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(. +3 (I. .1.vl.l l1»!- ’1:- ..v ..:-«31.32.93: 1.5.. .10 r»: (it! 90.): pf. 3 :5. call :...:. A 2.; 5. ’05321 . 5.0.3. A ..: 9r. ’1... .lttv :.- .(i‘... .h I y . My bumfinwwmfiwguud 19.. 3.3:!!- D r: u) Kai’s; X I; no {at 75.). (A II.- 11115. $1 3|...I4 6v...(1.l. ..: .r... I yo: I 01!: ilrrllt .l E TE UNIVERSITY LIBRARIE ”1111111111111111 11.11111111111 111 3 1293 00899 0842 This is to certify that the thesis entitled MAREK'S DISEASE VIRUS-MEDIATED ENHANCEMENT OF AVIAN LEUKOSIS VIRUS GENE EXPRESSION AND VIRUS PRODUCTION presented by James Thomas Edward-Stephen Pulaski has been accepted towards fulfillment of the requirements for Master's d . Science egree 1n (Qt-«amp Major professor Date lJOU- l3 51“ch— 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution 1" w ‘ "*‘N LIBRARY Michigan State University "A ,- PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE ’;!:)I:£I I l—TrTI MSU le An Affirmative AotlorVEquel Opportunity Institution chG-DJ MAREK'S DISEASE VIRUS-MEDIATED ENHANCEMENT OF AVIAN LEUKOSIS VIRUS GENE EXPRESSION AND VIRUS PRODUCTION By James Thomas Edward-Stephen Pulaski A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1992 ABSTRACT MAREK'S DISEASE VIRUS-MEDIATED ENHANCEMENT OF AVIAN LEUKOSIS VIRUS GENE EXPRESSION AND VIRUS PRODUCTION By James Thomas Edward-Stephen Pulaski Direct interaction between two viruses in coinfected cells may promote replication and/ or pathogenesis of one or both virus types. In birds, Marek's disease virus (MDV) may be an important cofactor in avian leukosis virus (ALV)—induced disease. Coinfection of susceptible cells with non-oncogenic serotype 2 MDV, an avian herpesvirus, and an oncogenic avian retrovirus, avian leukosis virus (ALV), resulted in enhanced transcription of retroviral genes. Consequently, in vivo assays show increased ALV reverse transcriptase activity and antigen production relative to input concentration of MDV. Interactive laser cytometry was used to detect accumulation of both MDV and ALV antigens within single cells from coinfected cultures. These results suggest a direct role for MDV-encoded or -induced factors in enhancement of ALV gene expression and demonstrate the importance of herpesviruses as cofactors in retrovirus replication and pathogenesis in coinfected cells. TABLE OF CONTENTS Page LIST OF TABLES vi LIST OF FIGURES vii LITERATURE REVIEW: AVIAN LEUKOSIS VIRUS Classification 1 Particle morphology 3 Endogenous virus 3 Genomic Structure 5 Replication 5 Neoplasms 7 Control 7 MAREK’S DISEASE VIRUS Classification 9 Virus-cell interactions 9 Lymphoproliferative diseases 11 Particle morphology 13 HVT and Genomic Structure 13 Replication 14 Control 15 RETROVIRUS-HERPESVIRUS INTERACTION 16 REFERENCES 19 THESIS BODY: INTRODUCTION 25 MATERIALS AND METHODS: Cells and viruses 27 RNA anal sis 28 RNA slot lot 29 Nuclear run-off transcription assay 29 Alv gs antigen assay 29 Reverse transcription activity assay 30 Interactive laser cytometry 30 DATA ANALYSIS 32 RESULTS Cells and viruses RNA anal sis RNA slot lot Nuclear run-off transcription assay ALV gs antigen assay Reverse transcription activity assay Interactive laser cytometry DISCUSSION ACKNOWLEDGMENTS REFERENCES CONTINUED RESEARCH SUGGESTED RESEARCH REFERENCES 32 32 39 39 43 45 46 49 51 52 ACKNOWLEDGMENTS To my mentor, Dr. Paul M. Coussens, for believing in me even when I found it hard to. To Dr. Crittenden, for guiding me into this field of study. To Matthew/ Amy Pulaski and Maria/ Bruce Corstange who were always there to support and encourage me. To Mom and Dad, thank you for supporting every decision I made. To all of my friends I grew with in the lab - Ron Southwick, Heidie Camp, Mindy Wilson, Jesse Marcus, Uldis Banders and Virginia Tieber. Finally, to my committee, for guiding me down the path of science. THANK YOU!! vi TABLES Page . Common laboratory strains of avian leukosis and sarcoma viruses according to predominant ne0plasm induced and virus subgroup. 2 . Names, phenotypes, and lines of endogenous avian leukosis virus (e0) 4 . Comiarison of e izootiolo 'c and pathologic features of Mare '5 disease ) and ymphord leukosis (LL). 8 . Herpes viruses subfamily division. 10 . Selected herpesviruses. 11 . Grouping herpesviruses on the basis of the properties of their genomes. 12 PPN?‘ v i i FIGURES The Retrovirus Genome Barn HI maps of HVT and MDV Analysis of ALV RNA in MDV/ALV coinfected cells Efffict of input MDV titer on levels of ALV RNA in coinfected e 5 Nuclear runoff transcription assay of MDV/ALV coinfected EFs Effect of input MDV titer on levels of ALV gs antigen in coinfected cells Effect of input MDV titer on ALV associated reverse transcriptase activity in media of coinfected cells Interactive laser cytometry of MDV/ALV coinfected cells HVT Barn I-II E Restriction Enzyme Map Page 6 15 33 35 37 41 42 50 LITERATURE REVIEW AVIAN LEUKOSIS VIRUS Retroviruses, viruses of the family retroviridae, are grouped into three subfamilies; oncovirinae ("tumor viruses"), spumavirinae ("foamy viruses"), lentivirinae ("slow viruses"); each having distinctive characteristics described by their latin prefix (Matthews, 1982). Retrovirus proteins have both type specific and group specific determinants. Oncovirinae is further subdivided into genera derived by morphological classification based on electron micrographs of distinctive virus particles. By this particle morphology, oncogenic retroviruses can be distributed into four categories: A- type, B—type, C-type, and D-type particles (Bernhard, Cancer Res. 20; 712). Avian leukosis virus (ALV) species are retroviruses classified as type C oncoviruses and have been divided into five subgroups, A—E, based upon host range, interference patterns with other subgroups, and serum neutralization tests to define envelope antigen type (Matthews, 1982). Strains of ALV are identified by the pathological lesions produced and their envelope subgroup. They are grouped with sarcoma viruses and given an abbreviated designation based on the neoplasm induced and/ or a person or laboratory that studied them. For example, "BH-RSV" is the high titer strain of Bryan, Rous sarcoma virus (Weiss et. a1. 