6L .;,‘."€1,§1.. : 1. F5‘1;.'11‘" :‘3..‘.\~11 3" ,"1~¢ 111 j '11 11'”le flz'i’hyjk‘g‘ 1| ‘ £\. -.;':- 1"3'1 ‘ ‘1 b,’ H :.§. 1‘3] 4‘ 9 l ”‘7' ["5131 . ~1‘_-'I!1*1walz'.,. 1:1 '1‘ 1.": 1v 1 l1 1 \ .1';1 t“ . . ‘1?"53) I 1L1 '2‘1‘.,;.“' 4.311,. 1“ I t 6.1“,” i 7 ~ 1.1., , .' 11.11111:1§‘,.,1 5; "1‘1 ;-l'-‘.‘. 1‘; 1: n... . 'n' ... _ 1- {4: 1,4,5: . n a» \1' ”1. ‘L'.;. "' 1‘ ‘15,..11fifl‘ as , a. _ x .Y' “1155'; :1; lg ' ‘ 1 - . 4 1.1 'r', :31. A; ”3 - ‘ 1', ”rudif’w‘vuu‘ 4:1" H? :1.: 1 -"$1.’"»31.531:R.li. . 1 , _1 _ 1 ‘ 3 .I' .1— 211:. 'L H ' .‘-' i011} 11 1.1;”lg1'41 ”411:: r? 1179i,l-:|!-511;{N1 . :1 .1. SanJ 1131,11. -' u 1 t, h 1 1 . - 11 ' ‘ ; E'-' 1 :1, "1er ”1%: 3:11;? I" 11% 1 :11. '1 {1111' 111111 #1111: "11'. ‘91 ‘1: "‘3'” .51’11'11I': 2.2.1.1 8’ 1': H {1131:113” : “1 ‘t‘a1'h'fi. ‘ 1.11;;l,:t:;:'11 . I 311 1‘?- W‘M'!‘ .' , . .1 '11! 13%| ' 31111113 ‘1 > TRL UL 1R1. [R3 Us TRS a,, b b’ a’II c’ c a IEIIIII} l"""IF""'}-—---iIIII:] Figm'e 1. The group B herpesvirus gerome structure standard group B genome based on HSV genome is written as a.b-UL-b’a..c’-Us-ca. TRL of group B genomes contain 11 copies of a sequence, whereas TRs contains one copy of a sequence. Numerous studies suggest that the 0 sequences present in the L-S junction and both termini of the HSV genome contain two cis-acting recognition signals which are involved in the inversion of L-S components relative to each other (Morse et al., 1977; Mocarslci et al., 1980; Chou and Roizman, 1985) and are also essential for proper cleavage and packaging of unit length viral DNA molecules (Deiss et al., 1986; Deiss and Frenkel, 1986). A potential a-like sequence has been identified in the L-S junctions and both termini of serotype 1 MDV and HVT DNA molecules (Kishi et al., 1991; Reilly and Sflva,1993) 7 Besides the standard group B genomic structure, serotype 1 MDV contains several sets of direct repeats consisting of more than 100-bp (designated DRl to DR5) scattered through the genome (Hirai, 1988). Physical maps of restriction endonuclease fragments have been constructed for the genomes of all three serotypes (Fukuchi et al., 1985; Igarashi et al.,1987; Ono et al., 1992) (Figure 2). The availability of RE maps and genomic clones of MDV DNA has greatly facilitated the cloning of MDV genes. Although all three MDV serotypes are antigenically related, their genomic RE patterns are very different (Ross et al., 1983). This A. ScotypelMDV ¥L -li-I5‘ U :h d- Q O '11 -v- N in» m - H ‘b H N u: go?" 7! D1 y—s ::_E > ::_.., .l B. Scotype 2 MDV (wlao (V.Z) (R1.S.1}I.X) P 51 0112 C NE “59H M G K D +_+_++++__f_i_'_'_fllllll l 1111 III I; I 11111 H II" III IT 1 C. Serotype 3 MDV (HVT) TRL UL IRL IRS US TRs E :EZI—lZI KI PN1 LllE DR B N211 0 \Mrch GI F0 A o l l 1 I ll 1 l L l I I I l I II I l r T I Figure 2. BamI-II restriction endonuclease maps of (A) Serotype 1 MDV (B) Serotype 2 MDV (C) Serotype 3 MDV difference can be used to identify new isolates (Silva and Barnett, 1991). Based on the 8 cross hybridization of cloned DNA fiagments, the genomes of MDV and HVT are related and similarly organized Recently, Ono et al. (1992) has shown that serotype 2 MDV is similar to serotype 1 MDV and HVT DNA molecules, with regard to genome structure and gene colinearity. III. DNA replication Mammalian DNA viruses have been usefirl model systems for the study of eucaryotic DNA replication (Challberg and Kelly, 1989; Stillman, 1989). Mammalian DNA viruses offer many advantages as models for DNA replication because their relatively simple genome structures allow easy manipulation at the molecular level Studies of Simian virus 40 (SV40) and adenovirus DNA replication, which rely mainly on the host-cell replication machinery, have revealed many essential eucaryotic cellular proteins which are involved in the viral DNA replication process (Challberg and Kelly, 1989). In contrast to SV40 and adenovirus, herpesvirus genomes are more complex and encode most of the proteins required for their DNA synthesis. Thus herpesviruses are an attractive model for studying the interactions between virus-encoded and cellular proteins involved in DNA synthesis . Study of MDV DNA replication is important for several reasons. First, Marek’s disease is highly contagious and results in tremendous losses to the poultry industry. In the interest of disease prevention, it is essential to understand the mechanism of MDV DNA replication. A better understanding of viral DNA replication may lead to better management of the disease in chickens. Second, an Imderstanding of the mechanism of MDV DNA replication may help to elucidate replication mechanisms in closely related 9 herpesviruses. In addition, knowledge of MDV DNA replication would contribute to the generation of a general model of DNA replication for or herpesviruses. Third, Marek’s disease is a good model for herpesvirus oncology because MD is a naturally occurring disease which can be reproduced experimentally using natural methods of exposure in the natural host. The mechanisms for establishment of latency and transformation by MDV infection are not clear at present. However, restriction of DNA replication has been implicated in DNA virus transformation. Also, integration of the virus genome into host cell chromosomes can lead to an imbalance of host gene expression and thereby contribute to the transformation process. Understanding MDV DNA replication will help to shed light on the mechanisms MDV uses for integration, and therefore provide insight to the transformation process by MDV. The literature review will be focused on comparisons of DNA replication from all three herpesvirus classes (alpha, beta, and gamma-herpesviruses). (i) Alphaherpesvirus DNA replication Herpes simplex virus (HSV) is an alphaherpesvirus and is the most extensively characterized of all alphaherpesviruses. As mentioned previously, MDV genome Structure is similar to that of HSV- 1. Location of the origin of replication within the complex HSV- 1 genome involved studies of both standard and defective viruses. Electron microscopic studies of replicating standard wild type HSV-l DNA isolated from infected cells have suggested that the genome contains two origins of replication, one near the middle of U1, and the other within the repeats flanking the Us (Friedmann et al., 1977; Hirsch et al., 1977). Studies of defective molecules of HSV- 1, which are generated during serial 10 passage of the virus at high multiplicities of infection, provided indirect evidence of two origins (Kaerner et al., 1979; Locker and Frenkel, 1979; Frenkel, 1981). Defective DNA molecules fall into two classes. Each of two defective DNA molecules consists of tandem duplications of small subsets of viral DNA sequence. Class I defective genomes contain sequences from the repeats which bracket Us (Kaemer et al. , 1979; Locker and Frenkel, 1979; Frenkel, 1981) whereas class II defective genomes contain sequences from UL (Kaemer, 1979; Frenkel, 1981). Direct evidence that the repeat units of class I and II defective genomes contain origins of replication was first provided by Frenkel and his colleagues (Vlazny and Frenkel, 1981; Spaete and Frenkel, 1982). These investigators demonstrated that monomeric Imits of class I and II defective DNAS are amplified to generate tandemly repeated DNA structures when cotransfected with wild type HSV-1 DNA which provides essential helper fimctions in trans. By using the cloned HSV-l fi'agments as seed units in the presence or absence of superinfecting wild type helper virus, the replication origins of HSV-1 were identified. From class I defective genomes, two copies of lytic origins have been identified within the inverted repeats flanking the S component (oris) (Figure 3) (Stow, 1982). A third origin, oriL, was localized to the center of L component (oriL) (Figure 3) (Spaete and Frenkel, 1982). Oris is located in a 90-bp fragment containing an imperfect palindrome with a central AT-rich region (Stow and McMonagle, 1983). The disruption of the palindrome abolishes the function of origin, suggesting that the palindrome is essential for origin function (Lockshon and Galloway, 1988). More accurate deletion mapping has refined the minimal sequence for origin fimction to a 75-bp fiagment ll onL 4 on, on8 WCMTATATATATAUAWAWAC site 111 site I site 11 adapted from Dabrowalri and Sdrafl‘er Figure 3. HSV-l origins and origin bindingprotein (UL9) recognition sites (Deb and Doelberg, 1988). This core region includes the 46-bp palindrome centered on AT -rich region and a region left of the left arm of palindrome. Replacement of AT base pairs in the AT-riCh region with GC base pairs eliminated origin activity (Lockshon and Galloway, 1988), suggesting that the AT-rich sequence at the center of palindrome is required for origin activity. OriL is located in a 425-bp fi'agment containing a perfect 144- bp palindrome (Weller, 1985). Deletion analysis revealed that the sequence within the palindrome is essential for origin fimction (Weller, 1985). Otis and orig, share extensive nucleotide sequence similarity, except for the sequences extending to rightward end from the AT-rich palindrome (Weller, 1985). To determine firnctional significance of the three separate origins, several mutant viruses were created. Mutant viruses lacking oriL or with one copy of oris replicated normally in vitro (Longnecker and Roizman, 1986; Polvino- Bodnar et al.,1987), while attempts to construct mutant viruses lacking both oris have not 12 been successful, implying that HSV-l DNA replication requires at least one copy of oris or, alternatively, at least two origin sequences (oris and orig, or two copies of oris ). Seven genes necessary for HSV-l DNA replication have been identified (Wu et al., 1988): a helicase and primase complex (UL5, UL8, and UL52), the origin-binding protein (OBP) (UL9), the major single-stranded DNA-binding protein (ICP8), DNA polymerase (UL30), and a polymerase accessory protein (UL42). The HSV-1 UL9 is an origin-specific binding protein and plays a role in initiation of HSV-1 DNA replication. Studies of OBP’S fimction indicate that it serves as an initiator for the HSV-1 DNA replication. A more extensive literature review on the description of HSV-1 OBP will be addressed in a later section entitled “origin-binding proteins of alphaherpesviruses”. Two high aflinity origin binding protein (OBP) sites (designated as sites I and II) at the ends of each arm of the oris palindrome have been identified (Figure 3) (Weller et al., 1985; Elias et al., 1986; Elias and Lehman, 1988; Olivo et al., 1988; Hazel et al., 1989; Martin et al., 1991). Site I has a 5 to 10—fold higher affinity for OBP than site II (Elias and Lehman, 1988). Using DNAase I footprinting, methylation interference, and electrophoretic mobility gel shift assay (EMSA) with mutant oligonucleotides for site I, the HSV-l OBP binding site was mapped to a domain of 11 nucleotides in site I (CGT'I‘CGCACTT) (Kofi‘ et al., 1988; Deb et al., 1989; Elias et al., 1990; Hazuda et al., 1991). The ll-bp element within site II is different in two positions fiom that within site I (Elias et al., 1990) (Figure 3). The presumed HSV-1 OBP binding site (1 l-bp element) is conserved in both oriL and orig of HSV-1 and HSV-2 as well as in VZV (Stow et al., 1986; Polvino-Bodnar et al., 1987; Baumann et al., 1989). It was proposed that HSV-l 13 OBP binds as a dimer to two inverted, overlapping pentanucleotides within site I (5’- GTTCGCAC-3’I3’-CAAGCGTG-5’) (Kofl‘ et al., 1988; Fierer and Challberg, 1995). An oscillating activity was observed with plasmids containing different copy numbers of the AT dinucleotide within the AT-rich region. This phenomenon suggested that OBP may be required to bind to the cognate binding sites located on the same side of the DNA helix (Lockshon and Galloway, 1988). Deletion and mutation analysis have shown that both sites I and II are required for the eflicient activity of orig (Deb and Deb, 1989; Weir and Stow, 1990; Hernandez et al., 1991, Martin et al., 1991). Thus, interaction of OBP with oris is critical to optimal HSV-l DNA replication. The Orig region contains a sequence which has strong sequence similarity to OBP- binding sites I and II, and has been designated as site 111 (Figure 3). Site H1 is located to the left of site I. No sequence-specific OBP binding, however, has yet been demonstrated to this Site (Elias et al., 1990; Weir and Stow, 1990). Analysis of origin sequences in HSV-2 have Shown that deletion of part of a sequence corresponding to site III in HSV-1 resulted in a dramatic loss in DNA replication (Lockshon and Galloway, 1988), while the deletion of site 111 in HSV-l oris affected replication only moderately (Weir and Stow, 1990; Martin et al., 1991). Thus, although OBP was not shown to bind to site 111 in vitro, in vivo studies suggested that all three OBP-binding sites were required for optimal replication of HSV- 1. Like other DNA-containing viruses, the three origins of HSV-1 DNA replication are flanked by sequences containing transcriptional regulatory elements. OriL is positioned between the divergent transcriptional start sites of the genes encoding the major DNA- l4 binding protein (ICP8) and DNA polymerase (Weller et al., 1985; Polvino-Bodnar et al., 1987). Oris, like oriL, is also located between divergently transribed genes. These genes encode the immediate-early proteins ICP4 and ICP22/47 (Stow and