation of RSV-LTR in vitro (Banders and Coussens, 1994). However, MDV ICP4-mediated transactivation on RSV-LTR was much less efficient than with intact MDV, suggesting that other MDV-encoded products are likely to play a role in MDV- mediated transactivation of RSV-LTR promoter. Interestingly, here we demonstrated that MDV ICP27 was also able to transactivate RSV-LTR promoter. Although MDV ICP27 and ICP4 mediated transactivation of RSVLTR promoter via a consensus region (-137 to +11), molecular mechanism underlying the trans regulatory activities of MDV ICP27 or MDV ICP 4 should be addressed in future studies. It is notable that our present studies provide another worthy candidate for studying the interaction between MDV and ALV. 93 ACKNOWLEDGMENTS We thank Dr. A. A. Abujoub for kindly providing pp14CAT and pp38CAT constructs, Mr. Ronald Southwick and Ms. Carolyn Cook for excellent technical assistance. This work was supported, in part through the Michigan Agricultural Experiment Station, the Center for Animal Production Enhancement, and USDA NRI Competitive Grant # 94 Figure 3.1 (A) Transcript maps of MDV ICP27 and MDV ICP4. The location and orientation of both gene transcripts are indicated by arrows. (B) Structures of MDV ICP27 and ICP4 transient expression constructs. A human cytomegalovirus immediate- early promoter (CMV, open bars) was fused with each gene coding region (cross-hatched bars) followed by their own 3’RNA region (ploy A, unbroken lines) 95 Ex 2: _llln:- >2uE2-2 <38 EB >92 22222-2 EB >92 8.82 >92 Laser. :35 secs: M l ._ _ _ fl 2 _ m _ _:§.E _ _ 556% 59.8% 556% o 83 so: D17 _ H. L. _ 96 Figure 3.2 Subcellular localization of MDV ICP27 by indirect immunofluorescence staining. Virus or Mock-infected CEF cells were incubated with GST-ICP27 polyclonal antiserum at a dilution of 1:40 and were stained with Goat anti-rabbit IgG conjugated with FITC at a dilution of 1: 20. The cells were visualized with Laser Scanning Confocal Microscope using 488 nm argon laser line and 520/560 barrier filter. (A) CEF cells infected with MDV-1 strain Mdl 1. 50X. (B) CEF cells infected with a fowlpox virus recombinant expressing MDV ICP27 (rFPV/ICP27). 50X. (C) Mock-infected CEF cells. 20X. (D) CEF cells infected with fowlpox virus wild type. 50X. 98 Figure 3.3 Characterization of MDV ICP27 polypeptides. (A) Western blot analysis of MDV ICP27. CEF cells infected with: Lane 1, Mock; Lane 2, MDV serotype 1 strain GA; Lane 3 and 4, MDV serotype 1 strain Mdll low and high passages respectively; Lane 5, rFPV/ICP27; Lane 6, MDV ICP27 in vitro translation product. Specific polypeptides were indicated by arrow. Molecular weight standards were indicated on the left. (B) Immunoprecipitation analysis of labeled MDV ICP27. CEF cells were infected with rFPV/ICP27 (lane2), FPV wild type (lane 3), and mock (lane 4). CEF cells were labeled in [a32P] orthophosphate for 6 hours. Cell lysates were precipitated with GST-ICP27 polyclonal antiserum as described in Materials and Methods. Lane], MDV ICP27 in vitro translation polypeptide was labeled with 358 methionine and used as positive control. (C) MDV ICP27 polypeptide dephosphorylation analysis. Protein samples are identical to that in (B), except the precipitates were treated with phosphatase (CIP). 99 kDa 123456 101 .0 '— 83.0 — 50.6 — 35.5 -- 29.1 '— 20.9 ‘— 100 mdN WON M...“ cdm QM” 0.2:. an: «9. mm IV I QON rdN rtmn 0.0m Qua 960.. an... 101 Figure 3.4 (A) Schematic structures of reporter constructs where different target promoters (unbroken lines) were fused with CAT gene (open box) followed with a SV40 small T 3’ intron and an early poly A signal. Restriction endonuclease sites used in the construction of reporter constructs are indicated with their positions of relative to transcriptional start site (arrow) or initiation ATG codon. (B) Effects of MDV ICP27 on different reporter constructs. For each 2X106 CEF cell mixture, 2 ug of reporter constructs (except pSph) were transfected along or cotransfected with 1.5 ug of M- ICP27CMV using electroporation. In pSph transfection, the same amount of effector was used as described above but only 0.2 ug of reporter (pSph) was used. The basal CAT activity of reporter promoter in absence of effector was arbitrarily set as 1.0. Fold changes of cotransfection of reporter construct with M-ICP27CMV was calculated relatively to the basal CAT activity. The standard division of mean was indicated. 