1984). Table 1 (Weiss et a1., 1984) shows the envelope subgroup classification and abbreviated names for different strains virus such as Rous sarcoma virus (RSV) and avian myoblastosis virus (AMV) listed across from their respective names. In the column marked 'defective' are the transforming viruses that require a 'helper' virus for propagation. These Table 1. Common Laboratory strains of avian leukosis and sarcoma viruses according to predominant neoplasm induced and virus subgroup Virus Class According Virus Class According to Subgroup No Subgroup to Neoplasm (Defective Virus) A B C D E Lymphoid Leukosis RAV-l RAV-2 RAV-7 RAV-SO RAV-60 virus (LLV) RIF-1 RAV-6 RAV-49 CZAV MAV-l MAV-2 RPL 12 HPRS— F42 Avian erythroblastosis AEV-ES4 virus (AEV) AEV-R Avian Myoblastosis AMV-BAI-A vinis (AMV) E 26 Avian Sarcoma virus SR-RSV- SR-RSV- B 77 SR-RSV- SR-RSV- BH-RSV (ASV) A B PR-RSV- D E BS-RSV PR-RSV- PR-RSV- C CZ-RSV PR-RSV- FuSV A B E PRC II EH-RSV HA-RSV PRC IV RSV 29 ESV Y 73 URI UR2 Myelocytoma/endo— MC 29 thelioma virus MH 2 CM II OK 10 Endogenous virus (EV) RAV-O ILV (Weiss et. al., 1984) helper viruses are listed under lymphoid leukosis viruses (LLV). LLV(s) are common field strains of virus that are associated with a variety of oncogenic diseases and substrains can be selected for the prevalence of one kind of disease (Frederickson et al., 1964; Smith and Moscovici, 1969). These viruses are collectively know as LLV, ALV, or transformation-defective viruses (Weiss et al., 1984). Morphologically, avian retroviruses of the subfamily oncovirus are spherical, enveloped, 80-120 nm in diameter, with two envelope (env) glycoproteins, projecting from the surface. Structurally, retroviruses have four internal group specific antigen (gag) proteins that make up an icosahedral capsid of nonglycosylated structural proteins, and a helical ribonucleoprotein. These core proteins are packaged with reverse transcriptase within the virus envelope (Baur, 1974; Norwinski et. al., 1973). Subgroup E viruses are known as endogenous avian leukosis viruses (abbreviated EV or ev), carried as a complete or defective proviral DNA integrated into different genomic sites of both somatic and germline cells (Crittenden, 1981, Smith, 1987). Thus, they are transmitted genetically and in a Mendelian fashion to progeny (Crittenden et. al., 1977). Phenotypic expression of these loci vary and is not well understood. When the complete ev genome is present, subgroup E virus may be produced. Expression of ev genes in cells can give positive reactions in enzyme-linked immunosorbent assays (ELISA) for group-specific (gs) antigen, compliment fixation tests for avian leukosis (COFAL), and the chick helper factor (chf) tests (Table 2). Importance of the existence of endogenous virus is not limited to false positives in exogenous virus assays. Crittenden et. al. (1987) demonstrated that an endogenous virus, RAV-O, can cause immune tolerance due to Table 2. Names, phenotypes and lines of endogenous avian leukosis viruses (w). e v Phenotype 1 gs- clif- 2 V-E+ 3 gs+ chf+ 4 gs- chf- 5 gS' CM- 6 gS' chf+ 7 V43“ 8 gS' ch? 9 gs- chf+ 10 V-E+ 11 V-E+ 12 V43“ 14 V-E+ 15(C) nme 16(D) none 17 g5“ chf 18 V-E+ 19 V—E+ (?)a 20 V-E+ Q)“ 21 V-E+ Line or Sourceb Most lines RPRL-72 RPRL—63 SPAFAS SPAFAS RPRL-lSI RPRL-15B K-18 K-18 RPRL-ISI4 RPRL-1514 RPRL-ISI H&N K-28 X K-16 K-28 X K-16 RC-P RI RW RW Hyline FP Note: an is associated with the gs- chf- phenotype but restriction fragments have not been characterized. a The presence of five ev loci in Reaseheath line W. birds precludes definitive assignment with the V-E+ phenotype. Definitive association requires further segregation of en genes. Hyline FP birds also carry an 1, en 3, and en 6. b Not exclusive to line or source. K: Kimber; R= Reaseheath; H&N= Heisdorf and Nelson. W No detectable virus product Expression of subgroup E envelope antigen Coordinate expression of group specific antigen and envelope antigens Spontaneous production of subgroup E virus (Smith, 1987) mm gs- ch? as chf” gst chf+ V-E+ m 1,4,5 5 envelope glycoproteins shared among endogenous and exogenous viruses. ALV's virion nucleic acid is an inverted dimer of linear positive sense RNA. Basic genetic information for production of infectious virions consists of three genes. In order from 5' to 3': group specific antigen (gag) codes for internal nonglycosylated virion proteins; polymerase (pol) codes for reverse transcriptase; and envelope (env ) codes for virion envelope glycoproteins. Redundant sequences, designated long terminal repeats (LTRs), flank each end of the genome and act as promoters for pro-viral DNA transcription initiation and termination (Figure 1). Proviral LTRs can be divided into three distinct regions: U3, R, and US. They are named so because of their location in the genomic RNA: respectively, uniquely at the 3' end, redundant sequence at either end, and uniquely at the 5' end (Figure 1). Notice they are referenced in upper case letters in the context of DNA, and lower case for RNA. Other genes for nonstructural components may also be present, but are not necessary for production of infectious virions (Matthews, 1982). A general outline for retrovirus replication has existed for some time (Ternin and Baltimore, 1972). Avian leukosis virus, as with other members of the family retroviridae, are distinguished from other enveloped single- stranded RNA viruses by use of reverse transcriptase and cellular DNA polymerase to produce a DNA intermediate step in replication (Matthews, 1982). After adsorption, penetration, and uncoating, single stranded virion RNA is transcribed into double stranded DNA "provirus" by virion reverse transcriptase. The LTR is formed during reverse transcription (See Figure 1) making retroviral DNA 500 to 1000 base pairs longer than the RNA genome (Fan, 1990). Proviral DNA is transported into the nucleus and integrated into the host genome in a semi-random manner. Cellular RNA polymerase II is responsible for viral RNA transcription from proviral DNA. Initiating in the pl aw (or ) E] I ERSE LA.Lb m In” on .30 l I can TAT U3 R U5 RNA SYNTHESIS LONG TERMINAL REPEAT Figure 1 The Retrovirus Genome upstream LTR at the U3-R border and terminating downstream at the R-US border, the proviral DNA is a complete transcription unit. U3 regions of retroviral or proviral LT Rs carry proximal and distal promoter elements, enhancer sequences, and sequences that control Pol II initiation sites, thus regulating viral transcription (Dynan and Tijian, 1985). Because LTRs carry all control sequences necessary for initiation of transcription, cleavage, and polyadenylation, the integrated provirus can be expressed as both progeny viral RNA and mRNA (Fan, 1990). These virion mRNAs are processed to resemble host mRNA and are transported to the cytoplasm where they are translated. Resulting structural proteins are assembled into virus particles, in which the progeny RNA, reverse transcriptase, and a cellular tRNA (to be used as a reverse transcriptase primer) is packaged. Envelope transmembrane proteins on the cell surface mark where particles bud, forming infective virus (Weiss et. al., 1984). ALV cause a wide variety of neoplasms in chickens and other birds. Although each strain has a characteristic neoplasm, the oncogenic spectrum of different strains may overlap (Beard, 1980). The major clinical disease caused by ALV in the field is called lymphoid leukosis, a B cell lymphoma originating in the bursa of fabricius of mature chickens (Weiss, 1984). Oncogenic patterns are influenced by viral and host factors such as origin (strain) (Fredrickson et. al., 1965), dose (Burmester et. al., 1959), route of infection (Fredrickson et. al., 1964), age (Burmester et. al., 1960), genotype, and sex of host (Crittenden, 1975, Burmester and Nelson, 