McMongle, 1983). The transcriptional regulatory elements within which oris resides are well characterized and collectively exhibit the properties of an enhancer element (Preston et al., 1984; Preston and Tannahill, 1984; Preston et al., 1988). A variety of recognized transcription factors have been shown to bind specifically to Otis-flanking sequences. These include the potent HSV-1 transactivator VP16 (apRhyS et al., 1989) and cellular transcription factors Spl, and nuclear factor II] (NF-III) (Bzik et al., 1986). It has been shown that the promoter-regulatory elements surrounding the oris act to increase the overall replication efliciency of orig-containing plasmids (Wong and Schafl‘er, 1991). Wong and Schafl‘er (1991) postulated that the trans-acting factors that bind to regulatory elements of immediate early genes may serve specifically to make oris more accessible to proteins of the initiation complex. Alternatively, the proteins which bind to and regulate the immediate-early genes may interact directly with DNA replication proteins and assist in promoting localized strand separation. Several studies have suggested that the linear genome of HSV-1 circularizes upon entry into susceptible cells and replicates predominantly by a rolling circle mechanism (Ben-Porat et al., 1976; Mocarski and Roizman, 1982; Roizman, and Sears, 1991). Based on pulse-chase experiments and electron microscope observations of HSV DNA replication intermediates from bouyant density centrifirgation, Jacob and Roizrnan (1979) observed that there were five types of HSV-1 DNA replication intermediates : (1) linear, 15 full size molecules with internal gaps and single-stranded regions at the termini; (2) molecules with a lariat structure; (3) circular, double-stranded molecules; (4) molecules with “D” loop; and (5) large, tangled masses of DNA By using restriction enzyme analysis of extracted HSV-l DNA replication intermediates, Jacob and Roizman (1979) reported that head-to-tail concatemers accumulated in the nuclei of infected cells. Based on these observations, it was proposed that HSV-1 DNA replicates by a rolling circle mechanism. It is possible that the linear HSV-1 DNA can fold back upon itself and the ends ligate together to form circular molecules through the a sequence. The resulting circular molecules may then serve as the templates to generate linear concatemers consisting of tandemly repeated genome size Imits of HSV-1 via a rolling circle mechanism. Recent studies on the mechanism of HSV-1 DNA replication firrther support the rolling circle mechanism for HSV-l DNA replication. Rabin and Hanlon (1990) performed in-vitro DNA syntheses with HSV- l-infected-cell extracts using a preformed replication fork that was made from a nicked, doubled-stranded, circular DNA molecule with a 5 ’ single-stranded tail The product of this in-vitro reaction was a linear concatemer, as demonstrated by electron microscopy. Skaliter (1996) performed an in-vitro DNA synthesis with HSV-l-infected-human cell extracts using pUC18 plasmid as the template to generate linear concatemers composed of tandemly repeated plasmid Size units of pUClS. Therefore, the rolling circle mechanism is involved in HSV-l DNA replication. However, other recent studies on the interactions between HSV-l origin binding protein (UL9) and other replication proteins have suggested that HSV-1 DNA initially replicates by an early origin-dependent theta-circle replication step followed by subsequent rolling 16 circle replication to generate concatemers that are packaged into viral particles. By immunoprecipitation, Lee et al. (1995) showed that the UL9 protein co- immrmoprecipitated with the 180 kDa catalytic subunit of cellular DNA polymerase a- primase but not with HSV-1 DNA polymerase, suggesting that the UL9 protein interacts with the cellular polymerase but not with the viral polymerase and initiation at viral origins may be accomplished by UL9 and a cellular polymerase. Skaliter and Lehman (1994) reported that extracts of insect cells infected with baculovirus recombinants containing seven HSV-1 genes required for replication but not the UL9 gene could promote rolling circle replication of pUC18 plasmid, suggesting that rolling circle replication is independent of the UL9 gene and the origins but does require other viral replication proteins. These results strengthens the model that HSV-1 DNA initiates replication at the viral origin and later replicates via the rolling circle mechanism. Several lines of evidence suggest that HSV viral genome maturation involves site- specific cleavage of viral DNA concatemers to unit size monomers of viral genome (Deiss et al., 1986a; Deiss et al., 1986b; Varmuza and Smiley; 1985). The cis-acting sequence required for cleavage is located within the a sequence (Varmuza and Smiley; 1985; Deiss et al., 1986; Deiss and Frenkel, 1986). The a sequence is present as a direct repeat at both termini and in inverted orientation at the L-S Motion. The a sequence is present in a single copy at the S terminus of the viral genome and in one to several copies at the L terminus and at the L-S jtmction (Mocarski and Roizman). Two separate cis-acting signals within the a sequence (called pac-l and pac-Z) appear to be essential for the cleavage/package process (Varmuza and Smiley, 1985; Deiss et al., 1986). The signals l7 essential for the packaging/processing reaction are structurally conserved among many herpesviruses (Davison, 1984; Matsuo et al., 1984; Albrecht et al., 1985; Bankier et al., 1985; Tamashiro and Spector, 1986; Marks and Spector, 1988). (ii) Beta-herpesvirus DNA replication Cytomegaloviruses (CMV) is a betaherpesvirus. In comparison to alphaherpesviruses which have relatively short reproductive cycles and spread rapidly in culture, betaherpesviruses have a long reproductive cycle and grow Slowly in culture. CMV has the largest genome among herpesviruses, equivalent to approximately 240 kliobase pairs (kb). The genome of human CMV also belongs to group B genome (Roizman, 1991). CMV infection is very complex Infection of the host by CMV can become latent or lead to productive infection that can either persist asymptotically, or cause disease. Based on a novel approach utilizing gancyclovir-induced chain termination, Hamzeh et a1 (1990) identified an authentic lytic origin (oriLyt) of human CMV DNA replication within the center of Imique long region (EcoRI-V fragment). Subcloning and deletion analyses of the region containing the authentic lytic origin defined a 2.4-kb core region containing elements required for the oriLyt firnction (Anders, et al. , 1992). In contrast to the alphaherpesvirus lytic origin of DNA replication, the human CMV lytic origin is large and complex The overall base composition of this region is similar to that for the entire genome, but is asymmetric. At the left boundary is an AT-rich region (up to 72%) while at the right boundary is a 62% GC-rich region (Anders et al., 1992). The region within and around the boundaries contain numerous repeated motifs, including 18 known transcription factor recognition sequences. OriLyt contains two kinds of lO-bp repeats, a 12-bp repeat, a 15-bp repeat and a 14-bp repeat (Anders et al., 1992). Numerous known transcription factor recognition sequences are present in the human CMV oriLyt, including ATF/CREB sequences, MLTF/U SF sequences, and Spl motifs (Anders et al. , 1992). TATA, CAAT and polyadenylation sequences are also present in the human CMV oriLyt (Anders et al., 1992). The lytic origin of simian CMV has also been identified and analyzed. Subcloning and deletion analyses defined a 1.3-kbp core region suflicient for origin fimction in the apparently noncoding region upstream of the single- stranded DNA-binding protein gene (dbp) (Anders and PImturieri, 1991). AS with the oriLyt of human CMV, the oriLyt of simian CMV is also complex Nucleotide sequence analysis has revealed four distinct domains : (1) a 9-bp repeated sequence; (2) an AT-rich segment; (3) an ll-bp direct repeat; and (4) a 47-bp direct repeat (Anders and PImturieri, 1991). Like alphaherpesvirus, CMV requires virus-encoded proteins for DNA replication. Eleven loci encoding trans-acting factors required for transient complementation of human CMV oriLyt-mediated DNA replication have been identified (Pari and Anders, 1993; Pari et al., 1993). In human CMV-infected cells, blocking expression of individual proteins expressed by members of this set of loci inhibited viral DNA replication (Ripalti et al., 1995; Smith and Pari, 1995), suggesting that these eleven loci are required for viral DNA replication in viva. Six of the defined loci encode homologs of HSV-1 replication genes (Pari and Anders, 1993). UL54 encodes a DNA polymerase that shows sequence similarity to a variety of alphaherpesvirus DNA polymerases. UL44 encodes a putative polymerase l9 accessory protein homologous to the HSV-1 UL42. UL57 encodes a single-stranded- DNA binding protein homologous to HSV-1 major DNA-binding protein (ICP8). UL70 encodes a homolog of HSV-1 UL52. HSV-1 UL52 encodes a primase activity which is a component of a three-submit primase-helicase complex UL70, UL101-102, UL105 are homologous to HSV-l helicase-primase subrmits. Five additional loci are required to complement human CMV DNA replication in transient assays. In contrast to HSV-1 DNA replication, the homologous proteins are not required to complement viral DNA replication in transient assays. Three of the five additional loci (UL36-3 8, UL122-123 and [RSI/TRSI) encode viral transactivators. It is not surprising that the viral transactivators are involved in DNA replication. It was established that origin efliciency was augmented or controlled by elements that also regulate transcription (Depamphilis, 1988), as found for HSV-1 (Wong and Schafl‘er, 1991). It has been shown that four of the eleven loci (UL36-38, UL112-113, IRSl/TRSI, and the major immediate early region UL122-123) required for transient complementation of human CMV DNA replication cooperate to activate expression of the replication genes (UL54, UL44, UL57, UL70, UL102 and UL1105) (Iskenderian, et al., 1996). The overall mechanism of CMV DNA replication is not clear. However, it is thought that after entering permissive cells, linear CMV genome circularizes (LaFemina and Hayward, 1983) and then replicates in the nucleus by a mechanism producing concatemers that are subsequently cleaved and packaged during virion assembly (Stinski, 1991). Transient transfection assays revealed that oriLyt containing plasmids can induce 20 the amplification of tandem oligomers (Anders et al. , 1992) and suggests that CMV DNA replicates via a rolling circle mechanism similar to HSV- 1. (iii) Gammaherpesvirus DNA replication Epstein-Barr virus (EBV) is a human herpesvirus belonging to the gammaherpesvirus family. Human B lymphocytes can be infected and immortalized by EBV (Henle, 1967; Epstein and Achong; 1979). EBV is associated with mononucleosis, nasopharyngeal carcinoma and Burkitt’s lymphoma (zur Hansen, 1981). The EBV genome is a linear, double- stranded 172 kb DNA molecules. The structure of EBV genome is composed of five unique sequence domains which are divided by four classes of internal repeats (Irs) and a variable number of directly repeated 0.5-kbp sequences (TR) located at both ends of EBV genome (Dambaugh, 1980). The overall EBV genome can be designated as TR-Ul-IRl-U2-IRZ-U3-1R3-U4-IR4-U5-TR. In cells immortalized by EBV, multiple copies of the EBV genome are maintained as 172 kb supercoiled plasmids (Lindahl et al., 1976; Gussander and Nonoyama, 1984). A cis—acting sequence that is required for plasmid replication and maintenance in immortalized cells has been identified. This sequence, designated oriP for origin of plasmid replication, is located on a 1.8 kb segment of the EBV genome which is located in the U1 region. (Yates et al., 1984; Lupton and Levine, 1985). Deletion mapping has identified two separate regions in oriP, each required in cis for plasmid maintenance (Lupton and Levine, 1985; Reisman et al., 1985). Region I is composed of 20 imperfect copies of a tandemly repeated 30 bp sequence. Region 11, located 960 bp away, consists of a 65 bp sequence forming a dyad symmetry. Region I essential for long-term maintenance of the 21 plasmid form of EBV (Lupton and Levine, 1985; Reisman et al., 1985) contains a termination site for replication (Gahn and Schildkraut, 1989) and fimctions as a transcriptional enhancer for RNA polymerase II-transcribed genes (Reisman and Sugden, 1986). Region I also plays a role in plasmid segregation during cell division. Region II is at the initiation site of latent cycle DNA replication and contains the actual origin of replication (Gahn and Schildkraut, 1989). In latently infected cells, oriP only replicates once during the S phase of the cell cycle in a coordinate fashion with cellular DNA synthesis (Hamper et al., 1974; Adams, 1987). The close proximity of initiation and termination sites in oriP results in replication of the plamd proceeding in a predominantly unidirectional manner (Gahn and Schildkraut, 1989). In latently infected B lymphocytes, infectious virus is not produced and only a small fiaction of the EBV genome is expressed. At least nine genes are expressed in latently infected cells. Six of these genes encode nuclear proteins (EBNA-l, -2,-3A, -3B, - 3C and -LP) . Of