102 0.34. \ / 1 _ Soto gamma Z «Sm ov>m .20 a. :55 mu . _+ com- OH< \ / _l _ _ Soho 95wa Z «:5 cv>m P82 NT. NS. OH< \ / _ _ 8 0 oh «we .56 so a. a .1 Z Em 3.5 of 5 E22. _- _ _+ hm— Sd a mod Illw\/I1 to a < O Em $> _+ 0: SN- 1 we a 92 >1— ._.m :5 an .. cwm+ .5 mm mum ed um o.m L (.1 56 Ema-ludl Em 25m «1 a 2 8e- 33:35-25 .. O 00 EUFNAHUM 2* H mm magn—o-HWEOU hfitOQOm m hNm0fi2 H285? SSE I coo? ooom ooom ooov ooom ooom (was) KIIAIIOV .LVO q mQthUHtE m5» NOBNAUTE _ LQBNAUEZ wnm H .l m5. >20than 2 < 110 kDa _ D 1 0 1 _ 0 1 8 <— 55.0 1: 50.0 46.0 <— 35.0 50.6 Chapter IV DISCUSSION lll 1. Summary and Conclusion Herpesvirus gene expression is temporally regulated in a cascade fashion and has been classified as immediate-early, early, and late genes. Most IE gene proteins are transcriptional regulators which are required for both early and late gene expression. IE genes have been extensively investigated in the a-herpesvirus, HSV-1. HSV-1 ICP27 is one of two essential IE genes for expression of viral genes and for viral replication. HSV-l ICP27 plays multifunctional roles during HSV-l lytic infection and is the only IE gene which is conserved in all three herpesvirus subfamilies. Molecular biology studies have indicated that MDV genomic structure and gene homology are extremely similar to that of HSV-1. Therefore, we speculated that HSV-l ICP27 homolog should be conserved in MDV. The overall aims of this project have been to identify the ICP27 homolog in MDV and to further investigate its regulatory functions. The MDV EcoRI-B fragment was chosen to initiate a search for the ICP27 gene, and two open reading frames (ORFs), ORF53 and ORF54, were identified within this region. By predicted amino acid sequence analysis, we determined that ORF53 encodes 354 amino acids and is homologous to the HSV-l glycoprotein K, whereas ORF54 encodes 473 amino acids and displays significant homology to the HSV-l immediate- early protein, ICP27. The predicted amino acid sequence of MDV ICP27 was compared to HSV-l ICP27 as well as VZV ORF4. The amino acid sequence identity and similarity between MDV ICP27 and HSV-l ICP27 are 25.8 and 42.5%, respectively and between 112 113 MDVICP27 and VZV ORF 4 are 29.5 and 49.4 %, respectively. The primary structure of MDV ICP27 can be divided into a highly hydrophilic N-terminal half and a relatively hydrophobic C-terminal half. Importantly, MDV ICP27 shares a very high structural domain conservation to HSV-l ICP27. First, a cystein-rich region and a potential zinc- finger motif which are highly conserved within ICP27 protein family are also found in the C-terminus of MDV ICP27. It has been demonstrated that this region is critical for HSV-l ICP27-mediated positive and negative regulatory activities. MDV ICP27 also contains a very basic domain from amino acids 151 to 200. A similar basic region, including two arginine-rich domains, was reported in the N-terminal half of HSV-1 ICP27 (Sandri-Goldin, 1991). A high acidic and serine-rich domain is also conserved in both MDV ICP27 and HSV-l ICP27. Based on amino acid sequence and structural similarities, we speculated that MDV ICP27 may have regulatory functions in MDV gene regulation. To detect MDV ICP27 gene transcript and determine the gene classification, cycloheximide was used to enhance IE gene transcription and assess if MDV ICP27 is expressed in immediate-early time. Using a BamHI-Kpnl DNA fragment probe (which maps within the ICP27 gene ORF), 1.6 and 2.9 kb transcripts were detected in MDV infected cells treated with cycloheximide. However, only the larger transcript (2.9kb) was detected using the probe Clal-Clal (which maps within the gK ORF). This suggested that MDV gK and MDV ICP27 transcripts overlap, sharing a common 3’RNA region, most likely due to read-through transcription of the MDV gK gene, since no poly(A) signal sequence was found between the two ORFs. 114 The bacteria fusion proteins trpE-ICP27 and GST-ICP27, which correspond to the C- and N-terminal half of MDV ICP27, respectively, were expressed and polyclonal rabbit antisera were produced. Using immunoprecipitation assays, the two antisera specifically reacted to individual MDV ICP27 in vitro translation products. Two specific polypeptides (55 & 52 kDa) were detected in the MDV 1 infected cells using GST-ICP27 antiserum. In contrast, only a 55 kDa polypeptide was detected in MDV serotype 1 infected cells using trpE-ICP27 antiserum. Failure to detect the smaller polypeptide with trpE-ICP27 antiserum