1945). ALV proviral sequences may insert within the cellular genome resulting in activation of a cellular pro-oncogene. During the normal transcription of the provirus, the 3' or 5' U3 region of the proviral LTR may promote transcription of downstream cellular genes (oncogenes) do to a lack of transcriptional termination at the 5' LTR (Hayward et al., 1981; Payne et. al., 1981). Because of the extended time before lymphoid leukosis (LL) develops (table 3), ALV is of primary concern to the egg layer industry. No efforts to eradicate the virus, ranging from attempts to produce attenuated strains for vaccines (Okazaki et. al.,1982) to selective breeding for genetic resistance in birds (Crittenden, 1975), have been unsuccessful to date. Exogenous virus has two routes of transmission: vertically from hen to progeny through the egg and horizontally from bird to bird by direct or indirect contact (Rubin et al., 1961, Ruben et. al., 1962). Because no reliable treatment has yet been devised, eliminating vertical transmission with strict breeding programs and thereby maintaining a virus free flock is the only method of controlling disease. Table 3. Comparison of epizootiologic and pathologic features of Marek's disease (MD) and lymphoid leukosis (LL). Characteristic M2 L1. Age of onset Peak time 2-7 mo. 4~10 mo. Limits >1 mo. >3 mo. Clinical signs Paralysis Comrm Absent Gross lesions Liver Common Confirm Nerves Cannon Absent Skin Canrmn Rare Bursa tumor Rare Cannon Bursa atrophy Cam'm Rare Intestine Rare Cammn Heart Canmon Rare Microlesions Pleomorphic cells Yes No Uniform blast cells No Yes Bursa tumor Interfollicular Intrafollicular Surface antigens MATSA 540% Absent IgM <5% 91-99% B cell 3-25% 91-99% T cell 60-90% Rare (Calnek and Witter, 1991) MAREK'S DISEASE VIRUS Viruses of the family herpesviridae are divided into three subfamilies, designated alpha-, beta-, and gammaherpesvirinae based on host range in vitro, cell tropism of latent infection, replication and cytopathology (Table 4 and 5) (Matthews, 1982). An alternative classification scheme, based upon orientation of repeat sequences in the genome has been proposed (Table 6) . Marek's disease virus is classified as a type B alphaherpesvirus and is further subdivided into three serotypes determined by irnmunodiffusion and immunoflourescence tests (Roizman et. al., 1992; Bulow and Biggs 1975 a,b). Serotype l MDV are mild to very virulent isolates and their attenuated variants. Serotype 2 MDV are naturally occurring nononcogenic isolates, and serotype 3 are isolates of herpesvirus of turkeys (HVT), which is not identical to MDV, but produces similar cytopathic changes in tissue culture and is antigenically related to MDV (Witter et. al., 1970a). Because they are related antigenically to oncogenic serotype 1 MDV and are nonpathogenic, serotype 2 and 3 MDV isolates are used in vaccines (Schat and Calnek, 1978; Zander et. aL,1972) 3 Four types of virus-cell interactions have been characterized in Marek's infections: Fully productive, semi-productive, non-productive neoplastic, and non-productive latent. In fully productive infections, replication of viral DNA occurs, antigens are synthesized, and fully infectious virus particles are produced resulting in cell death. Fully productive infections have only been found in feather follicle epithelium (Calnek et. al., 1970). Semi-productive infection occurs mainly in lymphoid and parenchymal tissue. This type of infection is exemplified by production of noninfectious naked nuclear virions. Infection is accomplished by cell to cell transmission and also leads 10 Table 4. Herpesviruses subfamily division. Family: Herpesviridae Subfamilies: Subfamily 1 (Alphaherpesvirinae) Host range: In vivo narrow, frequently restricted to the species or genus to which the host belongs. In vitro replicates best in fibroblasts although exceptions exist. Duration of reproductive cycle: Relatively long. Cytopathology: Slowly progressive lytic foci in cell culture. The infected frequently become enlarged, (cytomegalia) both in vitro and in vivo. Inclusions containing DNA frequently present in both nuclei and cytoplasm. Carrier cultures easily established. latent infection: Possibly in secretory glands, lymphoreticular cells, and kidney and other tissues. Subfamily 2 (Betaherpesvirinae) Host range: In vivo narrow, frequently restricted to the species or genus to which the host belongs. In vitro replicates best in fibroblasts although exceptions exist. Duration of replication cycle: Relatively long. Cytopathology: Slowly progressing lytic foci in cell culture. The infected cells frequently become enlarged (cytomegalia) both in vitro and in vivo. Inclusions containing DNA frequently present in both nuclei and cytoplasm. Carrier cultures easily established. latent infections: Possibly in secretory glands, lymphoreticular cells, kidney and other tissue. Subfamily 3 (Gammaherpesvirinae) Host range: In vivo usually limited to the same family or order as the host it naturally infects. In vitro all members of this subfamily replicate in lymphoblastoid cells and some also cause lytic infections in some types of epithelioid and fibroblastoid cells. Viruses in this group are specific for either B or T lymphocytes. In the lymphocyte, infection is frequently arrested either at a prelytic stage with persistence and minimum expression of the viral genome or at a lytic stage, causing cell death without production of complete virions. Duration of reproductive cycle: Variable. Cytopathology: Variable. Latent infection: Latent virus is frequently demonstrated in lymphoid tissue. (Matthews, 1982) 11 Table 5. SELECTED HERPESVIRUSES VIRUS NAME SUBFAMILY CLASS HUMAN HERPESVIRUSES Herpes simplex virus 1 a E Herpes simplex virus 2 a E Varicella-Zoster virus a D Epstein-Barr virus 7 C Cytomegalovirus B E HERPESVIRUSES OF NONHUMAN PRIMATE Ateline herpesvirus 2 y B HERPESVIRUSES OF BONEY FISHES Ictalurid herpesvirus 1 a A AVIAN HERPESVIRUSES Marek's disease virus (Serotype 1) 'Y E Marek's disease virus (Serotype 2) 7 E Turkey herpesvirus (Serotype 3) ‘y E to cell death (Calnek et a1. 1982). N on-productive neoplastic infection, in which the viral genome persists in lymphoid cells with limited tumor and viral antigen production, results in immortalized cells (Witter et al. 1975; Sharma, 1981; Ross, 1985). Non-productive l_at_egt infections, in which the viral genome persists in lymphoid cell without production of viral or tumor associated antigens. Virus can be rescued by inoculation of infected cells into chickens or onto cultured cells (Calnek et al., 1981). Observations of latent infections have been restricted to lymphocytes, primarily in T cells (Shek et. a1. 