these six nuclear proteins, EBNA-l is the best characterized protein. EBNA-l is the only EBV protein required for EBV genome replication during the latent cycle. (Yates et al., 1984; Lupton, S., and Levine, 1985; Yates et al., 1985). All other proteins required for plasmid replication are provided by the host cell. There are multiple EBNA-l binding sites within oriP. Each of the twenty 30 bp repeats within the region I of oriP contains one EBNA-l binding site (Ambinder et al. , 1991; Rawins et al., 1985) and region H contains four EBNA-l binding sites (Ambinder et al., Rawins et al., 1985). Multiple EBNA-l binding sites are required for the fimction of oriP (Wysokenski and Yates, 1989). Deletion analysis and site-directed mutagenesis of 22 oriP-containing recombinant plasmid revealed that at least six to eight copies of EBNA-l binding sites within region I are required for plasmid maintenance and only two EBNA-l binding sites are required for the firnction of region 11 (Chittenden et al., 1989; Harrison et a1. , 1994). EBNA-l is a rmrltifimctional protein. It cooperatively assembles on oriP as a dimer via a direct interaction (Summers, 1996). It is the only viral protein which is required for plasmid replication. The family of repeats within region I , when bormd by EBNA-l , can activate replication from region 11, enhance latent transcription and control the stable segregation of EBV episomes during cell division (Lupton and Levine, 1985; Reisman et al., 1985; Reisman and Sugden, 1986; Gahn and Schildkraut, 1989). DNase I and mm footprinting in vitra and in viva on oriP bound by EBNA-l has revealed that EBNA-l can induce distortion of region 11 but is unable to distort the duplex in region I (Hsieh et al., 1993 ). Investigation of the interaction of pure EBNA-l with oriP DNA has shown that EBNA-l dimers bound to region I and II interact emciently with each other, bringing the two elements together (Frappier and O’Donnell, 1991; Su et al., 1991; Middleton and Sugden, 1992). This interaction results in the generation of looped DNA molecules and cross-linking of multiple DNA molecules via EBNA- 1. Interactions between EBNA-l molecules bound to region I and H stabilize EBNA-l on region 11 and likely are an important part of the mechanism by which region I activates replication from region 11. Purified EBNA-l lacks helicase activities and does not appear to act by performing any enzymatic fimction (Yates and Camiolo, 1988a). Thus, initiation of replication at oriP is dependent upon a cellular DNA helicase for the initial unwinding of DNA. 23 FImctional domains of EBNA-l have been investigated extensively. Deletion analysis of the domain which consists of a repetitive array of glycine and alanine residues has revealed that this domain is not essential for function of EBNA-l (Yates et al., 1985; Polvino-Bodnar et al., 1988; Yates and Camiolo, 1988b). The domains essential for fimction of EBNA-l are all localized within the C-terminal domain, including the domains for nuclear localization, for dimerization, for DNA binding, for transactivation and for DNA-looping (Ambinder et al., 1991; Inoue at al., 1991; Laine and Frappier; 1995). Lytic EBV replication occurs in the mucosa] epithelial cells of the oropharynx and gential tract (Sixbey, 1989) and can be induced by treating latently infected B cells with lZ-O—tetradecanoyl-phorbol—13-acetate (TPA) (zur Hansen et al., 1978) or by introduction of the EBV Zta transactivator (Countryman and Miller, 1985). Lytic phase replication proceeds via a separate origin, oriLyt, which is difl‘erent fiom oriP and results in 100- to 1,000-fold amplification of the genome via concatemeric intermediates (Hammerschmidt and Sugden, 1988; Sato et al., 1990). There are two copies of oriLyt in the intact EBV genome. One copy is centrally located within the EBV genome, approximately 40 kb away from oriP and the other is located at the right end of EBV genome. EBV isolates containing one copy of oriLyt replicate as efliciently as EBV isolates containing two copies of oriLyt (Hammerschmidt and Sugden, 1988), suggesting that one copy is suficient for lytic phase replication. OriLyt is complex and contains arrays of direct and inverted repeats. OriLyt can be divided into three essential domains: (i) The first domain is the promoter and leader of the BI-ILFl gene. The BHLF l promoter contains four binding sites for the Zta (BZLF 1) transactivator and is strongly Zta 24 responsive in transient expression assays (Lieberman et al., 1989; Lieberman et al., 1990). (ii) The second domain is a central 225-bp region whose prominent features include two related AT-rich palindromes of 18 and 20 bp and an adjacent polyurine-polypyrimidine tract. Elements of this type may serve as sites for initiation of DNA replication or transmission of localized tmwinding in origins of replication (Kowalski, 1989; Wells, 1988). (iii) The third domain is an enhancer that responds to the Rta transactivator and contains two binding sites for Rta and one for Zta (Cox, 1990; Grufl‘at, 1990; Liberman, 1990) By using transient replication assays, Fixman et al. (1992) identified the EBV genes essential for transient complementation of oriLyt-mediated DNA replication, which inchided six EBV genes (BALF5, BMRFl, BALF2, BBLF4, BSLF], and BBLF2/3), the viral lytic-cycle transactivators, Zta, Rta and Mta, and an unidentified gene in the SalI F fiagment. These Six EBV replication genes are homologous to six of seven essential genes for HSV-1 DNA replication. BALF 5 shares 33% identity to the HSV-l DNA polymerase, BMRFl is a positional and functional homolog of the HSV-1 DNA polymerase processivity factor, BALF2 shares 25% identity with the HSV-l single stranded DNA- binding protein (ICP8), and BBLF4 as well as BSLF l have significant sequence identity with the HSV-1 UL5 and UL52 genes. BBLF2/3 is likely to be a homolog of HSV-1 UL8. The activity in Sail-F was shown to be encoded by BKRF3 which encodes an enzyme, uracyl DNA glycosylase. This enzyme is dispensable for replication (Fixrnan et al., 1995). One of the replication genes that has not been identified is an oriLyt origin-binding protein equivalent to the HSV-l origin-binding protein, UL9. Fixman et al. (1995) reported that 25 EBV does not encode an equivalent of HSV-1 UL9 and that Zta is the sole virally encoded protein that serves as an essential origin-binding protein. (iv) Marek’s disease virus DNA replication Replication origins for all three MDV serotypes have been identified. A functional origin of replication for serotype 2 MDV was identified (Camp at al., 1991) using a defective MDV genome and transient replication assay. The serotype 2 MDV replication origin is located in the inverted repeats flanking the unique long region (Figure 4), suggesting that there are at least two copies of the origin. The replication origin of serotype 2 MDV was located to a 90-bp region (Figure 4). Like HSV-1 oris and oriL, it contains an imperfect palindrome with 30 bp of alternating AT sequence located at the enter. The structure and sequence of the serotype 2 MDV replication origin is very similar to HSV-1 oriL and Otis, VZV origin, and equine herpes virus type 1 (EHV-l) origin (Camp at al., 1991). In addition, the serotype 2 MDV replication origin contains a 9-bp motif which is located both at the left and right arms of the palindrome (Figure 4). This 9- TRL UL m, 112., vs mg i: a 11. E: ACGCGTW CGAACCAATA TMGATTATA TATATMTAT A‘I'I‘A’I'I‘GGCG WCGTCCG CGCAATCGGG Figure 4. The origin ofserotype 2 MDV DNA replicatiar. Th 9-bp motifwhich is cmserved ammg alphaherpesviruses is indicated by the bars under seqrence bp motif is highly conserved among alphaherpesviruses. The 9-bp motif sequence is a 26 subset of a ll-bp motif that is recognized by HSV-1 origin binding protein (UL9). The presence of two copies of the 9-bp motif suggests that there are two potential binding sites for MDV origin-binding protein. Based on nucleotide sequence analysis, a putative replication origin of serotype 1 MDV was reported (Bradley et al., 1991). The putative serotype 1 MDV replication origin is located in the inverted repeats flanking the unique long region of serotype 1 genome and contains several transcription factor binding sites (spl and Oct-1 binding sites) within the flanking sequence. As with HSV-l, CMV and EBV, these auxiliary elements may play a role in MDV DNA replication. Morgan et a1 (1991) reported that there was a decrease in MDV plaque formation when cells were cotransfected with viral DNA and plasmids containing the auxiliary components flanking the putative serotype 1 MDV replication origin. Based on nucleotide analysis and DpnI resistance assays, an HVT replication origin was identified (Smith et al., 1995). The HVT replication origin is located in the Motion between UL and IRL and is very similar to the origins of replication of MDV- 1, -2, HSV— 1, VZV and EHV-l. It also contains a 9-bp motif which is conserved among alphaherpesviruses. As found for the MDV-1 replication origin, there are CAAT sequences, spl binding motifs and an Oct-1 binding motif present in the HVT replication origin. The mechanism of MDV DNA replication has not been determined. However, based on the structural similarity and gene colinearity between HSV-1 and MDV, it is likely that MDV replicates via rolling circle mechanism Based on nucleotide analysis, 27 Camp et al (1993) reported that an HSV a-like sequence was identified within the 5’-end of 4 kb MDV replicon (a single monomeric repeat unit of serotype 2 defective MDV genome). 3. Origin binding protein (OBP) of alphaherpesviruses I. The OBP of herpes simplex virus type 1 (HSV-1) HSV-1 UL9 is a multifimctional origin-binding protein. HSV-1 UL9 binds directly and cooperatively to two UL9 binding sites (sites I and H) within oris (Olivo et al., 1988; Elias et al., 1990). It also exists as a dimer in solution (Bruckner et al., 1991), unwinds the partial DNA duplex in vitra (Fierer and Challberg, 1992), exhibits DNA-helicase activity (Bruckner et al., 1991; Boehmer et al., 1993) and has DNA-dependent nucleoside 5'-triphosphatase activity (Dodson and Lehman, 1993). In addition, purified HSV-1 UL9 forms a high order nucleocomplex with plasmids containing HSV-l oris via inter- and intra-molecular interactions (Rabkin and Hanlon, 1991). Based on DNase I and MO; footprinting with purified HSV-1 UL9, Kofl‘ et a1(1991) reported that HSV-l UL9 loops and distorts HSV-l oris. Since HSV-l UL9 is an origin binding protein, it is not surprising that HSV-l UL9 interacts with other replication proteins. Several studies have revealed that HSV-1 UL9 interacts with single-stranded DNA binding protein (ICP8) in vitra and in viva (Boemer et al., 1993; Boemer et al., 1994; Gustafsson et al., 1995) and that this interaction is influenced by DNA ligands (single-stranded or double- stranded DNA). HSV-1 UL9 interacts with ICP8 and double-stranded DNA to form a triple complex but the complex between HSV-1 UL9 and ICP8 can be destroyed by single- stranded DNA, suggesting that 28 the interaction between HSV-1 UL9 and ICP8 serves to position the single stranded DNA-binding protein with high precision onto the single-stranded DNA at the replication fork or at the origin of replication. By using immunoprecipitation, Lee et a1 (1995) showed that the HSV-1 UL9 interacts with the catalytic subunit of DNA polymerase or. HSV-l UL9 also has been shown to interact specifically with HSV-l UL8 (Mclean et al., 1994). By using the indirect irmmmofluoresence, Liptak et a1 (1996) showed that the HSV-1 replication proteins assemble in an orderly fashion to form a prereplicative complex, and suggested that the HSV-l UL9 is a component of the replication complex The structure and fimction of HSV-1 UL9 have been investigated extensively. The N-terminal domain of HSV-1 UL9 encodes helicase activity (Martinez et al., 1992) and is responsible for dimerization and cooperativity (Hazuda et al., 1992; Elias et al., 1992). The helicase activity of HSV-1 UL9 is consistent with the presence of six helicase conserved motifs within the N-terminal domain. Single amino-acid substitutions at conserved residues in each of six helicase motifs inactivated HSV-l UL9 in transient origin-dependent replication assays (Martinez et al., 1992). Insertion nnrtagenesis of the HSV-l amino-termirral leucine zipper domain dramatically afi‘ected cooperativity and dimerization of HSV-1 UL9 in solution (Hazuda et al., 1992), suggesting that the leucine zipper within the N-terminal domain was essential for cooperativity and dimerization. Stabell and Olivo (1993) reported that the truncated form of HSV-1 UL9 which contains only the C-terminal origin binding domain, binds to oris but does not induce conformational changes in orig, suggesting that besides the helicase activity and dimerization, the N-terminal of HSV-1 UL9 is necessary for a UL9-induced 29 conformational change on oris. The C-terminal domain of HSV-1 UL9 encodes DNA- binding activity (Deb and Deb, 1991; Arbuckle and Stow, 1993; Martin et al., 1994). It contains two known structural motifs (a pseudo-leucine zipper and a helix-tum-helix motif). By insertion and deletion mutagenesis, the pseudo-leucine zipper was shown to be important for DNA-binding activity (Deb and Deb, 1991; Arbuckle and Stow, 1993; Martin et al. , 1994). However, importance of the helix-turn-helix motif for DNA-binding