may be due to the relative poor titer of this antibody, since the fragment used for production of this polypeptide was derived from a region of predicted poor antigenicity in the C-terminal region of MDV ICP27. The most likely explanation for the presence of two distinct ICP27 species is post-translational modification. We also demonstrated that MDV ICP27 is a phosphoprotein by immunoprecipitation and protein dephosphorylation analysis. Like most herpesvirus IE proteins, MDV ICP27 is predominantly located in the cell nucleus. The predominantly nuclear localization of MDV ICP27 in MDV serotype 1 infected cells and in CEF cells infected with rFPV/ICP27 suggested that MDV ICP27 may contain signal peptides which direct the protein to the cell nucleus during MDV infection. We noted that a strong nuclear localization signal (NLS) and a nucleolar localization signal (NuLS) have been identified and mapped within the N-terminal half of HSV-1 ICP27 (Mears et al., 1995). The NLS of HSV-1 ICP27 is similar to the bipartite NLSs found in Xenopus laevis nucleoplasmin (Robbins et al., 1991). The NuLS in HSV-l ICP27 includes a strong NLS, and a RGG box that is a putative mRNA binding 115 motif. (Mears et al., 1995). Although neither the bipartite elements nor RGG box has been observed in MDV ICP27, MDV ICP27 contains a very basic amino acid region, including 24% of Arg and 16% & His and Lys from amino acid residues 151 to 200. Highly basic amino acid conservation in this region of MDV ICP27 implies that may play a role for its predominant nuclear localization property. However, more defined mutagenesis studies should be required to verify this possibility. Transient expression assays were used to determine if MDV ICP27 is a transcriptional regulator. The MDV ICP27 gene was placed under control of the CMV promoter and expressed in CEF cells by transient DNA transfection. Initially, seven gene promoters were used as the target promoters, including six homologous promoters derived from all three MDV kinetic classes, and one heterologous promoter from the RSV-LTR U3 region. We reported that MDV ICP27 can significantly transactivate the MDV pp14 and pp38 gene promoters in our transient transfection experiments. MDV pp14 and pp38 genes are located within MDV genomic repeat regions (IRL and TRL). MDV pp14 and pp38 genes share a common control region which has been defined as a bi-directional promoter (Cui et al., 1991; Chen and Velicer, 1992; Hong and Coussens 1994). There are several cis-acting elements including two TATA boxes, two Spl sites, two CAAT sits and one Octamer motif within this control region (Cui, et al 1991). However, the MDV ICP27 in vitro translation product did not specifically retard any small DNA fragments derived from the pp38 and pp14 promoter region determined by mobility-shift assays. This suggested that MDV ICP27-mediated transactivation did not directly involve a DNA-protein interaction. We have not determined which cis-acting 116 site is critical for MDV ICP27-mediated transactivation at this time, but this should be addressed in future studies. MDV ICP27 also possesses a negative regulatory activity on target promoters. An MDV early promote, the TK gene promoter was strongly repressed by MDV ICP27. This result suggested that MDV ICP27-mediated transrepression of the MDV TK promoter is functionally distinct from either the HSV-l ICP27 or VZV ORF4 protein. As described above, the HSV-l ICP27 showed negative effects on the HSV-l TK promoter but was dependent on the presence of ICP4 and /or ICPO (McCarthy et al., 1989; Rice and Knipe , 1990), while the VZV ORF4 protein can independently transactivate both the homologous TK promoter and the HSV-l TK promoter (Inchauspe and Oatrove, 1989). In addition, the MDV TK promoter sequence also displayed some features different from those of HSV-1 TK promoter. There is a C/EBP (CCAAT box), two Spl, and an Oct-1 (octamer motif) cis-acting elements in the HSV-l TK promoter region but not in the MDV TK promoter. Whether this promoter difference leads to the functional results of MDV ICP27-mediated transrepression on the TK promoter is, at present, not clear. Deletion mutant experiments indicated that amino acids from 207 to 378 are critical for MDV ICP27-mediated repression of the MDV TK promoter. The lack of transactivation activity of all three deletion mutants suggested that MDV ICP27- mediated positive regulation is not a single region, but seems to require multiple regions. Western blot analysis indicated that deletion mutants of MDV ICP27 are expressed with the comparable yields and molecular sizes. 