1983). Marek's disease is the most common lymphoproliferative disease of chickens and is characterized by mononuclear infiltration of one or more of 12 Table 6. Grouping herpesviruses on the basis of the properties of their genomes Number of isometric Group Arrangement of repeated sequences arrangements3 A A single set repeated at termini in the same 1 orientation B Numerous repeats of the same set of sequences at 1 both termini in the same orientation C (0 Numerous repeats of the same set of sequences at 1 both termini in the same orientation; (ii) A variable number of tandem repeats of a different sequence internally w—I—fi me D (i) A single set of sequences from terminus repeated 2 internally; (ii) A subset of terminal sequences repeated at all termini in the same orientation M E (i) A single set of sequences from both termini 4 repeated in inverted form internally; (ii) A subset of terminal sequences repeated at both termini in the same orientationlr2 H m—z F Terminal reiterations in the genome of class F have 1 not yet been described 1. Although the genome of Marek's disease virus is characteristic of the E group,it's L and 5 components do not invert. 2. The presence of a terminal sequence repeated at all termini has yet to be proven in some herpesviruses. 3. Defined by the number of genome populations differing in the location of sequences in the unique regions relative to the termini (Modified from Matthews, 1982; Koch et al., 1986) 13 the following: peripheral nerves, gonad, iris, viscera, muscle, and skin (Calnek and Witter, 1991). Symptoms of the disease are variable, and the gross lesions are difficult to distinguish from those of ALV infections (Table 3). Nerve lesions are the most common gross lesions observed in infected birds (Payne,1985) and lead to paralysis of the extremities (Biggs, 1968). T-cell lymphoma is the ultimate response to serotype 1, possibly progressing to tumor development (Calnek and Witter, 1991). Lymphoma composition is complex, consisting of neoplastic, inflammatory, and immunologically active cells (Rouse et. al., 1973). Based on studies of a large number of cell lines, T cells are the usual targets for transformation (Powell et. al., 1974). Neoplastic cells carry MDV DNA, are continuous, and are usually nonproductive in- vivo (Calnek and Witter, 1991). Virion structure, as revealed by electron micrographs of negative stained preparations, is an enveloped nucleocapsid surrounding a nucleoid varying in shape from spherical to toroid (Nazerian 1974). Virus particles isolated from feather follicle epithelium have envelopes measuring 273-400 nm and appear as irregular, amorphous structures (Calnek et. a1. 1970). Negative stained preparations also illustrated a cubic, icosahedral nucleocapsid 150-160 nm (Calnek et. al. 1970) made up of 162 hollow centered capsomeres (N azerian 1973). The nucleic acid winds around a central structure connecting to two inner capsid poles forming a nucleoid (N azerian 1974). As described before, herpesvirus of turkeys (HVT) is a serotype 3 herpesvirus with genomic structure (Cebrian et. al., 1982) and antigenic properties similar to Marek's disease virus (Witter et. al., 1970a). Because of these characteristics, plus it's nononcogenic nature in chickens (Schat and Calnek, 1978), HVT is frequently used as a vaccine against MDV (Zander et. 14 al., 1972). In addition to similarities in structure and antigenicity, reports have also demonstrated a degree of homology between the two herpesviruses (Igarashi et. al., 1987). Igarashi et. al. have shown the similarities between MDV and HVT genome structure and sites of homology. MDV DNA is a linear, double stranded molecule with a size between 166 to 184 kilobase pairs (85-110 x 106 da MW) (Wilson and Coussens, 1992; Hirai et. al., 1979; Lee et. al., 1971). Both MDV and HVT are "E” type genome structure consisting of a long unique region and a short unique region, each flanked by one internal and one terminal repeat (Cebrian et. a1. 1982). All three serotypes differ in homology (Hirai et. al., 1984, Hirai et. a1. 1981) and restriction endonuclease digestion patterns (Ross et. al. 1983). Figure 2 compares restriction enzyme maps of serotype 1 and serotype 3 viruses; a map of serotype 2 viruses has not yet been generated. Transcription of MDV and HVT has a typical a herpesviruses "cascading" pattern ((Fenwick and Owen, 1988; Kato and Harai, 1985). Studies of this cascading cycle are complicated by the fact that MDV and HVT are highly cell associated. Infection is normally cell associated and accomplished by formation of intracellular bridges (Kaleta and N eumann, 1977). To fully study replication, cell free virus must be collected from the feather follicle epithelium (Calnek et. al., 1970) or sonicated cells (Paul M. Coussens, personal communication). The transcription cascade is divided into three distinct temporally regulated phases: Immediate early (IE or a), early (E or B), and late (L or y) (Maray et. al. 1988). After adsorption and penetration of cell free virus, IE genes begin the transcription cycle. IE gene products regulate E and L gene expression, and do not require prior viral protein production(I D 2 u. > a m 3 o 3 O < 2 < MW(kb) 8.6—- «- «1.0—- 2.2—- FIGURE 3: Analysis of ALV RNA in MDV/ALV coinfected cells. Primary Line 0 CEF cells were prepared and maintained as described in Materials and Methods. Cells (1 x 106) were infected with 1 x 105 IU of RAV-2 ALV alone (Lane 2), 1 x 105 PFU of serotype 2 MDV, strain SB-1 alone (Lane 3), or 1 x 105 PFU and [U of MDV and RAV-2, respectively (Lane 4). Total cellular RNA was isolated from infected cells and control, uninfected cells (Lane 1) at 105 hours post-infection by the proteinase K/SDS method (Sambrook et al., 1987) and electrophoresed through denaturing agarose gels (1.2 %) containing 2.2 M formaldehyde. Separated RNA was transferred to supported nitrocellulose membranes (Optibind, Schleicher & Schuell, Inc., Keene NH), prehybridized and hybridized to ALV-specific probes as described in Materials and Methods. RNA bands hybridized to ALV probe were visualized by autoradiography using Dupont Cronex lightening-plus intensifying screens. Approximate RNA band sizes were determined by comparison to an ethidium-brornide stained lane of RNA standards (Bethesda Research Laboratories, Inc., Bethesda MD) run on the same gel. 34 kb species observed in MDV/RAV-2 coinfected cells (Figure 3, Lane 4) have been observed previously in northern blots of ALV infected cells (Payne et al., 1981). RNA isolated from CEF cells infected with strain SB-1 MDV alone did not hybridize to ALV specific probes (Figure 3, Lane 3). Thus, increases in RAV-Z-associated RNA in MDV/RAV-Z coinfected cells were not a product of cross reactive MDV or cellular RNA species. These results demonstrate that coinfection with MDV and ALV augments accumulation of retroviral RNA. Levels of total ALV RNA in MDV/ALV coinfected cells are directly related to input MDV titers. To define more precisely the effect of MDV on RAV-2 RNA levels in coinfected cells, CEF cells were infected with a constant amount of RAV-2 (1 x 105 IU) and decreasing amounts of MDV. Total RNA was extracted at 105 hours post-infection and subjected to slot-blot analysis using an ALV specific probe. Consistent with results of northern blot analysis (Figure 3), coinfection of CEF with 1 x 105 PFU of MDV and 1 x 105 IU of RAV-2 increased total ALV RNA expression by approximately S-fold, relative to cells infected with RAV-2 alone (Figure 4). As the amount of input MDV was reduced, total RAV—2 RNA concentration decreased to levels observed in cells infected with ALV alone (Figure 4). Presumably, decreasing the amount of input MDV reduces the number of cells coinfected with MDV and RAV—Z. Alternatively, reduced levels of MDV may limit the quantity of MDV encoded or induced factors available for interaction with RAV-2. In either case, our results demonstrate a direct relationship between MDV infection and ALV RNA levels in co-infected cells. MDV-mediated enhancement of ALV gene transcription in MDV/ALV coinfected cells. Though transactivation of ALV LTR promoters by factors encoded or induced by MDV was the most likely explanation for increases in 35 TOTAL ALV RNA A COMPARISON OF ALV/MDV CO-ulNrfiEZC’EiI) CE? 8 150 . 