activity remains to be elucidated (Deb and Deb, 1991; Arbuckle and Stow, 1993; Martin et al., 1994). A highly conserved region is formd within the very C-terminal end of HSV-1 UL9. This sequence, termed the VZV homology region (Martin et al., 1994), is very similar to a corresponding sequence in VZV gene 51 ( a homolog of HSV-1 UL9). Despite a lack of similarity to any known structural motif, the VZV homology region is critical in maintaining DNA-binding activity of HSV-1 UL9 (Deb and Deb, 1991; Arbuckle and Stow, 1993; Martin et al., 1994). In addition to DNA-binding activity, the C-terminal domain is also needed for association with ICP8. Boehmer et a1 (1994) showed that a UL9 mutant protein that lacks the C-terminal 27 amino acids exhibits the normal origin-specific DNA binding and retains its DNA-dependent ATPase and helicase activities, but has greatly reduced afinity for ICP8 (Boehmer et a1, 1993), indicating that the C-terminal 27 amino acids are essential for ICP8 association. The biochemical activities of HSV-1 UL9 are similar to those of large T antigen of simian virus 40 (SV40). In SV40, large T antigen not only binds to the replication origin as two hexamers and unwinds it locally for recruitment of other replication proteins but 30 also serves as the DNA helicase for subsequent steps in replication (Boroweic et al. , 1990). It play an essential role in initiation of SV40 DNA replication. The precise role of HSV-1 OBP in HSV-1 DNA replication has not been established but it is suggested that it plays a role in initiation of HSV-1 DNA replication similar to the role of SV40 large T antigen serving as an initiator protein for viral DNA replication. II. The OBP of varicella-zoster virus (V ZV) VZV gene 51 is a homolog of HSV-1 UL9. The predicted amino acid sequence of gene 51 protein was shown to be 44% identical to HSV-l UL9 (Chen and Olivo, 1994). VZV gene 51 protein binds to three sites (A, B and C) within the VZV origin but with different aflinity for each of these three sites (Stow et al., 1990). All three binding sites for VZV gene 51 protein are very similar to the HSV-l UL9 binding site I. They all contain an ll-bp motif which is recognized by HSV-1 UL9. Based on deletion analyses, Chen and Oilvo (1994) showed that the C-terminal domain of VZV gene 51 protein encoded DNA binding activity. By alignment of the predicted amino acid sequences of HSV-1 UL9 and VZV gene 51, Chen and Olivo (1994) showed that VZV gene 51 contained all the motifs essential for firnction of HSV-1 UL9, including six helicase conserved motifs as well as a leucine zipper within the N-terminus and a pseudoleucine zipper as well as a helix-tum- helix motif within the C-terminus. The spatial arrangement of all three conserved motifs within VZV gene 51 protein are the same as that within HSV-1 UL9. The similarity between HSV-1 UL9 and VZV gene 51 suggests that the VZV gene 51 has biochemical activities similar to HSV-1 UL9. Consistent with this prediction, Webster et al. (1995) reported that VZV OBP can substitute for the HSV-1 OBP in a transient origin-dependent DNA replication assay in insect cells using the recombinant baculoviruses which can 31 express the seven genes essential for HSV-1 DNA replication. In addition, VZV gene 51 can support the replication of a HSV-l UL9 null mutant (Chen et al., 1995). The ability of VZV gene 51 to complement UL9 suggests that alphaherpesviruses have a highly conserved mechanism of initiation of viral DNA synthesis. III. The OBP of equine herpes virus type 1 (EHV-1) Based on the predicted amino acid sequence, the EHV-1 gene 53 was shown to have a strong similarity to HSV-1 UL9 (Martin and Deb, 1994). Using PCR to clone the EHV-1 gene 53 and in-vitra coupled transcription-translation to express the EHV-l gene 53, Martin and Deb (1994) Showed that EHV-l gene 53 protein binds to HSV-1 UL9 binding site I as well as to the EHV-1 origin and that a 9-bp motif which is a subset of the 11-bp motif recognized by HSV-l UL9 is essential for binding the EHV-1 gene 53 protein. By deletion analysis, Martin and Deb (1994) reported that the C-terminal domain encodes the DNA-binding activity. By alignment of the predicted amino acid sequences of HSV-1 UL9, VZV gene 51 and EHV-l gene 53, Martin and Deb (1994) showed that all the structural motifs which are essential for fimction of HSV-1 UL9 are conserved among all three OBPS and that the spatial arrangement of all conserved motifs is the same for all three OBPS. The similarity between HSV-1, VZV and EHV-l OBPS suggests that OBPS are highly conserved among alphaherpesviruses and the OBP of MDV should be structurally similar. Chapter 2 Cloning and sequence analysis of a Marek's disease virus origin binding protein (OBP) reveals strict conservation of structural motifs among OBPS of divergent alphaherpesviruses Ting-Fang Wu, Wei Sun, Mekki Boussaha, Ronald Southwick and Paul M. Coussens“ Virus Genes in press 32 Abstract Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus. In it's natural host, MDV induces Marek's disease (MD), a lethal condition characterized by malignant lymphoma of T cells Although symptoms of MD may be prevented by vaccination, no practical pharmacological method of control has been widely accepted. Viral replication represents a point at which pharmacological control of herpesvirus infection may be most successfirl However, this requires detailed knowledge of viral replication proteins. Studies in HSV-l DNA replication implicate the UL9 protein as a key initiator of replication. For example, binding of UL9 to HSV-l origins is a prerequisite for assembly of additional replication proteins. In this study, a protein, whose apparent molecular size iS similar to that of HSV-1 UL9, was identified in extracts of MDV infected cells by western blot analysis with anti-HSV-l UL9 antibody. A putative MDV UL9 gene was subsequently identified through sequencing of MDV genome fragments (BamHI G and C). Extended DNA sequence analysis revealed an open reading frame (ORF) which could encode a protein homologous to HSV-1 UL9. The MDV UL9 ORF encodes 841 amino acids, producing a sequence 49 % identical to HSV-1 UL9 and 46% identical to VZV gene 51 product (VZV UL9). MDV UL9 shares numerous structural motifs with HSV-1 and VZV UL9 proteins, including six conserved N-terminal helicase motifs, an N- terminal leucine zipper motif, a C-terminal pseudo-leucine zipper sequence, and a putative helix-tum-helix structure. 33 Introduction Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus. In chickens, MDV is the etiologic agent of Marek's disease (MD), characterized by malignant lymphoma of T cells (Calnek et al., 1991). MDV cell tropism and pathology are similar to those of gammaherpesviruses (Roizman and Knipe, 1990). However, based on overall genomic structure and colinearity of many MDV genes to those of alphaherpesviruses, such as herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV), MDV has been classified as an alphaherpesvirus (Cebrian et al., 1982; Bucmaster et al., 1988; Brunovski et al., 1992; Roizman, 1992). Consistent with this classification, MDV genomes are linear double-stranded DNA molecules 160 to 180 Kbp in length, consisting of two unique regions (Us and UL) flanked by inverted repeats (IRS, IRL, TRS, and TRL). Furthermore, two fimctional replication origins, with high similarity to HSV-l replication origins, have been identified in different MDV serotypes (Camp at al., 1991; Smith etal., 1995) Viral replication represents a point at which pharmacological control of herpesvirus infection may be most successful. However, this requires detailed knowledge of viral replication mechanisms and proteins involved ill the replication process. Thus, better management of MD in poultry requires an understanding of MDV DNA replication mechanisms. In addition, information on MDV DNA replication may help elucidate similar mechanisms in closely related herpesviruses and contribute to generation of a general model of DNA replication for herpesviruses. Much of what is currently known regarding herpesvirus replication stems from studies of HSV-1. HSV-1 encodes seven proteins that are both necessary and suficient for 34 35 origin-dependent DNA synthesis (Wu et al., 1988). The seven genes encode a two- subrmit DNA polymerase (UL30 and UL42), a single-stranded DNA binding protein (UL29 or ICP8), a three protein complex with helicase-prirnase activities (UL5, UL8 and UL52) and an HSV-1 origin-specific DNA binding protein (UL9). The UL9 protein binds to specific sequence elements within the viral DNA replication origin, a critical event which represents the first step toward initiation of DNA replication. There are two types of HSV-1 DNA replication origins: (1) oris, located in inverted repeats flanking the Us region (Stow and Mcmonagle, 1983); and (2) mil, centrally located within the unique long region (Weller et al., 1985). Both origins feature palindromic DNA sequences with central A+T-rich regions and share considerable homology (Weller et al. , 1985). Disruption of palindromic sequences abolishes origin fimction (Stow, 1984; Lockshon and Galloway, 1988), suggesting that these sequences are essential for replication. Two high-aflinity UL9 binding sites occur within regions of dyad symmetry in both oris and oriL (Weller et al., 1985; Elias et al., 1988; Olivo et al., 1988; Martin et al., 1991). Mutagenesis experiments indicate that UL9 binding sites are also critical for origin function (Deb and Deb, 1989; Weir and Stow, 1990). HSV-l UL9, purified fiom recombinant expression systems, binds cooperatively to the two UL9 binding sites (sites I and H), within oris (Olivo et al., 1988; Elias etal., 1990), exists as a dimer in solution (Bruclnler et al. , 1991), exhibits DNA-helicase activity (Bruckner et al. , 1991; Boehmer et al., 1993) and DNA-dependent nucleoside 5'-triphosphatase activity (Dodson and Lehman, 1993). 36 There are six conserved helicase motifs within the N-terminus of HSV-1 UL9 (Martinez et al., 1992) and a C-terminal domain encodes DNA binding activity (Deb and Deb, 1991). UL9 homologs have been identified in other alphaherpesviruses, such as VZV and equine herpesvirus type 1 (EHV-1) (Chen and Olivo, 1994; Martin and Deb, 1994). Replication origins and binding sites for UL9 homologs of VZV and EHV-1 are highly similar to HSV-1 oris (Stwo and Davison, 1986; Bauman et al., 1989; Chen and Olivo, 1994; Martin and Deb, 1994). Similarly, domain structures of VZV and EHV-1 origin binding proteins are conserved relative to those of HSV-1 UL9 (Chen and Olivo, 1994; Martin and Deb, 1994). thctional MDV replication origins have been identified in serotype 2 MDV defective virus (Camp et al., 1991) and in serotype 3 MDV (Smith et al., 1995). The serotype 2 MDV replication origin is located within inverted repeats flanking the Imique long region (IRL) in intact genomes (Camp at al., 1991). This origin is highly similar to HSV-l oris and oriL and contains a 9-bp sequence (CG'I‘TCGCAC) which is highly conserved in alphaherpesvirus replication origins. This 9-bp sequence is a subset of an ll-bp motif (CGTTCGCACTT) shown to be required for HSV-l UL9 binding (Deb and Deb, 1989; Elias et al., 1990). Similarity of MDV replication origins to those of HSV-1 and VZV, combined with preliminary data indicating MDV encodes a protein capable of sequence- specific interaction with these origin sequences (Camp, 1993), suggested that MDV encodes a fimctional UL9 homologue. In this study, a protein whose apparent molecular size is similar to that of HSV-1 UL9, was identified in extracts of MDV infected cells by western blot analysis with anti-HSV-l 37 UL9 antibody. A putative MDV UL9 gene was subsequently identified through partial sequencing of genome fiagrnents. An open reading frame (ORF) within a transcriptionally active region of the MDV genome, which encodes a protein homologous to HSV-1 UL9 was identified. The MDV UL9 ORF encodes an 841 amino acid polypeptide, which bears 49 % identity to HSV-1 UL9 and 46% identity to the VZV gene 51 product (VZV UL9). MDV UL9 shares several structural motifs with HSV-1 and VZV UL9 proteins. For example, the amino terminal domain of MDV UL9 contains six conserved helicase motifs and a leucine zipper motif, which is important for cooperativity and dirnerization of HSV- 1 UL9 (Elias et al., 1992; Hazudz et al., 1992). In addition, MDV UL9 contains a pseudo-leucine zipper motif and a helix-tum-helix motif in the carboxyl-terminal domain. A similar pseudo-leucine zipper motif, found in the C-terminal portion of HSV-1 UL9, is important for sequence-qrecific DNA binding activity (Deb and Deb, 1991; Arbuckle and Stow, 1993; Martin and Deb, 1994). Material and Methods Cells and Viruses Chicken embryo fibroblast (CEF) cells were prepared, maintained and infected with MDV according to previously described procedures (Glaubiger et al., 1983). Two MDV serotype 1 strains, GA (passage 14), Mdll (passage 16) and Mdll (passage 89) were used for this study. CEF cultures were grown in Leibovitz-McCoy medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 4% calf serum at 37°C ill a humidified atmosphere containing 5% C02. Calf serum concentration was reduced to 1% following MDV infection. Western blot analysis Mock-infected and MDV-infected CEF cell lysates were prepared by sonication (Dabrowski and Schafl‘er, 1991). Lysates were separated on 12% SDS-PAGE minigels (Bio-Rad, Hercules, California) and transferred to nitrocellulose (NC) membrane (Schleicher & Schuell, Keene, New Hampshire). NC membranes were blocked using 5% dry milk Rabbit antiserum against HSV-1 UL9 (generous gift of Dr. M. Challberg, National Institute of Allergy and Infectious Disease, National Institues of Health) was used at 1:200 dilution and donkey anti-rabbit IgG conjugated with horseradish peroxidase was used as secondary antibody. Proteins bound by antibody were visualized using an Amsherm ECL system according to the manufacturer's specification. 