117 Previous observations indicated that MDV infections were able to enhance avian leukosis virus-induced lymphoid leukosis (Bacon et al., 1989). The in vitro studies demonstrated that MDV gene products or cellular proteins induced by MDV infection, efficiently transactivate RSV-LTR promoters and a 28 bp segment from -109 to -l37 within RSV-LTR is an MDV-responsive element. MDV ICP4 is partially responsible for the activation of the RSV-LTR promoter (Tieber et al., 1990; Banders and Coussens, 1994). Here we demonstrated that MDV ICP27 was also able to transactivate RSV-LTR promoter. Although MDV ICP27- and ICP4-mediated transactivation of RSVLTR promoter via a consensus region (-137 to +11), the molecular mechanism underlying the trans regulatory activities of MDV ICP27 or MDV ICP4 should be addressed in future studies. MDV ICP27 may also be involved in the interaction between MDV and ALV. To determine if 3’RNA processing signals affect MDV ICP27-mediated gene- modulation as described in HSV-lICP27, we created two alternative reporter constructs, RSVCAT27PA and TKCAT27PA, which were derived from pSph and TKCAT, respectively. Each plasmid differed from its parent only in the 3’RNA processing signals. Transient expression assays indicated that substitution of 3’RNA processing signals did not affect MDV ICP27-mediated transactivation of RSV-LTR promoter. In contrast, the TK promoter showed a negative response to MDV ICP27 when the SV40 small T 3’splicing site and the early poly(A) signal were replaced with the MDV ICP27 gene poly(A) signals in the TK promoter construct. These results taken together suggested that MDV ICP27-mediated transactivation and transrepression activities are involved in the different mechanisms of modulating of gene expression. 118 We also noted that MDV ICP27 displayed a negative effect on either MDV immediate-early promoters (MDVICP4 and MDV ICP27), or on the MDV late gene gB promoter. Functional roles of MDV ICP27 in MDV IE genes and late gene expression remain to be determined. The MDV ICP4 was also able to transactivate the MDV ppl4, pp38 promoters as well as RSV-LTR promoter. However, addition of MDV ICP27 had no demonstrable effect on promoter activation by MDV ICP4 with any three target promoters. In conclusion, both MDV ICP27 and ICP4 independently transactivate target promoters but exhibit no co-operation. 2. Future Research Directions Identification and characterization of the MDV ICP27 homolog adds more information about gene expression, especially for studying and understanding the regulation of MDV gene expression. We primarily investigated MDV ICP27 regulatory activities at the transcriptional level , but many questions remain to be answered and should be addressed in future studies. It would be very important to extend our experiments to in vivo studies. Construction of MDV mutants which are functionally defective in MDV ICP27 will greatly contribute to the following issues. First, construction of viral mutants defective in ICP27 will answer if MDV ICP27 is essential for viral growth and for expression of viral genes. Second, the functional role of MDV ICP27 will be precisely elucidated for expression of different viral genes. Since MDV can only be grown in primary cell culture, it was difficult to mutate the essential genes from MDV. Recently, a chicken embryo fibroblast cell line, OU2, was used for MDV infection and MDV OU2 cell lines infected with MDV serotypel Mdll were established (Abujoub and Coussens, 1995). MDV OU2 cell lines are similar to certain lymphoblastoid cell lines, and are capable of transferring MDV infection to primary CEF monolayer cultures. However, MDV OU2 cell lines are also capable of supporting a cytolytic infection of MDV. Thus, MDV OU2 cell lines can be used for creation of either “knock out” or insertion virus mutants which display functionally defective MDV ICP27. It is also necessary to create cell lines which can stable express MDV ICP27 in OU2 cells to provide complementary cells. 