777i ,,, 145 — 140 - 135 - 1.30 - '75 — '70 — 115 - 110 — 105 - 100 — 95 — NSITY 90 — 85 - 80 — I L_. :2' 'II {\1 75 - 7O - 65 - 60 - O 000 O (x 10°) 0.. 9 e e 0 9’9’ r—i H. o ’0 50- 45- RLLATEVL' 35- 50- 20 - 15 - 10 - O T T T 1 r 1 1 Y. CEF MDV ALV ALV+MDV ALV+UDV ALV+UDV ALV+ MDV ALV+ NW A V- Ul‘v (5x105) (1x105) (brio‘) (ax-.03) (:x'55) (xx-sw- iNF—ECTION GROUPS FIGURE 4: Effect of input MDV titer on levels of ALV RNA in coinfected cells. Total ALV RNA levels in Line 0 CEF cells, coinfected with 1 x 105 IU of RAV-2 and various amounts of MDV, was isolated as described (Sambrook et al., 1987, Materials and Methods). PFU of MDV added to each plate of culture sets is denoted in parentheses below the appropriate x-axis position. RNA from coinfected cultures as well as control cultures (uninfected, ALV infected, and MDV infected) was denatured in 2.2 M formaldehyde and spotted unto supported nitrocellulose. All samples were analyzed in triplicate (n=3). Total ALV RNA was detected by hybridization to a 3.9 kb ALV-specific probe as described for Figure 3. Following auto- radiography, RAV-2 RNA hybridized to ALV probe DNA was quantitated by scanning densitometry. 36 ALV specific RNA following coinfection with MDV, it was possible that MDV increased RAV-2 RNA stability. To distinguish between these possibilities, nuclear run-off transcription assays were performed. Equivalent amounts of radiolabeled nuclear run-off transcription products were used to probe linearized and denatured ALV (Hughes et al., 1987), cloned serotype 1 MDV gp57-65 gene (Coussens and Velicer, 1988), chicken beta-actin (Cleveland etal., 1980), and pBR322 control DNA immobilized on supported nitrocellulose. As expected, run-off transcription of chicken beta-actin remained relatively constant throughout all treatment groups (Figure 5). In contrast, run-off transcription of ALV-specific RNA in nuclei of MDV/RAV-2 coinfected cells wasover 3-fold more efficient than that observed in nuclei from cells infected with RAV-2 alone (Figure 5). Consistent with data from northern blot hybridizations, run-off transcripts from uninfected CEF or CEF infected with MDV alone did not hybridize to ALV DNA. Though we cannot completely rule out the possibility that MDV increases RAV-2 RNA stability, our results clearly demonstrate that factors encoded or induced by MDV infection can activate transcription of ALV RNA in coinfected cells. MDV-mediated activation of ALV gene transcription results in increased ALV protein expression and virus production. Herpesvirus-mediated enhancement of retroviral transcription may lead to augmentation of retroviral pathogenesis and virus production, provided that ALV specific RNA is efficiently translated in coinfected cells. To determine the effect of MDV-mediated transactivation on retrovirus protein expression incoinfected cells, extracts of coinfected cells and media were analyzed for RAV-2 group-specific (gs) antigen. Cells coinfected with 5 x 105 PFU of MDV and 1 x 105 IU of RAV-2 contained approximately 10-fold more gs antigen than those 37 NRO nose I fl ALV a car ALV MDV MDV r ALV - - MDV < z D pBR322 ,Acrm - -' - - Figure 5: Nuclear run-off transcription assay of MDV/ALV coinfected CEF. Nuclei were collected 105 hours post-infection from: control uninfected CEF (CEF), CEF infected with 5 x 105 IU of RAV-2 (ALV), CEF infected with 5 x 105 PFU of strain SB-l MDV (MDV), and CEF infected with 5 x 105 PFU of strain SB-l MDV plus 1 x 105 IU of RAV-Z (ALV & MDV). Run-off transcription reactions and hybridization of radiolabeled transcripts to supported nitrocel- lulose membranes were performed essentially as described by Stewart et al. (1987). Each nitrocellulose strip contained 5 ug each of: plasmid RCAS (cloned ALV genome, Hughes et al., 1987) (ALV, row 1), p19MDA2.35 (Coussens and Velicer, 1987) (MDV, row 2), pBR322 (row 3), and pAl (chicken beta-actin, Cleveland et al., 1980) (actin, row 4). All plasmid DNA was linearized with appropriate restriction enzymes prior to immobilization on membranes. Due to the low homology between serotype 1 MDV and serotype 2 MDV at the locus represented by p19MDA2.35, hybridization of run-off transcripts to serotype 1 DNA could only be detected following prolonged exposure of membranes to film (data not shown). GROUP SPECIFIC ANTIGEN PRODUCTION A COMPARISON OF ALV/MDV CO—INFECTED CEFs a “°°“ Li I g ‘°°°‘ 7 % I E :22: %Z W /%2 2:: %%%} 02% yer/a I $,,%a//ze@ INFECTION GROUPS FIGURE 6: Effect of input MDV titer on levels of ALV gs antigen in coinfected cells. Quantities of gs antigen in cell culture lysate were measured by ELISA as described in Materials and Methods. Control cultures included uninfected CEF cells (CEF), CEF infected with 1 x 105 IU of ALV alone (ALV), and CEF infected with 1 x 105 PFU of MDV alone (MDV). Coinfected groups (ALV & MDV) are labeled with the appropriate input PFU of MDV in parentheses below the x-axis. All coinfected plates contained 1 x 105 IU of RAV-2 ALV. Quadruplicate samples of each group were analyzed (n=4). Concentrations of gs antigen were quantitated by comparison to a standard absorbance curve prepared using a known concentration of RSV subgroup C gs antigen (generous gift of Dr. Eugene Smith, USDA-ADOL, East Lansing, MI). 