38 39 DNA sequencing Five internal EcaRI subclones from the MDV strain GA BamHI C fiagment were generated in pUC 19. DNA sequencing was performed on double-stranded plasmids by dideoxy chain termination using [35 S]ATP (NEN Research Products, Williamston, DE) and sequenase DNA sequencing kits (United States Biochemical Corp., Cleveland, OH). DNA sequences were analyzed using Genepro Software (Riverside Scientific enterprises, Bainbridge Island , Washington) and Wisconsin Sequence Analysis Package (Genetics Computer Group, University Research Park, Madison, WI). Directed oligonucleotide primers and nested Imidirectional deletion clones were used to complete sequencing of the MDV UL9 gene in both directions. EcaRI clone junctions were confirmed by direct sequencing from the BamHI C clone. BamHI G-C jtmction sequences were confirmed by sequencing a PCR clone generated using opposing primers which amplified this region from intact MDV DNA The up stream primer (GGATGGTTACTATGGTGAGAA) was located approximately 50 bp within the BamHI G fragment. The downstream primer (TI'I‘CTAGACCAACGTI‘CAGAGCCGCTGATGC) was located within the C-terminal domain of MDV UL9. PCR reactions were performed in 100-uL using a GeneAmp® PCR System 9600 DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT). Each reaction contained 50mM KC], 10mM Tris-HCl pH 8.3, 1.5mM MgClz, 0.001% gelatin (WN), 5.0 pmole of each primer, 200uM of each deoxyribonuceloside triphosphate (dNTP), 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer) and lug DNA template. Template for PCR reactions was total cellular DNA isolated from CEF infected with MDV GA strain. Cycling conditions were 30 cycles of: 95°C for 1min; 55°C for 1 min; and 72°C for 1 40 min. The resulting PCR product was cloned into the pGEM-T vector (Promega, Madison, WI) prior to DNA sequence analysis using commercially available primers. Total cellular RNA isolation and Northern blot analysis Total cellular RNA was isolated from mock-infected and MDV strain GA or Mdl 1- infected CEF cells using a guanidium-phenol: chloroform method as described by Chomczynski et a1 (1987) (Chomczyski and Sacchi, 1987). The isolated RNA was treated with RQl DNase (Promega, Madison, WI) at 37°C for 30 min in DNase 1 buffer containing 40 mM Tris-HCL (pH 7.9), 10 mM NaCl, 6 mM MgClz and 10 mM CaCl 2 , followed by phenol/chloroform extraction to remove the enzyme. Total RNA (15 pg) was loaded onto 1.2% agarose gels containing 5% formaldehyde and electrophoresed for 12 hr at 30 V. RNA was transferred onto Hybond-N membrane (Amersham Corp., Heights, IL) as described by Sambrook et a1 (1989) (Sambrook etal., 1989). A 0.3Kbp BamHI-BgIH subfragment fiom the MDV strain GA BamHI-G fragment, a 0. 59Kbp EcaRI-A va 1, a 3.1Kbp EcoRI and a 1.3 Kbp EcaRI-BamHI subfiagment fiom the MDV strain GA BamH I-C fragment were labeled using [32P]dCTP (NEN research Products) and used as probes. Northern blot hybridization was performed using standard procedures (Sambrook et al., 1989). Transcript size was determined by comparison to an ethidium bromide stained RNA ladder marker (Life Techonologies, Inc, Gaithersburg, MD). Genebank accession number Nucleotide sequence data presented in this paper has been asigned Genebank Accession Number U28785 Results Identification of a protein similar in size and immunologically related to HSV-1 UL9 in MDV infected cells Based on gene colinearity between MDV and other alphaherpesviruses, as well as origin similarities, it seemed likely that MDV encodes a UL9 homolog which is critical for initiation of DNA replication. Conservation of a potential UL9 binding sites within the MDV core origin of replication and specific recognition of a 22-mer oligonucleotide probe representing these sequences by both MDV and HSV-1 proteins further suggested that MDV UL9 may be highly similar to HSV-1 UL9 proteins (Camp, 1993). To analyze this possibility, we performed Western blot analyses of MDV-infected and uninfected cell extracts using a polyclonal anti-HSV-l UL9 protein antibody (generous gift of Dr. M. Challberg, National Institute of Allergy and Infectious Disease, National Institues of Health). All 85 kd protein was specifically detected in extracts from CEF infected with MDV, strain Mdll (Figure 1). A similar protein was not detected in mocked-infected CEF. The apparent molecular size of this protein is consistent with that reported for HSV-1 UL9 (83 kd) (Bruckner etal., 1991). Identification and nucleotide sequence analysis of an MDV UL9 gene Sequence-specific interaction of an MDV-encoded protein with the serotype 2 origin sequence (Camp, 1993), and antigenic recognition of a protein similar in size to HSV-l UL9 by heterologous polyclonal antisera strongly suggested that MDV encodes a 41 42 Figure 1: Detection of MDV UL9 in MDV-infected cells. Western blot hybridization was performed as described in Material and Methods. Mock-infected CEF cell lysate (CEF) was used as negative control. Position of the putative MDV UL9 protein identified in lysates of CEF cells infected with MDV strain Mdllpl6 (CEF/Mdl lpl6) is indicated by an arrow to the right. Protein sizes were determined by comparison to standards of known size. 43 kDa CEF/Md1 1 p16 LL UJ O 4— 85 kDa 44 UL9 homolog. We therefore proceeded with localization and identification of an MDV gene capable of encoding this polypeptide. Identification of a functional MDV replication origin (Camp at al., 1991) and initial mobility shift assay results (Camp, 1993) were obtained with extracts from cells infected with serotype 2 MDV. However, subsequent Western blot analysis, cloning, and DNA sequence analysis were performed with serotype 1 MDV strains. Serotype 1 WV has been more extensively characterized with regard to gene content and colinearity with HSV-l than serotype 2, thus providing substantially more reference points for genome comparisons. Using relative map imits, it was possible to predict that an MDV UL9 homolog would be arranged within a cluster of replication protein genes near the leftward end of the unique long region, specifically ill BamHI fiagments G and C (Figure 2). Preliminary sequence analysis of the BamHI G fi'agment fi'om serotype 1 MDV (strain GA), indicated that the leftward terminus of BamHI G could encode the N-terminal portion of a MDV UL5 homolog. Using this as an anchor, a major portion of MDV UL9 was predicted to be located within the leftward end of MDV BamHI C. To precisely locate the MDV UL9 gene, five internal EcaRI subclones from BamHI-C were constructed in pUC19 and terminal sequences determined using standard forward and reverse pUC19 primers. Comparison of deduced terminal amino acid sequences with that of HSV-1 UL9 revealed one subclone, with an insert size of 3.1 kb, whose deduced amino acid sequence displayed 57% identity with the N-terminal portion of HSV-1 UL9. Directed oligonucleotide sequencing and EonI nested deletion clone sequencing were used to expand our analysis (Figure 2) fiom the 3.1 Kb EcaRI subclone into an adjacent 45 Figure 2: Location of the MDV UL9 gene within the MDV genome of' the GA strain. (A) BamHI restriction map of the MDV genome Showing approximate location of several major genes, including those encoding thymidine kinase (TK), glycoprotein B (gB), glycoprotein C (gC), and ICP4. Restriction map is adapted from Fukuchi et al. (1984). (B) Predicted localization of the MDV UL9 gene within the BamHI G and C fragments. Locations of several restriction enzyme cleavage sites used ill cloning and sequencing are also shown, as are the bormdaries of MDV BamHI G and C fragments. (C) An EcaRI- AvaI subfiagrnent located at the N-terminal domain of the MDV UL9 gene which was used as a probe for Northern blot hybridization. Drawing is not to scale. 46 are as u... as. are. . u a a e. an e. 2 L 0 Ease he were” DEER” e care. may “l" 5..» m: we: .5: .5 U 47 subclone and the BamHI G fiagment. The EcaRI junction region was sequenced fiom an intact BamHI C clone and the BamHI C-G junction sequence was confirmed by analysis of a 600bp PCR fi'agment (see Materials and Methods). The MDV UL9 gene extends for 2104 bp within the serotype 1 MDV BamHI C fragment and extends 419 bp into the adjacent BamHI G fragment (Figure 2). MDV UL9 is transcribed from right to left with respect to the MDV genome and, as expected, is consistent with direction and location of the HSV UL9 gene. There are several potential translation start codons (ATG) located at nucleotides 3, 17, 21, 33, 52, 354 and 502 (Figure 3). Based on identification of termination codons between nt 3 and at 502, comparison with HSV-1 UL9, and Kozak's consensus sequence for eukaryotic translation (Kozak, 1989), the translation start codon of MDV UL9 has been assigned at at 502 (Figure 3). There are four potential TATA boxes located 62, 239, 286, and 297 bp upstream fiom the putative ATG of MDV UL9 (Figure 3). At present, it is unclear which, if any, of these may function as a site for TBP interaction. Several binding sites for transcription factors could be found upstream from the translation Start codon. One CAAT box and five GC boxes are located 406, 39, 85, 120, 342 and 376 nt, respectively, upstream from the translation start codon. There are no potential poly(A) signals within the known sequence downstream of the MDV UL9 open reading frame (ORF). In HSV- ], the poly(A) signal for UL9 gene transcripts is located 2490 nt downstream fiom the UL9 translation stop codon, creating a bicistronic transcript encoding UL9 and UL8 (Mcgeoch et al., 1988). A similar situation may exist ill IVfl)V UL9 transcription. The ORF predicted for MDV UL9 could potentially encode an 841 amino acid polypeptide 48 Figure 3: Nucleotide sequence of a 3108-bp segment of MDV strain GA DNA from BamHI fragments C and G. Predicted amino acid sequence is indicated Imder the DNA sequence. Four potential TATA boxes are denoted by asterisks beneath the DNA sequence. The CAAT box and five GC boxes are indicated by dotted lines under the DNA sequence. To provide continuity between Figures 3 and 4, conserved structural motifs are indicated by solid bars under the amino acid sequence and conserved regions without homology to known structural or firnctional domains are indicated by open boxes. 49 AAATGCACCACTCCCCATGTATGCGCACCCACATGTTTCTTGCTCGATATAATGCACCAA 60 CAGTAATATAACATACGAGAAACACGAAAAQAATAQTCGTTGCATAGAAGAAAAAAACTA 120 “CAAT box" QQQQQITTGTCTGGAAGAATAATACTGGAACCACGQCCQCCAGCCTGGGATAGGTGAGAC 180 ‘GC box" ‘GC box" CCGTGTGCACTAGTGTCGTATTAACTGTTCTATAATCTACAACGGCTGCGAAGAAGCACG 240 ******* ****** GGAAACCCTCAGTTAAATTTATAGACGCGGCAACAAGTGTTCCAAAGAATACCAGTACGG 300 **** CGATCCCGAAGCACACCGCTTGGACTACCCACATCTTTTTGTGCACGTAGTCTATGTGAC 360 TCAGTCCTCTGTAATTCCGQTCCATTCGTGCTCGACTGGCCATAGCGACGAggggggGTA 420 ‘GC box" “GC box" GTTTTCTGATCAGTGTTATACGATAGACACTCAGCCAGGGCQACTTTCTTCCGTAACGTT 480 **** “GC box" AGTAATCAGAGACAACTCAAAATGATAGACTATGCATCCAGCGCCTCCTTGTCTAGAATG 540 M I D Y A S S A S L S R M 13 TTATATGGAGAGGATCTTATAGACTGGATTATCAAGAACAGACCGGGAATAACAACAGAG 600 Ir Y G E D L I D W I I K N R P G I T T E 33 CGTCAATCCGACGGTCCCGTTACTTTTCCGTCACCTTTGTACCCGAGAACGCGCAATGTC 660 R. Q S D G P V T F P S P L Y P R T R N V' 53 CTTATAGTACGTGCACCTATGGGATCTGGCAAGACTTCTGCTCTCATGAACTGGTTGCAG 720 L I V R A. P M G S G K T S A L M N W L Q 73 motif I TGTATTTTATGCAATTCAAATATGAGCGTTTTGATTGTGTCTTGTAGACGCAGTTTTACC 780 C I L C N S N M S V L I V S C R R S F T 93 AATACATTATCCGAAAAAATTAATAGGGCTGGCATGTCAGGATTTTGTACCTATCTGTCA 840 N 'T L S E K I N R A G M S G F C T Y L S 113 TCCAGCGATTATATTATGCGAGGTAGAGAGTTTTCTAGACTCTTAGTACAAATTGAATCT 900 S S D Y I M R G R E F S R L L g Q I E S 133 Leucine CTACATCGCGTAGATTCGAAACTTCTTGATAATTATGACATCGTTATATTGGATGAAATC 960 I. H B V D S K L L Q N x 2 I y I L D E I 153 Zipper motif ATGTCGACCATCGGCCAGCTTTTCTCTCCAACTATGAAACATTTATGTCAGGTCGATAAT 1020 11 S T141 G Q L F S P T M K H L C Q V D N 173 II ATATTGACATCTCTTCTCAGATATCGTCCGAAGATTGTAGCAATGGACGCTACTATAAAT 1080 I L T S L L R Y R P K I V A M D .A T I N 193 motif ACTCAATTGATAGATATGTTAGCCATCATGAGAGGTGAAGAAAATATACATGTTATCGTA 1140 T‘ Q L I D M L A I M R G E E N I H V I V' 213 ---TTI---- GGTGAATATGCAGCATCCGGGTTTTCTAGAAGGTCATGTACGATCCTCCGTAGTTTGGGA 1200 Gr E Y .A .A S G F S R R S C T I L R S L G .233 ACTAATATCCTTCTTTCGGTGATGAATGAATTCAAACAACTTCCATCTCATACTCAGCCT 1260 T‘ N I L L S V' M N E F K Q L P S H T Q P 253 ATATTTAAACAGAGTACAGGCGTCAACGGTTCTTTGGATATAAGTCTCCATGATCGGACG 1320 I F K Q S T G V N G S L D I S L H D R T‘ 273 TTTTTTTCAGAACTCACTAGACGGCTTGAGGGGGGGTTGAATATTTGTTTGTTTTCCTCA 1380 F F S E L T R R L E G G L N I C L F S S 293 motif 50 ACTATATCATTTTCAGAGATTGTGGCACGTTTCTGCCTCGCATACACGGATTCGGTTTTA T I S F S E I V A R F C L A Y T D S V L —_'_Iv GTGTTAAATTCCACAAGAAATACGCCAATAGATATAAATTCATGGTCTAACTACCGCGTC V' L N S T R N T P I D I N S W S N Y R V GTAATTTATACTACCGTAGTAACCGTTGGTCTTAGTTTTAACGATTCCCATTTTCATAGT V’ I Y T T V V T V