119 120 We demonstrated that MDV ICP27 can transactivate pp38 and ppl4 promoters in transient expression assays. Since it has been reported that both pp38 and ppl4 genes are expressed in the MDV transformed lymphoblastoid cell line, MSB-l, it would be interesting if we could introduce the M-ICP27CMV plasmid into MSB-l and determine if MDV ICP27 displays the same activities. The defined deletion mutant studies should be considered in the future to identify the nuclear localization signals in the MDV ICP27 polypeptide. These studies should focus on the N-terminal region which has a highly basic domain, similar to that of HSV-1 ICP27. Our previous studies indicated that MDV ICP27 selectively modulated different target promoters. We also mentioned that MDV ICP27 did not directly bind to target promoter DNAs. Information from HSV-l and VZV implied that ICP27 may be involved in protein-protein interactions. Thus, in vitro experiments should attempt to identify which MDV encoded proteins or MDV induced cellular factors are involved in MDV ICP27-mediated transactivation or transrepression activities. These studies should focus on the ppl4, pp38 and TK promoters using deletion or insertion approaches. First, pp14 and pp38 promoters contain several cis—acting element binding sites such as SP1, CAAT, and Oct-1. Using deleted ppl4 and pp38 promoters as targets may help to determine which cellular factors are involved in MDV ICP27-mediated transactivation of promoters. The MDV TK gene promoter is distinguished from HSV-l TK by its lack of SP1, CCAAT box (C/EBP) and Oct-lsequences. Introducing certain cis-acting elements into the MDV TK promoter or using the HSV-l TK promoter as a target for MDV ICP27 121 will provide clues for the explanation why MDV ICP27 displays functional differences on the TK promoter. APPENDIX Supplementary Data 122 123 Expression of GST fusion protein in E. coli The vector system used to express MDV ICP27 in E. coli is plasmid pGEX2T (Pharmacia, Alameda CA) which contains a glutathione S-transferase (GST) gene under the control of an isopropylthiogalactopyranoside (IPTG)-inducible tac promoter. As escribed in Chapter III, a BamHI-Kpnl fragment, corresponding to amino acids 23-206 of MDV ICP27, has been cloned into pGEX2T vector and transformed into E. coli, DH501 cells. The insertion DNA was confirmed by sequencing. 3 mM IPTG was used for GST induction. Expression and purification of GST-ICP27 have been performed according to the manufacture’s specification. New Zealand white rabbits were initially immunized with 20 ug of purified GST-ICP27 fusion proteins in Titer-Max adjuvant (Cthx Co., Norcross, GA). The rabbits were boosted with the same amount proteins in Titer-Max after 4 weeks and bled 10 days following the last injection. Figure A.l (A). SDS-PAGE analysis of GST-ICP27 fusion protein. Lane 1, protein standard marker; Lane 2 & 4, GST control; Lane 3 & 5, GST-ICP27 fusion proteins; A 47 kDa fusion protein was purified using glutathione-sepherose 4B (lane 5) Figure A.l (B). Cleavage of GST-ICP27 by thrombin. A 20 kDa polypeptide (lane 3) of MDV ICP27 was specifically cleaved from GST-ICP27 fusion protein with thrombin. Lane 1, protein standard marker; Lane 2, GST-ICP27; Lane 3, ICP27 polypeptide cleaved from fusion protein; Lane 4, GST cleaved from fusion protein with thrombin; Lane 5, GST control. 124 Unpurified u 1.9. ': 3 0. kDa -<-GST-ICP27 _ + GST 29.1 — 20.9 —- * kDa 1 2 3 4 5 101.0__., 83.0 —‘. 50.6 _:' 35.5 —‘?‘I 29.1 —' .. 20.9 — ‘ICP27 (N) 4GST-ICP27 125 Figure A.2 Specificity analysis of MDV ICP27 polyclonal antisera As described in Chapter 11, two polypeptides of MDV ICP27 were synthesized using in vitro and labeled with 358 methionine. Using TNT/1‘ 7 in vitro translation system (Promega), a 55kDa polypeptide was translated from plasmid pBlue-ICP27 that contains the entire MDV ICP27 ORF (lane 1), while a 35 kDa polypeptide was translated from pRSET-BK derived from N-terminal half of MDV (lane 2). Two synthetic polypeptides were precipitated with two different polyclonal antiserum. Both synthetic polypeptides were specifically precipitated using GST-ICP27 antiserum (lane 3, and lane 4). In contrast, only 55 kDa polypeptide was precipitated using trpE-ICP27 antiserum (lane 5, and lane 6). As expected, both synthetic polypeptides were not precipitated by preirnmun serum (lane 7 & 8). These results indicated that two different antisera specifically reacted with polypeptides which were generated from distinct coding region of MDV ICP27. 126 12345678 kDa 101.0 ‘— 83.0 ‘— 55.0 4— 35.0 29.1 20.9 LIST OF REFERENCES 127 128 Abujoub, A. A., T. Tesmer, and P. M. Coussens. 1996. Unpublished data. Anderson, A. S., and R. W. 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