39 infected with RAV-2 alone (Figure 6). As with RAV-2 RNA levels, gs antigen production in coinfected cells was directly related to the amount of input MDV, falling to near basal levels at MDV concentrations of 5 x 102 PFU per 106 CEF cells (Figure 6). Thus, MDV-mediated enhancement of retroviral RNA levels leads to increased production of retroviral proteins in coinfected cells, relative to cells infected with ALV alone. Retrovirus production requires proper packaging of genomic RNA, T assembly and packaging of retrovirus proteins, and budding of mature viral ! particles from the plasma membrane. To determine if MDV-mediated increases in ALV RNA and protein expression could lead to augmentation of i ALV production in coinfected cells, RAV-2 viral particles were isolated from media of CEF cultures infected with RAV-2, MDV, or RAV-2 plus MDV, as well as control uninfected cultures. Virus particles were pelleted by ultracentrifugation, resuspended in a constant volume, and disrupted for assay of reverse transcriptase (RT) activity. RT is carried in the capsid of mature retroviral particles and is thus an indirect measure of retrovirus production. In contrast to gs antigen production and RAV-2 RNA expression, RT activity did not exhibit a direct relationship with respect to amount of input MDV (Figure 7). The highest concentrations of RT activity (approximately 3-fold over RAV-2 alone) were associated with lower amounts (5 x 103 to 1 x 103 PFU) of input MDV. Increasing input MDV to 5 x 105 PFU reduced RT activity to near basal levels (Figure 7), suggesting that higher concentrations of MDV may interfere with RAV-2 virus production, perhaps by competition for host factors involved in virus release. Alternatively, higher concentrations of input MDV may cause extensive cell death, thereby reducing the number of viable cells capable of releasing 40 matureretroviral particles. Nevertheless, our results clearly indicate that coinfection of cells with MDV and ALV augments retrovirus production. Accumulation of MDV and ALV antigens in single cells. Presumably, direct virus-virus interactions would require both virus types to be resident in a single cell. Alternatively, factors encoded or induced by one virus would need to accumulate within a cell infected with another virus type. To determine if MDV and ALV proteins were capable of accumulating within a single cell, cultured CEF cells coinfected with 1 x 105 PFU of SB-1 MDV and 1 x 105 IU of RAV-2 were permeablized by acetone fixation on glass multi-chambered slides. Fixed cells were treated with anti-ALV and anti-MDV antibodies followed by appropriate second antibodies conjugated to either phycoerythrin (RAV-Z) or fluorescein isothiocyanate (FITC) (MDV). Following extensive washing, cells were visualized using an interactive laser cytometer (Meridian Instruments, Okemos, MI). Fluorescence due to both phycoerythrin and FIT C was visible in coinfected cells (Figure 8), indicating that both MDV and RAV-2 antigens were present in single cells. Cells containing antigens of only one virus type were also visible in the same field (Figure 8). Control uninfected cells exhibited no detectable fluorescence. Cultures infected with MDV alone or with ALV alone displayed fluorescence due only to FIT C or phycoerythrin, respectively (data not shown). In selected fields, between 28% and 88% of cells contained ALV antigens only, while 5% to 28% of cells contained only MDV antigens and 5% to 28% of cells contained antigens of both viruses. Results of interactive laser cytometry suggest that MDV and ALV antigens are able to accumulate in the same cell, thus supporting a direct interaction between the two viruses. 41 REVERSE TRANSCRIPTASC ACTIVITY A COMPARISON OF ALV/MDV COINFECTED CE? 3 so. 55:— so} INN I l 1 I CEF ALV MDV ALV-Haw ALV+MOV ALV+UDV ALVHJ 5 5 4 :5 (5x10 ) (1x10 ) (5x10 ) (5x10 V+UW A.V+U'_)V p 5 2. ‘x10 ) (sins ) A INFECTION GROUPS FIGURE 7: Effect of input MDV titer on ALV associated reverse transcriptase activity in media of coinfected cells. RT activity associated with retroviral particles purified from culture media of MDV/RAV-Z coinfected cells was measured as described in Materials and Methods. Quadruplicate samples of each control and infected group were analyzed (n=4). Controls and infected groups are as detailed for Figures 3 and 4. All coinfected cultures contained 1 x 105 IU of RAV-Z and the amount of SB-l MDV indicated in parenthesis below the appropriate x-axis position. 42 Figure 8: Interactive laser cytometry of MDV/ALV coinfected cells. Double immune fluorescence assays quantitated by the adherent cell analysis and sorting (ACAS) cytometer (Meridian Instruments, Okemos, MI). Secondary Line 0 CEF cells , were infected with 1 x 105 IU of RAV-2 and 5 x 105 PFU of 83-1 MDV, diluted 1:10 and transferred to tissue culture chamber slides (1 ml per chamber) (Nunc, Inc., Naperville, IL). Control cells infected with 1 x 105 IU of ALV alone, 1 x 105 PFU of MDV alone, or uninfected were also plated in chamber slides. Cells were maintained and fixed with acetic acid/ acetone prior to antibody incubations as described in Materials and Methods. Rabbit anti-p27 antibodies (SPAFAS, Inc., Storrs, CT) were employed as primary antibody against ALV. A mouse monoclonal antibody, Y-5 (generous gift of Dr. Lucy Lee, USDA-ADOL, East Lansing, MI), specific for serotype 2 MDV (Lee et al., 1983), was used as primary antibody to detect MDV. Secondary antibody for detection of ALV was goat anti-rabbit conjugated to phycoerythrin. Secondary antibody for MDV detection was goat anti-mouse conjugated to fluorescein isothiocyanate (FITC). Different combinations of antibody solution were used as controls to calculate background fluorescence and interference. Fluorescence intensity was measured using an ACAS 470 interactive laser cytometer (Meridian Instruments, Inc., Okemos, MI). Units set to color values are photons measured per unit time. Fluorescent labels were excited with an argon laser at 488nm. Emissions of FITC (detector 1, MDV) at 520 nm and phycoerythrin (detector 2, ALV) between 570 nm and 580 nm were measured by two photomultiplier tubes (PMT) bound by detection limits of 515 nm and 675 nm, respectively. Figure 8 represents a digitized image of detector output. DISCUSSION Herpesviruses produce transcription factors capable of transactivating retroviral LTR promoters in a promiscuous fashion (Casarele et al., 1989; Ho et al., 1990; Skolnik et al., 1988; Tieber et al., 1990). Herpesvirus-mediated augmentation of retroviral induced disease by coinfection of susceptible hosts (Bacon et al., 1989), further suggests that herpesviruses may be important cofactors in some retroviral-induced diseases. We have presented evidence which clearly indicates that herpesvirus and retrovirus antigens can accumulate in the same cell, consistent with results of Nelson et al. (1988) which indicate that hCMV and HIV may infect the same cells in AIDS patients. In the case of MDV and RAV-2, MDV-encoded or -induced factors transactivate RAV-2 gene transcription, ultimately leading to increased production of infectious retrovirus from coinfected cells, relative to cells infected with ALV alone. Enhanced expression of RAV-2 RNA and gs antigen is dependent upon the quantity of input MDV. These results suggest that MDV is directly responsible for enhanced ALV gene expression. Results presented in this report do not distinguish between cellular transactivating factors induced by MDV infection and those encoded by MDV. However, subgenomic fragments of MDV are capable of efficiently transactivating the RSV LTR promoter (Tieber et al., 1990; Coussens et al., Manuscript in preparation), suggesting that MDV may encode factors responsible for