G, L__§__£__fl_ D S H F H S motif V ATGTTTGCATACATCAAGCCTACGATAAATGGTCCCGAAATGGTATCGGTTTACCAATCT 14 P A Y I K P T I N g E E .M, V S V Y o S motif TTGGGTCGAATTAGATCACTGCGTCTTAATGAAGTGCTAATTTATATCGATGCATCGGGA Li 9 3 I 3 § L E L N E V L I Y I D A S G] VI GCTGGGTCGGAGCCGGTTTTTACGCCCATGCTACTAAATCACGTAATCGCCAATGGAGGA [A (3 S E P v F T’ P MG’L L ‘N’ H V l A NT G G] Conserved Box II GGATGGCCAACGCGCTTTTCTCAAGTTACCAATATGTTGTGTCACAATTTCAGAAGAGAT [(5 ‘W: P T R F S Q V T’ N M‘ L CJ H N F R R D TGTATACCAACGTTCAGAGCCGCTGATGCATTATACATATTCCCACGATTTAAGTACAAA C I P T F R A A D A L Y I F P R E' K Y K] CATTTATTTGAGCGATGTACACTAAACAATGTAAGTGATAGTATTAATATTCTACATGCT [II L F E’ R C’ T L N"N v S D s r N 1 L ”H ”A1 Conserved box III CTCCTTGAATCAAATTTAATACACGTGCGCTTCGATGGCTGTGATCTCCAATTAAATGCT [I4 L E"S’ N L I’ H”v R F D G C’ D L Q L N Aj GAAGCCTTCTGTGATTTTTTAGTAATTCTTAGAGCAGATTCTATAACTGCCCAACGCGAT LEAFCUFLVILRADSITAQRD] ATGAAAACTCTGCGCAAAAATGCTACCTGCCCTTTACCGGTAGAGGTCGACGTAATTGAT HT K T’ L R] K N A T C P L P V E V D V I D AGTGATGCGGTAGCGTGTTTTGTTCAGAAATATCTAAGACCTACTGTGCTCGCCAATGAT s D .A v .A C F v Q K Y L R p T v L A N D CTCACAGAACTCCTAACAAAATTAGCGGAACCCATTACTAGAGAACAGTTCATAAACATC I. T E L L T x L A E p I T R E Q P I N I ACTATGCTGGAAGCATGTCGTGCAACCCCGGCGGCTCTTTACAGTGAAGCGGTATTTTGT 'T M L E A C R A T [P A .A L Y s E A v F C] CGTATATATGATTATTATGCATCTGGAAATATACCTATAATTGGACCAAGTGGAACCTTA [R I Y D Y Y A s G N I P I I G P s G T L] conserved box I GATACAACGATACTGACATGCGATTTTAATACATCTGGAAGATGGGACTTATACAGGGTA [D 'T T I L] T C D F N T S G R w D L Y R v TGTTGTAAATGGGCCGAATTATTGGGCATCAACCCTTTAGAAGGACCCAATGCTGATATA (I C K W A E L L G I N P L E G P N A D I helix turn 1440 313 1500 333 1560 353 1620 373 1680 393 1740 413 1800 433 1860 453 1920 473 1980 493 2040 513 2100 533 2160 553 2220 573 2280 593 2340 613 2400 633 2460 653 51 GATCCAACAAAACTGTTGCACGTCATGAAAGACGACTATGATATTTATGCTCGTTCTGTG 2520 D P T K L L H V M K D D Y D I Y .A R S V’ 673 helix CTGGAAATTGCGCGATGTTACTTGATTGACGCCCAAACTGCCTTAAAACGCCCTGTGCGA 2580 Ir E I A R C Y L I D A Q T A L K R P V R. 693 Leucine Eipper GCAACAAAATGTGCCTTGAGTGGGATCCAAAATTCTCACCATAGTCAACCATCCACTCAG 2640 A 'r K C A L S G I Q N S H H S Q P S T Q 713 AGCCATGCAGTGTCTTTATTTAAAGTCACATGGGAGATTCTCTTCGGACTCCGCCTCACA 2700 S H .A V S L F K V IT W E I L F G L R L T‘ 733 AAGAGTACAACAACATTTCCGGGTAGAACAAAAGTAAAGAATTTACGGAAGGCGGAGATA 2760 l( S T T T F P G R T K V K N L R K A E I 753 GAAGCTCTGTTAGACGGAGCGGGTATTGATAGAACGTCATGCAAAACTCACAAGGATCTC 2820 IE .A L L D G A. G I D R T S C K T H K D L] 773 noflbrgy TACACCCTCTTGATGAAAAGCAAGTCATTATTTCGCAATATGCGCTATGATATTCGACGC 2880 Y 'T L L M K S K S L F R N M R Y D I R R. 793 [CCGAAGIGGIACGICCTATTAAGATCTCGTTTAGACAAAGAGTTGGGTATATATCATGAT 2940 P K W Y D L L R S R L D K E L G I Y H D 813 CTGGTAGATTTGGAATCTGTGTTGGCGGAAATTCCGTCAGCACTCTGGCCACGCGTAGAA 3000 Ir V D L E S V L A. E I P S A L W P R V E 833 GGTGCTGTAGATTTTCATCGTTTATAATTATTGGAACCGAATGCGTCAAACCATATCAAC 3060 (3 A V D F H R L 841 GATGGCAGCATCGTCAAAAACTAATATCATGCAGATAATGCGAGGATG 3108 52 with a calculated molecular weight of 95 kd, similar to that predicted for the HSV-l UL9 ORF, which encodes 851 amino acids. Predicted amino acid sequence of MDV UL9 demonstrates strict conservation of structural motifs. Overall, the predicted amino acid sequences of HSV-1 and MDV UL9 share 49% identity and 66% similarity while those of VZV and MDV UL9 share 46% identity and 63% similarity (Figure 4). MDV UL9 thus represents the second most highly conserved MDV protein identified to date, following glycoprotein B (gB), which had a mean identity of 50% between MDV, HSV-1 and VZV (Ross et al., 1989). Furthermore, all three UL9 proteins: HSV-1 UL9 (851 amino acids); MDV UL9 (841 amino acids); and VZV UL9 (835 amino acids) are of similar size. Alignment of MDV, HSV-l, and VZV UL9 protein predicted amino acid sequences clearly demonstrates this sequence conservation (Figure 4). Consistent with location of important fimctional motifs, the N-terminal domains of MDV, HSV-l, and VZV UL9 proteins are more highly conserved than the C-terminal domains (Figure 4). Similarity among alphaherpesvirus UL9 proteins fiom divergent sources supports the notion that UL9 plays a central role in alphaherpesvirus DNA replication. Much of the similarity between MDV, HSV-1, and VZV UL9 protein N-terminal sequences occurs within several highly conserved motifs. For example, all putative helicase motifs found within the N-terminal portion of HSV-1 and VZV UL9 proteins are present in MDV UL9. Furthermore, the spatial arrangement in these motifs is conserved between the three origin 53 Figure 4: Aligment of the predicted amino acid sequences of MDV, HSV-1 and VZV UL9 proteins. The top sequence shows the predicted amino acid sequence of MDV UL9 protein (mdvul9), HSV-1 UL9 amino acid sequence is listed ill the second row (hsvul9) and the VZV UL9 sequence is listed in the third row (vzvul9). A consensus sequence derived from examination of the three UL9 proteins is presented in the fourth row (within the shaded box). Upper case letters in the consensus sequence indicate an amino acid is the same in all three proteins while lower case letters represent amino acids which are identical in two of the three proteins. Structural motifs and conserved regions are labeled and indicated by boxes surrounding the appropriate amino acids. 54 1 50 mdvul9 .................... ...MIDYASS ASLSRMLYGE DLIDWIIKNR hsvul9 MPFVGGAESG DPLGAGRPIG DDECEQYTSS VSLARMLYGG DLAEWVPRVH vzvul9 .......... ....MSPNTG ESNAAVYASS TQLARALYGG DLVSWIKHTH A - a. 5‘55... ., a, 5.4.5.}... - ”-55-.‘5- . . - o n u n . 2 :3 I ‘ ‘21:::::§ <. I . .‘u ._.....' r . s s e r 'n . '.'.' ‘ 4.21.39. .... ' ._.'.;.;.....: _ : ;.:.-. ........... ........................................ . n...” ... ......-... - ........................... .......................................... ' .‘. 6‘9. 0. . . .5 . .' '.‘$'. .5 . . as. ' " . . . . . . . q . . . . . .~.'. .-.'.’. . . .'. _._ . . ..........,...~.-.‘.‘.'.'."------ ”Ham-mawawauawaw-maaM -.-.-~.-.v.->M-e~mHW»A-Wmmm~.v¢~a.-a 51 mdvul9 PGITTERQSD GPVTFPSPLY hsvul9 PKTTIERQQH GPVTFPNASA vzvu19 PGISLELQLD VPVKLIKPGM .(motif I) 101 mdvul9 NSNMSVLIVS CRRSFTNTLS EKINRAGMSG FCTYLSSSDY hsvul9 SPDTSVLVVS CRRSFTQTLA TRFAESGLVD FVTYFSSTNY vzvul9 KADISVLVVS CRRSFTQTLI QRFNDAGLSG FVTYLTSETY IM... 151 200 mdvulg lVQIESLHRV DSKLLDNYDI VILDEIMS I GQLFSPTMKH LCQVDNILTS hsvul9 VQVESLHRV GPNLLNNYDV LVLDEVMS I GQLYSPTMQQ LGRVDALMLR vzvu19 VQLESLHRV SSEAIDSYDV LILDEVMS I GQLYSPTMRR LSAVDSLLYR Leucine Zipper motif II 201 250 mdvul9 LLRYRPKIVA MDATINTQLI V LAIMRGEE NIHVIVGEYA ASGFSRRSCT hsvul9 LLRICPRIIA MDATANAQLV LCGLRGEK NVHVVVGEYA MPGFSARRCL vzvul9 LLNRCSQIIA MDATVNSQFI ISGLRGDE NIHTIVCTYA GVGFSGRTCT 3.... ._._. ._._. \ o_. u o v '.'.' '-'~' ' o 5.5... I.I . ”-3.5..- 1...! ._._u n - fig... - -_ -'-'s ............................................................................................................................................................................................... ................................................................................... . . . . . . a. .x- - u - - - - - - - - - s - r - .'--v-.. ................................................... ......................................................................................................................... 251 300 mdvul9 ILRSLGTNIL LSVMNEFKQL PSHTQPIFKQ STGVNGSLDI SLHDR..TFF hsvu19 FLPRLGTELL QAALR ............. PP GPPSGPSPDA SPEARGATFF vzvul9 ILRDMGIDTL VRVIK ............... .RSPEHEDVR TIHQLRGTFF 55 301 350 mdvul9 SELTRRLEGG VICLFSSTI SFSEI ARFC LAYTDSVLVL NSTRNTPI.D hsvul9 GELEARLGGG ICIFSSTV SFAEI ARFC RQFTDRVLLL HSL..TPLGD vzvul9 DELALRLQCG ICIFSSTL SFSE AQFC AIFTDSILIL NSTR..PLCN mdvul9 INSWSNY IYTTVVTVGL SF SHFHSM FAYIKPTI hsvul9 VTTWGQY IYTTVVTVGL SF PLHFDGM FAYVKPMN vzvul9 VNEWKHF L VYTTVVTVGL FHSM FAYIKPMS PEMVSVYQSL PDMVSVYQSL PDMVSVYQSL 401 450 mdvul9 GRIRSLRDNE IIYIDASGA GSEPVFTPML LNHVIANGGG WPTRFSQVT' hsvul9 GRVRTLRKGE IIYMDGSGA RSEPVFTPML LNHVVSSCGQ WPAQFSQVT‘ vzvul9 GRVRLLLLNE YVDGSRT RCGPLFSPML LNFTIANKFQ WFPTHTQIT‘ mdvul9 hsvul9 vzvul9 FKYKHLFERC FRYKHYFERC FKYKHLFERC ...... .;.:..._'. n .. ........ 501 550 mdvul9 ILHALLESNL IHVRFDGCD. .LQLNAEAFC DFLVILRADS ITAQRDMKT hsvul9 LHMLLTLNC IRVRFWGHD. .DTLTPKDFC LFLRGVHFDA LRAQRDLRE vzvul9 ILQTLLASNQ ILVVLDGMGP ITDVSPVQFC AFIHDLRHSA NAVASCMRS ................................................................................ 3.32;.- ' - - - - - - - ' - - - - ' -.- v - . - ' - - O. s. 0.x nu. ma... t. a. I“ . . s.v. .-.-.-.-.‘ ' ~. -.- as»: .\-.\-.\-.-.-.-.|.-.\v. ‘ 3.3.2.3.} .5... ...... ............................. ............................................................................................................................................................................................... ......................................... v.2 . .1. . . ...................................................................................................................................................................................................... ......... mdvul9 hsvul9 vzvul9 VACFVQKYLR PTVLANDLTE LLTKLAEPIT RDPEASLP AQAA..ETEE VGLFVEKYLR SDVAPAEIVA.pMRNLNSLMq ODNDSCLTD FGPSGFMADN ITAFMEKYLM ESINTEEQIK VFKALACPIE Leucine . - xv . ......... ~ ...... 3 45“.. ............... ............... ................... ...... ............... ............. ................ .................... .............................................................................................................. 56 601 650 mdvul9 REQFINITML EACRA.PAAL YSEAVFCRIY DYYASGNIPI IGPSGTLDT hsvul9 ETRFIYLALL EAFLR 'MAT RSSAIFRRIY DHYATGVIPT INVTGELEL vzvul9 QPRLVNTAIL GACIRIPEAL EAFDVFQKIY THYASGWFPV LDKTGEFSI Zipper conserved 651 Helix Turn Helix 700 mdvul9 I CDFNTSG RWDLYRVCqFWAELLGINPL EGPNA. DIDP‘fiRLLHVMKQD hsvul9 vzvul9 701 Leucine Zipper mdvul9 YDIYARSVFIE IARCYLIDAQ TALKRPVRATT‘ KCALSGIQNS H.HSQPSTQS hsvul9 YDRYMQLVE‘E‘. LGHCNVquL LLSEEAVKRV ADrFsEsGC. .P P.RGSM§ETq vzvul9 qIELAQLIEE VMRFNVTDAK IILNRRVWRT TG DGCHNQ qEREIPTKHE v: Helix h, v- Turn mdvul9 HAVSLF TW EILFGLRLTK STTTFPGRTK VKNLRKAEIE ALLDGAGIDV hsvul9 VALF IW GELFGVQMAK STQTFPGAGR VKNLTKQTIV GLLDAHHID vzvul9 YNEALFRLIW EQLqGARVTK STQTFPGSTR VKNLKKKDLE TLLDSINVD Varicella Zoster Virus 801 850 mdvul9 SCKTHKDLY TLLMKSKSLF RNMRYDIRRP KWYD LRSRL DKELGIYHDL hsvul9 ACRTHRQLY ALLMAHKREF AGARFKLRVP AWGRCLRTHS SSANP..NAD vzvul9 ACRTYRQLY NLLMSQRHSF SQQRYKITAP AWARHVYFQA HQMHLAPHAE Homology 851 877 mdvul9 VDLESVLAEI PSALWPRVEG AVDFHRL hsvul9 IILEAALSEL PTEAWPMMQG AVNFSTL vzvul9 AMLQLALSEL SPGSWPRING AVNFESL 57 binding proteins (Figures 4 and 5). Average amino acid identity within helicase motifs in MDV, HSV-l, and VZV UL9 proteins is 73%. Most striking among these conserved domains is an ATP-binding motif (motif 1), which is virtually identical in these three origin binding proteins (Figure 4). HSV-l UL9 possesses an intrinsic helicase activity (Boehmer, 1993). Single amino-acid substitutions at conserved residues in five helicase motifs inactivated the firnction of HSV-1 UL9 in transient origin-dependent replication assays (Martinez et al., 1992). Conservation of these motifs in MDV UL9 suggests that, like HSV-1 UL9, MDV UL9 possesses an intrinsic helicase activity within the N-terminal portion. A leucine zipper region, also within the N-terminal domain of MDV UL9 (overlapping conserved helicase motif H), has 67% and 59% homology with corresponding sequences in HSV-1 and VZV UL9, respectively (Figure 4). Insertion mutagenesis of the HSV-1 amino-terminal leucine zipper domain dramatically affects cooperativity and dimerization of HSV-1 UL9 in solution (Hazuda et al., 1992). As with the helicase motifs, spatial arrangement of the leucine zipper motifs is conserved between MDV, HSV-1, and VZV UL9 proteins (Figures 4 and 5). In addition to conservation of known structural/fimctional motifs, there are two highly conserved regions in MDV, HSV- l, and VZV UL9 (conserved boxes H and IH) which do not show clear homology to previously identified fimctional domains (Figure 4). The C-termirral domain of MDV UL9 contains two structural motifs in common with the C-terminal domain of HSV-1 UL9, a pseudo-leucine zipper (not clearly defined ill VZV UL9) and a helix-tum-helix motif (Figure 4). By insertion and deletion mutagenesis, 58 Figure 5: Schematic summary comparing conservation and spacial arrangement of structural motifs in HSV-1, MDV and VZV UL9 proteins. Solid black boxes indicate conserved helicase motifs. A large hatched box highlights the VZV homology domain. Positions of conserved leucine zippers and helix-tum-helix domains are indicated by text and highlighted by solid bars. Small hatched boxes represent conserved regions (boxes I, H, and IH). DNA-binding domains for HSV-l and VZV UL9 are also indicated. cases snacking 730652 >N> W“ m E H Hi! — _ 33>N> E a mummw. P 9 5 59:0“. oc_uc_mr<20 fl 30.080; >N> _ I\\\\\\\\\\\\\\\\ W m m— — — i W Pi 33-5: x__e.._ _ seen a a team. F3... 