augmentation of ALV gene expression in coinfected cells. Expression of retrovirus genes occurs following integration of proviral DNA into the host cell genome (review, Weiss et al., 1984). Thus, it seems likely that increased levels of ALV RNA and gs antigen observed in this report result from 44 MDV-mediated transactivation of integrated provirus LTR promoters. Experiments to determine if MDV is capable of transactivating the LTR promoters of endogenous retroviral loci are in progress. It is possible that MDV-mediated transactivation of endogenous retroviral loci may augment expression of cellular genes adjacent to the provirus insertion site. In the case of ALV insertion near the myc proto-oncogene locus, immortalization and/ or oncogenic transformation may result. In this scenario, a non- oncogenic herpesvirus and a benign retrovirus (i.e. RAV-O) may combine to produce a lethal neoplasm. In the case of HIV and CMV, activation of HIV gene transcription increases production of the tat protein (Sklonik et al., 1988; Ho et al., 1990). Subsequent tat-mediated activation of CMV gene expression results in a truly synergistic coinfection. In the case of MDV and ALV, however, increased levels of ALV gene expression and virus production result in reduced titers of MDV, relative to cells infected with MDV alone (Frankel and Groupe, 1971). Evidence from our laboratory suggests that ALV proviruses integrate into MDV genomes in coinfected cells. In many cases, ALV integration produces a lethal mutation, thus reducing MDV titers (Wilson and Pulaski, unpublished observations). Interactions between ALV and MDV thus resemble a parasitic, rather than a synergistic relationship. 45 ACKNOWLEDGMENTS We thank S. Conrad, H. Roehl, L.P. Provencher, M. Flesverb, and J. Sell for helpful advice and assistance, A. Fadly for helpful discussions and for providing RAV-2 ALV, and L. Lee for providing Y-5 monoclonal antibody. We also thank H.A. Tucker and J.J. Ireland for helpful suggestions and critical review of the manuscript. This work was supported, in part, by grants # 88-37266-3983 and 90-34116-5329 awarded to RM. Coussens under the Competitive Research and Special Research Grants Programs, respectively, administered by the US. Department of Agriculture, by the Michigan Agricultural Experiment Station, and the Research Excellence Fund, State of Michigan. LIST OF REFERENCES LIST OF REFERENCES Bacon, L. D., Witter, R. L., Fadly, A. M. (1989). Augmentation of retrovirus-induced lymphoid leukosis b Marek's disease herpes-virus in white leghorn chickens. . Virol. 63, 504 Casareale, D., Fiala, M., Chang, C. M., Cone, L. A., Mocarski, E. S. (1989). Cytomagalovirus enhances lysis of HIV-infected T lymphoblasts. Int. J. Cancer. , 124-130. Calneck, B. W., and Pa e, L. N. (1976). Lack of correlation between Marek's disease tumor in uction and expression of endogenous avian RNA tumor virus genome. Int. J. Cancer. 17, 235-244 Campbell, W. F. and Frankel, J. W. (1979). Enhanced oncornavirus expression in Marek's disease tumors from specific-pathogen-free cluckens. J. Natl. Cancer Inst. 62, 323-328. CampSell, W. F., Kufe, D. W., Peters, W. P., S iegelman, S., Frankel, J. . (1978). Contrasting characteristics 0 Marek's disease herpesvirus isolated from chickens with and without avian leukosis virus infection. Intervirol. 10, 11-23. Cleveland, D. W., Lopata, M. A., MacDonald, R. J., Cowan, N. J., Rutter, W. J., Kirschner, M. W. (1980). Number and evolution conservation of alpha- and beta-tubulin and Icytoplasmic ta- and arlrlirzrba-gaisctligsgenes using specific cloned cD A probes. e , - . Coussens, P. M. and Velicer, L. F. (1988). Structure and complete nucleotide se uence of the Marek's disease herpesvirus gp57-65 gene. J. Virol. 62, 237 2379. Crittenden, L. B. and Fadly, A. M. (1985). Responses of chickens lacking or expressing endogenous avian leukosis virus genes to infection with exogenous virus. Poult. Sci., 64, 454-463. Crittenden, L. B., McMahon, S., Halpern, M. S., Fadly, A. M. (1987). Embryonic infection with the endogenous avian leukosis virus Rous-associated virus-0 alters responses to exogenous avian leukosis virus infection. J. Virol., 61, 722-725. Davis, M. G., Kenne , S. C., Kamine, ., Pagano, J. S., Huang, E., (1987). Immediate-ear y gene re ’on 0 human cytomegalovrrus trans- activates the romoter 0 human immunodeficiency virus. Proc. Natl. Acad. Sci. US . 84, 8642-8646. Dworkin R. and Drew W. L. (1990). Therapy for herpesvirus infections in AIDS. Current Opinion in Infectious Disease. 3, 108. 47 Fields, B. N. et al. (1991). Fundamental Virology. Raven Press, New York, ed. 2 pp. 839-949 Frankel, J. W., Farrow, W. M., Prickett, C. O., Smith, M. E., Campbell, W. F., Groupe, V. (1974). Responses of isolator-derived and conventional chickens to Marek's disease herpesvirus and avian leukosis virus. J. Natl. Cancer Inst. 52, 1491-1496. Frankel, J. W. and Groupe, V. (1971). Interactions between Marek's disease herpesvirus and avian leukosis virus in tissue culture. Nature New Biology. 234, 125-126. Gendelman, H. E., et al., (1986). Trans-activation of the human immunodeficiency virus long terminal re eat sequence by DNA viruses. Proc. Natl. Acad. 50. USA 83, 97 9-9763. Glaubiger, C., N azerian, K., Velicer, L. F. (1983). Marek's disease herpesviruses. IV. Molecular characterization of Marek's disease herpesvirus A antigen. J. Virol. 45, 1228-1234. Hirumi, H., Frankel, J. W., Prickett, C. O., Maramorosch, K. (1974). Coexistence of particles resembling he esvirus and tytrxi‘e-c virus in feather follicle epithelium of chickens. . Natl. Cancer st., 52, 303-306. Ho, .W.-Z, Harouse, J. M., Rando, R. F., Gonczol, E., Srinivasan, A., Plotkin, S. A. (1990). Reci rocal enhancement of gene expression and viral replication between uman cytomegalovirus and human immunodeficiency virus type 1. J. of Gen. Vir. 71, 97-103. Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J., Sutrave, P. (1987). Adaptor plasmids simplify the insertion of forei DNA into helper-independent retroviral vectors. J. Virol. 1, 3004-3012. Ignjatovic, J., and Bagust, T. J. (1985). Variation insusceptibility to avian sarcoma viruses and expression of endogenous avran leukosis virus ar71§3gens in specific pathogen-free chicken lines. J. Gen. Virol., 66, 1 1731. Jakovleva, L. S. and Mazurenko, N. P. (1979). Increased susceptibili of leukemia-infected chickens to Marek's disease. Neoplasm, 26, 93-396 Kenney, S., Kamine, J., Markovitz, D., Fenrick, R., Pagano, J. (1988). An Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat. Proc. Natl. Acad. Sci. USA. 85, 1652-165 Lee, L. F., Liu, X., Witter, R. L. (1983). Monoclonal antibodies with sppcificity for three different sero es of Marek's disease viruses in c 'ckens. J. of Immun., 130, 1003- . 48 Nelson, J. A., Re olds-Kohler, C., Oldstone, M. B. A., Wile , C. A. (1988). HIV and H MV coinfect brain cells in patients with DS. Virology, 165, 286-290. Ostrove, J. M., Leonard, J., Weck, K. E., Rabson, A. B., H. E. Gendelman, H. E., (1987). Activation of the human immunodeficiency virus by herpes simplex virus type 1. J. Virol., 61, 3726-3732. Payne, G. S., Courtneidge, S. A., Crittenden, L. B., Fadly, A. M., Bishop, J. M., Varmus, H. E. (1981). Analysis of avian leukosis virus DNA and RNA in bursal tumors: Viral gene expression is not required for maintenance of the tumor state. Cell, 23, 311-322. Pedersen C., Gerstoft, J., Tauris, P., Lundgren, J. D., Gotzsche, P. C., Buhl, M., Salim, Y., Schmidt, K. (1990). portunistic infections and malignancies in 231 Danish A patients. AIDS 4, 233-228. Peters, W. P. (1973). Biological and biochemical evidence for an interaction between Marek's disease he esvirus and avian leukosis virus in vivo. Proc. Nat. Acad. Sci. USA 70, 175-3178 Resnick L., Herbst J. 5., Raab-Traub N. (1990). Oral hairy leukoplakia. J. Am. Acad. Dermatol., 6, 1278-1282. Sambrook, J., Fritch, E. F., Maniatis, T. (1989). Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory : New York. Skolnik, P. R., Kosloff, B. R, Hirsch, M. S. (1988). Bidirectional interactions between human immunodeficiency virus type 1 and cytomegalovirus. J. Infect Dis. 157, 508-514. Smith, E. J., Fadl , A., Okazaki, W. (1979). An enzyme-linked immunosor ent assay for detecting avian leukosis-sarcoma viruses. Av. Dis., 23, 698-707. Stewart, C. J., Ito, M., Conrad, S. E. (1987). Evidence for transcriptional and ost-transcriptional control of the cellular thymidine kinase gene. 01. Cell. Biology, 7, 1156-1163. Tereba, A. and Murti, K. G. (1977). A very sensitive biochemical assay for detecting and quantitating avian oncornaviruses. Virology, 80, 166-176. Tieber, V. L., Zalinskis, L., Silva, R F., Finkelstein, A., Coussens, RM. (1990). Transactivation of the Rous Sarcoma Virus lon terminal repeat promoter by Marek's disease virus. Virology, 1 9, 719-727. Weiss, R., Teich, N ., Varmus, H., Coffin, J. (eds.) (1984) RNA Tumor Viruses. 2nd Ed. Cold Spring Harbor Press, New York, Witter, R. L., Sharma, J. M., Chase, W. B., Halvorson, D. A., Sivanandan, V., (1985). Field trials to test the efficacy of 01 alent Marek's disease vaccines in la er and broiler breeder Slick‘elns. Poultry Science, 64, 2280-22 . 49 CONTINUED RESEARCH As described before, herpes virus of turkeys (HVT) not only is similar to MDV in genomic structure (Cebrian et. al., 1982) and antigenic properties (Witter et. al., 1970a), but also has displayed homology in hybridization studies (Igarashi et. al., 1987). Because it also had demonstrated the ability to enhance CAT expression in RSV-CAT constructs (Tieber et. al. 1990), it is a viable alternative to MDV for characterization of transactivating genes. CAT assays were used by Dr. Paul Coussens to characterize the site in HVT that expresses the gene responsible for enhancing gene expression. Transient assays using an RSV LTR-CAT construct were performed on different HVT Barn HI restriction fragment clones. Clones were separated into five groups according to where they mapped on the HVT genome. Cotransfection with RSV—CAT established the HVT Bam HI E clone to be responsible for the highest level of CAT enhancement (Personal communication, Dr. Paul Coussens). HVT E is approximately 11.5 Kbp in length and is cloned into a 6.4 Kbp vector, pHC 79 (Fukuchi et. al. 1984). It lies on the right hand side of the UL region of HVT's genome (Figure 9) and has exhibited homology to Bam H1 restriction fragments Q1 and G in southern blot analysis of MDV serotype 1 GA strain (Igarashi et. al. 1987). To further ascertain the enhancer expression site on the HVT E fragment, a restriction endonuclease map was made (Figure 9). Subclones were later to be constructed and employed in transient assays with RSV-CAT. To construct a restriction endonuclease map, purified HVT E clone and HVT E fragment were digested with Bam HI, Bgl II, Eco RI, Sal I, and Sph I in different combinations. The digests were subjected to electrophoresis on a 50 L 11.5 kb U L m s 13L U 3 TR s ‘ ‘ :1:l——I—_—J HVT E Barn HI Bum HI I : I i t t 4 s 5.11 Sall Sphl 3310 3.11 33111 Sal I L10 I 0.6 I 6.9 [ 3.0 I Bgl II [ 4.0 I 6.9 I 0.6 ] Sph I I 3.0 l 8.5 I Sizes in kilobase pairs (Kb) Figure 9 HVT Barn H1 E Restriction Enzyme Map 0.8% agarose gel at 50 V next to a A Hind III ladder. Ethidium bromide stained gels were then studied for fragment sizes and restriction patterns. Sal I digests formed four fragments ranging in size from 0.6 to 6.9 Kbp. Three fragments of 0.6, 4, and 6.9 Kbp were generated by Bgl II digestion. Cutting with Sph I yielded two fragments 3.0 and 8.5 Kbp in length. Because Eco RI did not cut the E fragment it was used in double digests of the Barn HI E clone to position other restriction sites. See figure 9 for the restriction enzyme map. Used in CAT assays, the subclones and restriction enzyme map will help localize the gene(s) responsible for ALV enhancement. This manner of limiting the number of base pairs to sequence and study will accelerate further research. 51 SUGGESTED RESEARCH Continued research should begin with investigation of Marek's disease virus infection in avian leukosis virus subgroup E infected cells. It could be of great interest to the poultry industry if vaccine strains of MDV were activating latent ALV. ELISA, reverse transcriptase assays, RNA quantitation, and nuclear run-off transcription assays could all be used to detect any changes in endogenous virus activity. Research investigating the transactivator gene down to as small an area as possible. Because ALV enhancer expression has already been located in the HVT E. Using chloramphenicol acetyl-transferase (CAT) assays, we can test for transactivator gene expression in HVT E fragment subclones. When the site of expression is found, it should be sequenced. Once open reading frames are identified, they can be compared to other known herpes virus sequences for similarity. To further characterize the transactivating protein, running SDS-PAGE on HVT E transfected whole cell extracts could determine it's size and number of subunits it contains. Comparison with similar extracts run on a non-denaturing gel (PAGE) could partially determine mechanism of enhancement if the transactivating protein binds to other cellular proteins. Protein sequence data derived from nucleotide sequence should also be compared to other protein sequences for similarity. LIST OF REFERENCES LIST OF REFERENCES Igarashi, T., Takahashi, M., Donovan, J., Jessip, J., Smith, M., Hirai, K., Tanaka, A., N onoyama, M., (1986). Restriction enzyme ma of Herpesvirus of turkey and it's collinear relationship with arek's disease virus DNA. irology 157, 351-358. Fukuchi, K., Sudo, M., Lee, Y-S, Tanaka, A., N onoyama, M. (1984). Structure of Marek's Disease Virus DNA: Detailed restriction enzyme map. J. of Vir., 51, 102-109. Tieber, V. L., Zalinskis, L., Silva, R. F., Finkelstein, A., Coussens, RM. (1990). Transactivation of the Rous Sarcoma Virus lon;9 terminal repeat promoter by Marek's disease virus. Virology, 1 , 719-727. 52 "IIIIIIIIIIIIIIIIIIIII