28:3 3.03%. can: Season >N> I\\\\\\\\\\\\\\\ _ E — _ H nine. 3% ..oaqul an H E : admin w scone... kw...» 650:3 60 the pseudo—leucine zipper was shown to be important for DNA-binding activity of HSV-1 UL9 (Deb and Deb, 1991; Arbuckel and Stow, 1993). However, importance of the helix- tum-helix motif for the DNA-binding activity remains to be elucidated (Deb and Deb, 1991; Arbuckel and Stow, 1993 ). In addition to these two recognizable structural motifs, there are two, less obvious regions, which may be important for UL9 DNA-binding activity. From comparison of MDV, HSV- l, and VZV UL9 proteins, a region from amino acids 583 to 618 (conserved box I) was found to be highly conserved between all three proteins (Figures 4 and 5). Another highly conserved region is found within the very C-terminal end of MDV UL9. This sequence, termed the VZV homology region (Martin et al., 1994), is highly similar to corresponding sequences in HSV-1 and VZV UL9 proteins (Figure 4). Despite a lack of Similarity to any known DNA binding motif, the VZV homology region is critical in maintaining the DNA-binding activity of HSV-1 UL9 (Deb and Deb, 1991; Arbuckel and Stow, 1993). Detection of MDV UL9 transcripts Northern blot hybridization was performed to detect transcripts arising from the MDV UL9 gene region. An EcoRI-Aval DNA fragment (0.59 kb) (Figure 2), which maps within the 5'-end of the MDV UL9 gene, was used as probe for detection of transcripts. The EcoRI-Aval probe hybridized to two transcripts of 4.4 and 2.1 kb in RNA derived from CEF cells infected with MDV strain GA (Figure 6A, lane 2). No transcripts were detected by the AvaI-EcoRI probe in mock-infected CEF cells (Figure 6A, lane 1). An EcoRI subfragment (3.1 kb) clone, originally used to identify the MDV UL9 gene within 61 Figure 6: Northern blot analysis of MDV UL9 gene transcription. Total RNA was isolated from uninfected CEF cells and cells infected with MDV strain GApl4 as described in Materials and Methods. Panel A: RNA was hybridized with a 0.59 Kbp EcoRI-A vaI probe from MDV BamHI fragment C. Panel B: RNA was hybridized with a 3.1 Kbp EcoRI clone from MDV BamHI fragment C. In both panels, Lane 1 contains RNA isolated from mocked-infected CEF while Lane 2 contains RNA from CEF infected with MDV strain GA. The large diffuse band of hybridization below 1.0 kb in MDV- infected cell lanes of both panels likely represents viral DNA not completely removed by RQl DNase treatment (see Materials and Methods). 62 3 § Lu 0 CEF/GAp14 ‘ 4'4”” <— 4.4kb ‘— 2.6kb ' 2'1 kb ‘— 2.1 kb 63 the BamHI C fragment, was also used as probe for detection of transcripts. The 3.1 Kb EcoRI probe hybridized to three transcripts of 4.4, 2.6 and 2.1 kb (Figure 6B, lane 2). In HSV-l, the UL9 coding region is 2.5 kb while the predominant transcript of the HSV-1 UL9 gene is about 5.2 kb, being 3'-coterminal with UL8 gene transcripts (Baradaran etal., 1994). HSV-l UL10 is transcribed from a gene immediately downstream of HSV-1 UL9 oriented in the opposite direction, relative to the HSV-l genome. The transcription start Site for HSV-l UL10 is within the HSV-1 UL9 coding region and the coding region of HSV-1 UL10 is 1.4 kb (Mcgeoch et al., 1988; Baradaran et al., 1994). Given the Similarity of MDV and HSV-l gene organization, it is likely that a Similar organization of replication protein genes exists in MDV. Based on analogy to HSV-l and our Northern blot analysis, it is reasonably possible that the 4.4-kb transcript represents the primary MDV UL9 gene transcription product, possibly co-terminal with an MDV UL8 gene transcript, while the 2.1-kb transcript represents the MDV UL10 gene. To confirm the presumption that the 4.4-kb transcript represents the primary MDV UL9 gene transcription product, while the 2.1-kb transcript likely represents the MDV UL10 gene, an BamHI-BgIII subfragment from BamHI G fragment (Figure 2) was used as probe for northern blot hybridization. This probe contains sequence from the 3 ’-end of the MDV UL9 gene and would not be expected to hybridize with MDV UL10 gene sequence. The BamHI-Bglll subfragment hybridized to two transcripts (4.4 kb and 2.0 kb) (Figure 7A, lane 2). An EcoRI-BamHI subfragment from BamHI C fragment (Figure 2), representing central portions of the MDV UL9 gene, was also used as probe for detection of transcripts. The EcoRI-BamHI fragment hybridized to only one transcript (4.4kb) (Figure 64 Figure 7: Northern blot analysis of MDV UL9 transcription. Total RNA was isolated from uninfected CEF cells and cells infected with MDV strain Md11p89 as described in Materials and Methods. Panel A: RNA was hybridized with a 0.3 Kbp BamHI-Baglll probe fi'om MDV BamHI G fragment. Panel B: RNA was hybridized with a 1.3 Kbp EcoRI-BamHI probe from MDV BamHI fragment C near the jimction between BamHI C and G fragments. In both panels, Lane 1 contains RNA isolated from mocked-infected CEF while Lane 2 contains RNA from CEF infected with MDV strain Mdl 1. 65 8a r 55:qu “mo awn F 55:qu “mo fl 4— 4.4 kb +— 2.0kb 66 7B, lane 2). These two results suggest that 4.4-kb transcript represents the primary MDV UL9 gene transcription product and it is reasonably possible that the 2.1 kb band represents MDV UL10. Discussion We have identified and sequenced the MDV serotype 1 UL9 gene, which is located within BamHI—G and BamHI-C fragments of MDV strain GA DNA. Predicted amino acid sequences of MDV UL9 is highly Similar to UL9 sequences fiom other alpha- herpesviruses, particularly HSV-1 and VZV. A summary of structural motifs and conserved regions within MDV, HSV-l and VZV UL9 proteins is presented in Figure 5. The N-terminal domain of MDV UL9 contains six conserved helicase motifs and a putative leucine zipper sequence which, by analogy to HSV-1 UL9, may be important for activity and dimerization, respectively, of MDV UL9. Spatial arrangement of both the helicase motifs and leucine zipper within MDV UL9 are Similar to that within origin- binding proteins of HSV-1 and VZV (Martinez et al., 1992; Chen and Olivo, 1994; Martin et al., 1994) (Figure 5). Using a model DNA substrate, Boehmer et a1. (1993) has Shown that HSV-1 UL9 possesses intrinsic helicase activity, consistent with a hypothesis that UL9 protein serves as an initiator of viral DNA replication. Presumably, UL9 unwinds origin regions, allowing entry of DNA replication machinery. Conservation of helicase motifs and a putative leucine zipper iii MDV UL9 suggests that the N-terminal domain of MDV UL9 may encode a helicase activity and be necessary for dirnerization and cooperativity of MDV UL9 monomers. Thus, MDV UL9 may serve as an initiator for MDV viral DNA replication. In addition to conservation of helicase and leucine zipper elements within the UL9 amino terminal one-half; two regions of high homology (conserved boxes I] and III), occur in UL9 proteins from MDV, HSV-1, and VZV. 67 68 Sequences within conserved boxes [I and 111 do not correspond to any recognizable structural motifs and, to date, have no assigned or obvious function. Sequence-specific DNA binding activity of HSV-1 UL9 is localized to amino acids 564 to 832 (Deb and Deb, 1991) while that of VZV UL9 is localized to amino acids 551 to 835 (Chen and Olivo, 1994). Although the C-terminal domain of MDV UL9 exhibits lower similarity than other regions, when compared with HSV-l and VZV UL9, it contains several elements which are important for DNA-binding activity of HSV-1 UL9 (Arbuckle and Stow, 1993; Martin et al., 1994), a pseudo-leucine zipper sequence, a helix-tum-helix motif, and a VZV homology region (Martin et al., 1994). In contrast to the amino-terminal helicase and leucine zipper motifs, Spatial arrangement of C-terminal pseudo-leucine zipper and helix-turn-helix domains within MDV UL9 is difl‘erent from that within HSV-l and VZV UL9 (Figure 5). At present, consequences of altered spatial arrangement for C-terminal elements are unknown. However, presence of essential motifs for DNA-binding activity suggests that the C-terminal portion of MDV UL9 may function in this capacity. In addition to elements mentioned above, a region from amino acids 583 to 618 within the C-terminal portion of MDV UL9 is highly conserved among UL9 proteins from diverse alphaherpesviruses. This conserved region does not resemble any known structural motifs, however, It may be important for DNA-binding activity (Martin et al., 1994). Overall, MDV UL9 appears to retain the same domain structure as UL9 proteins from HSV-1 and VZV, that is the N-terminal portion encodes dirnerization Signals, helicase activity and an ATP binding site, while the C-terminal portion encodes 69 DNA-binding activity. Experiments designed to test this hypothesis using in vitro translated MDV UL9 protein are currently in progress. In HSV- l, UL9 plays a central role in initiation of viral replication. UL9 protein binding to an origin sequence is the first committed step for initiation of replication. Following UL9 binding, other replication proteins bind to the origin and form a fimctional replication complex. Reflecting this central role, MDV UL9 is the second most highly conserved MDV protein, relative to HSV-l and VZV, behind the essential glycoprotein B (Ross et al., 1989). One MDV replication origin, which is highly similar to origins of other alphaherpesviruses has been identified (Camp etal., 1991). This origin contains two sequences which are highly Similar to HSV-l UL9 recognition sites. Each of these putative UL9 recognition sites contains one 9 bp sequence (CG'I'I‘CGCAC) which is a subset of an ll-bp motif (CGT‘TCGCACT'I‘) shown to be recognized by the HSV-1 origin binding protein. A viral specific band was detected in electrophoretic mobility shift (EMS) assays with MDV-infected cell extracts using an oligonucleotide containing a potential MDV UL9 binding site (Camp, 1993). Identity of MDV encoded proteins which bind to this site have yet to be determined. However, given the conservation of UL9 binding Sites in HSV-l and VZV and the high degree of Similarity between HSV-1, MDV and VZV UL9, it is likely that each of the 9-bp repeats serves as a site of MDV UL9 recognition. EMSA assays with in vitro translated MDV UL9 will address this issue. In other alphaherpesviruses, replication protein genes are clustered within the unique long region. Transcription of these genes often leads to bicistronic or polycistronic transcripts (Mcgeoch et al., 1988). Based on Northern blot hybridization, the MDV UL9 70 gene appears to be transcribed in a Similar manner. Two transcripts (4.4 kb and 2.1 kb) were detected in RNA of cells infected with MDV, using a probe fiom within the UL9 coding region. A third transcript (2.6 kb) was observed when a large (3.1 Kb) probe containing sequences immediately downstream of MDV UL9 was used The 4.4 kb transcript was also observed in RNA of cells infected with MDV when two probes, one from the 3 ’-end of MDV UL9 gene and another centrally located along the WV UL9 sequence, were used. Based on gene colinearity between MDV and HSV-, it is a reasonable possibility that the 2.1-kb transcript, whose 5'-end overlaps the N-terminal domain of MDV UL9 coding region, is transcribed from the opposite direction and represents the MDV UL10 gene transcript. The 4.4-kb transcript likely represents the predominant MDV UL9 encoding message. In support of these predictions, open reading fi'ame analysis of known DNA sequence revealed that predicted amino acid sequences of one ORF, located upstream from the MDV UL9 ORF and oriented in a direction opposite the UL9 gene, is homologous to N-terminal sequences of HSV-1 UL10 (47% similarity). Thus, gene organization of at least UL9 and UL10 within the MDV genome appears similar to that within the HSV-1 genome. Lack of a consensus polyadenylation Signal downstream of MDV UL9 supports the suggestion that the 4.4 kb UL9 transcript either contains a long 3' cotranslated region or is bicistronic. Acknowledgements We thank Jean Wilms-Robertson, Carolyn Cook, and Ronald Southwick for excellent technical assistance. This work was supported, in part by the Research Excellence Fund, State of Michigan, by the Michigan Agricultural Experiment Station, and grants #95- 02063 and 94-37204-0935 awarded to P.M.C. through the National Research Initiative, Competitive Research Grants Program, USDA Chapter 3 Marek’s disease virus (MDV) DNA replication : a MDV gene homologous to herpes simplex virus type 1 (HSV-1) UL9 gene encodes an origin binding protein (OBP) Ting-Feng Wu, and Paul M. Coussens“ 72 Abstract In our previous studies, we identified an MDV UL9 homolog gene which lies between the BamHI C and BamHI G fragments of the serotype 1 Nfl)V strain GA genome. Computer analysis of the predicted amino acid sequences of MDV and HSV-l UL9 proteins revealed that MDV UL9 is highly similar to HSV-1 UL9 and shares numerous motifs with HSV-1 UL9. It is suggested that MDV UL9 may have the similar biochemical activities to HSV-l UL9 protein, specifically the origin-binding activity. In this study, a MDV UL9 protein of 95 kd was detected in nuclear extracts prepared from chick embryo fibroblast (CEF) cells infected with serotype 1 MDV by western blot analysis with antiserum against the MDV UL9 protein. PCR was used to clone the MDV UL9 gene. In vitro transcription-translation of this gene generated a protein of 95 kd as measured by sodium dodecyl sulfate-polyacrylamide analysis. Further characterization of this protein was accomplished via the use of Electrophoretic mobility shill assays (EMSA). EMSAS with in-vitro expressed MDV UL9 protein or MDV-infected CEF nuclear extracts showed that the MDV UL9 protein could bind to the HSV-1 UL9 binding site I and one of two potential OBP binding Sites within the serotype 2 MDV origin. A mutant MDV UL9 site 11 DNA containing a point imitation which render the MDV UL9 binding Site II to become MDV UL9 binding site I was used in competitive EMSAS to showed that the last nucleotide (T) within MDV UL9 binding site II was essential for the binding of MDV UL9 protein to MDV UL9 binding Site II. A series of oligonucleotides containing substitutions within the flanking sequence around the MDV UL9 binding site I was used in competitive 73 74 EMSAS to showed that the last two nucleotides (TT) within HSV-l UL9 binding site I were essential for the binding of MDV UL9 protein in vitro. Introduction Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus. In chickens, MDV is the etiologic agent of Marek's disease (MD), characterized by malignant T-cell lymphomas(Calnek et al., 1991). Originally MDV was classified as a gammaherpesvirus based on cell tropism and pathology (Roizman, 1990). However, based on overall genomic structure and colinearity of many MDV genes to those of alphaherpesviruses, such as herpes Simplex virus type 1 (HSV-l) and varicella-zoster virus (VZW, MDV has been reclassified as an alphaherpesvirus (Bnmovskis and Velicer, 1992; Buckmaster et al., 1988; Cebrian et al., 1982; Roizman, 1992). Consistent with other alphaherpesviruses, MDV genomes are linear double-stranded DNA molecules 160 to 180 Kbp in length, and consist of two imique regions (Us and UL) flanked by inverted repeats (IRS, IRL, TRS, and TRL). Replication of viral genomes depend on cis-acting elements which fiinction as origins of viral DNA replication, and trans-acting proteins. The replication origin of HSV-1 have been functionally identified by using assays based on amplification of plasmid DNA molecules containing suspected origins in transfected tissue culture cells expressing viral replication proteins (Weller et al, 1985; Stow et al, 1983). Two HSV-1 origins have been identified, one (oriL) lies close to the center of unique long (UL ) region (Weller et al, 1985), while two identical copies of the second (Otis) are located within the inverted repeats flanking the unique short (Us) region (Stow et al, 1983). Both origins feature palindromic DNA sequences with central A+T-rich regions and Share considerable sequence homology (Weller et al. , 1985). Functional MDV origins of replication have 75 76 been identified in serotype 2 MDV defective virus (Camp et al., 1991) and in serotype 3 MDV (Smith et al., 1995). The serotype 2 MDV origin of replication is located within the inverted repeats flanking the imique long region (UL) (Camp et al., 1991). The serotype 2 MDV origin of replication has a structure Similar to the replication origins of HSV- l, VZV and EHV-1. The HVT replication origin is located in the junction between UL and IR, and is highly Similar to the origins of replication of MDV-2, HSV-l, VZV and EHV-l HSV-1 encodes seven proteins that are both necessary and sufficient for origin- dependent DNA synthesis (Wu et al., 1988). HSV-l UL9, one of seven essential HSV-1 genes, encodes the origin binding protein (OBP) (Olivo et al , 1988). OBP has been Shown in several studies to be essential for viral DNA replication and to bind specifically to sequences within HSV-1 orig (Elias et al, 1990; Weir et al, 1990; Deb et al, 1989; Weir et al, 1989; Elias et al, 1988; Kofl‘ et al, 1988; Olivo et a1 , 1988; Elias et al, 1986). HSV-1 orig contains three OBP-binding sites, Site I and II are located within the right and left arms of palindromic sequences flanking the central A+T core and site III is located to the left ofsite I (Elias et a1 1990; Weir et al, 1990; Weir et al, 1989; Elias et al, 1988; Deb et al, 1988; Lockshon et al, 1988). All three OBP-binding Sites are highly homologous however, Site Ihas 5 to 10-fold higher infinity for OBP than site II (Elias et al, 1990; Weir et al, 1990; Elias et al, 1988). All three OBP-binding sites have been shown to be essential for HSV-l viral replication in viva (Hernandez et a1, 1991; Weir et al, 1990). Using DNAase I footprinting, methylation interference, and filter binding assay with mutant oligonucleotides for Site I, the HSV-1 OBP binding site was mapped to within a domain of 11 nucleotides of Site I (CGT'I‘CGCACTT) (Hazuda et al, 1991; Elias et al, 1990; Deb 77 and Deb, 1989;1(ofl‘et al, 1988). The ll-base-pair (bp) element within Site II is different in two positions fi'om that within site I, while the motif within Site 111 difl‘ers only in one position (Elias et al, 1990). The presumed HSV-l OBP binding site (ll-bp element) is conserved in both mi and orig of HSV-1 and HSV-2 as well as in a VZV origin of replication. It was proposed that the HSV-1 OBP binds as a dimer to two inverted, overlapping pentanucleotides within site I (5’-GTTCGCAC-3’/3’-CAAGCGTG-5’) (Kofl‘ et al, 1988). Two distinct regions which are highly homologous to the conserved ll-bp element are identified within the serotype 2 MDV origin. These two regions are located within the left and right arms of a serotype 2 MDV origin palindrome. Each region contains a 9-bp sequence (CGT'I‘CGCAC) which is highly conserved in the oriL and orig of HSV-1 and HSV-2 as well as the origins of VZV and EHV-1 (Camp et al, 1991). This 9-bp element is a subset of the conserved ll-bp element. Thus, there are two potential OBP binding Sites within MDV serotype 2 origin. The HVT replication origin also contains a 9-bp motif which is conserved among the alphaherpesviruses. In our previous studies, we identified an MDV UL9 homolog gene which lies between the BamHI C and BamHI G fragments of the serotype 1 MDV strain GA genome. The predicted amino acid sequences of the HSV-l and MDV UL9 proteins Share 49% identity and 66% Similarity while those of VZV and MDV UL9 Share 46% identity and 63% similarity. Computer analysis of the predicted amino acid sequences of MDV and HSV-1 UL9 proteins revealed that MDV UL9 shares numerous motifs with HSV-1 UL9 and VZV gene 51 ( a HSV-1 UL9 homolog). Conserved motifs include several helicase 78 moieties and a leucine zipper in the N-terminus, a C-terminal pseudo-leucine zipper, and a putative helix-tum-helix structure. In this study, we detected MDV UL9 protein in nuclear extracts prepared fiom chick embryo fibroblast (CEF) cells infected with serotype 1 MDV strains GA and Mdll by western blot analysis with antiserum against the MDV UL9 protein. Electrophoretic mobility Shifl assays (EMSA) with in-vitro expressed MDV UL9 protein showed that the MDV UL9 protein could bind to the oligonucleotide for the HSV-l UL9 binding site I and also bind to one of two potential OBP binding sites within the serotype 2 MDV origin. EMSAS with MDV-infected CEF nuclear extracts showed that the MDV UL9 protein present in the infected CEF nuclear extract could bind to the HSV-1 UL9 binding site I and one of two potential OBP binding Sites within the serotype 2 MDV origin. Materials and Methods Cells and Viruses Chicken embryo fibroblast (CEF) cells were prepared, maintained and infected with MDV as previously described (Glaubiger et al., 1983). Two serotype 1 MDV strains, GA (passage 14) and Mdll (passage 89) were used for this study. CEF cultures were grown in Leibovitz-McCoy medium (Life Technologies, Inc., Gaithersburg, NED) supplemented with 4% calf serum at 37°C in a humidified atmosphere containing 5% C02. Calf serum concentration was reduced to 1% following MDV infection. Preparation of nuclear extracts CEF cells were infected with serotype 1 MDV strains GA and Mdll. Cells infected with MDV strain GA or Mdll were harvested 48 hr or 24 hr later, respectively. The harvested cells were lysed in bufl‘er A (15mM KCI, 10mM HEPES pH 7.6, 2mM MgClz, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40) for 10 minutes on ice. Nuclei were pelleted by centrifugation at 500 xg at 4°C for 10 min. Pelleted nuclei were lysed in buffer B (1 M KCl, 25mM HEPES pH 7.6, 0.1 mM EDTA, 1 mM DTT), at 4°C for 15 minutes with frequent vortexing, and then centrifuged at 14000 rpm for 20 minutes. The supematants were collected and dihited with buffer C (20% glycerol, 25 mM HEPES pH 7.6, 0.1 mM EDTA, 1 mM DTT) at 1:3.75 ratio. Western blot analysis Mock-infected and MDV-infected CEF cell nuclear extracts were separated on 10% SDS-PAGE minigels (Bio-Rad, Hercules, California) and transferred to nitrocellulose 79 80 (NC) membrane (Schleicher & Schuell, Keene, New Hampshire). NC membranes were blocked using 5% dry milk Rabbit antiserum against MDV UL9 protein was used at 1:100 dilution and donkey anti-rabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody. Proteins boimd by antibody were visualized using an Amsherm ECL system according to the manufacturer's specification. Expression of GST fusion protein The pGEX-Sx-3 vector system (Pharmica Biotech, Uppsala, Sweden), which encodes a 27 kDa bacteria GST ORF under the control of an inducible Lac promoter, was used to express the GST-MDV UL9 fusion protein. A BamHI-Kpnl fi'agment from the 3’ end of MDV UL9, which encodes amino acids 702 to 841 of MDV UL9 and has a significantly hydrophilic characteristic, was treated with T4 polymerase in the presence of dGTP to remove the 3’-protruding end of the KpnI Site. The digested fragments were cloned between the BamHI and EcoRI Sites of pGEX-Sx-3, had the EcoRI site blimt-ended with klenow enzyme. DH50c cells containing the GST-MDV UL9 ORF fusion protein plasmid were cultured at 37°C until the OD.“ reached between 1.0 and 2.0. Then Isoprople-D- thiogalactoptranoside (IPTG) was added to a final concentration of 0.3 mM at 37°C for 3 hrs to induce fusion protein expression and then centrifuged. Pelleted cells were suspended in 25ml ice-cold PBS and GST fusion protein was purified by glutathione beads following the manufacturer’s recommendation (Parmacia). Purified GST fusion protein was analyzed on a 12% SDS-polyacrylamide gel (PAGE). Purified GST-MDV UL9 fusion protein was 81 mixed with adjuvant (Titer-max; Cthx Co., Norcross) and used to minimize New Zealand rabbits subcutaneously. Cloning and expression of MDV UL9 MDV UL9 gene was amplified from total cellular DNA isolated from MDV-GA- infected CEF cells by polymerase chain reaction (PCR) reaction. The upstream PCR primer (5’-GGGGTACCCCGGT'I'I‘GACGCAT'I‘CGGT'I‘CC-3’) was located 27 bp upstream of the ATG codon of MDV UL9 gene and the downstream primer (5’- GGGGTACCCCAGGGCGACTTI‘CTTCCGT-3’) was located 5 bp downstream of the termination codon. Primers were designed to contain Kpnl Sites at the 5’-ends (Fig. 2). PCR reactions were performed in 100-uL volume using a GeneAmp® PCR System 9600 DNA Thermal Cycler(Perkin-E1mer, Norwalk, CT). Each reaction contained 50mM KCI, 10mM Tris-H01 pH 8.3, 1.5mM MgClz, 0.001% gelatin (WN), 5.0 pmole of each primer, 200uM of each deoxyribonuceloside triphosphate (dNTP), 2.5 U AmpliTaq DNA polymerase (Perkin-Elmer) and lug total celhilar DNA isolated from CEF infected with MDV GA strain. Cycling conditions were 20 cycles of: 95°C for 1 min; 65°C for 1 min; and 72°C for 1 min. PCR fragments were purified by low-melting-point agarose gel electrophoresis and digested with KpnI. Digested PCR fragment were cloned into the KpnI site of the pBKCMV vector (Stratagene, Ja Jolla, CA. ). The plasmid was designated pBKMDVUL9. 82 MDV UL9 protein was expressed in coupled transcription-translation reactions (TNT reticulocyte lysate; Promega, Inc., Madison, Wis.) fi'om plasmid pMDVUL9. An aliquot of 35 S-labeled, in-vitro translation products were mixed with an equal amormt of loading buffer containing 6M urea, 100mM Tris pH6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.1% bromophenol blue and 1.8 M 2-mercaptoethanol and separated on an SDS- polyacrylamide gel electrophoresis (PAGE). Gels were treated with an intensifying agent (Amplify; Amersham Life Science, Arlington Height, IL), dried and exposed to autoradiography film (XAR-S; Kodak, Mass). Immunoprecipitation An aliquot of transcription-translation lysate prepared from pBKMDVUL9 was incubated with preimnnme or antiserum against MDV UL9 on ice for 1 hr. A aliquot of transcription-translation lysate prepared without plasmid DNA was also incubated with antiserum against MDV UL9. The imrmmocomplexes were collected by 10% protein A- agarose bead suspension. Alter formation of immunocomplexes, the protein A-agarose bead was added to the reticulocyte lysate. The suspension was incubated at 4°C for 1 hr with rocking and then centrifuged. The pellet was washed with lysis bufl‘er (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.5 mM PMSF) three times, resuspended in protein sample loading buffer containing, 6M urea, 100mM Tris pH6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.1% bromophenol blue and 1.8 M 2- mercaptoethanol, boiled for 5 min and loaded onto a 10% SDS-PAGE. 83 Electrophoretic mobility shift assay (EMSA) CEF nuclear extract (10 pg) or MDV strain GA or Mdl l-infected CEF nuclear extracts (10 pg) were incubated with 32P-labeled oligonucleotides (1-2 ng) and 1 pg poly(dI-dC) at room temperature for 30 minutes in 20 pl of reaction buffer. Oligonucleotides were labeled with T4 polynucleotide kinase in presence of [32P]y-ATP. In experiments involving in vitro transcription-translation products, 32P-labeled oligonucleotides were incubated with an aliquot of transcription-translation lysate prepared without or with plasmid DNA The reaction buffer consisted of 12mM N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)—NaOH (pH7 .6), 4mM Tris- HCl (pH7.6), 100mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 60 pg of bovine serum albumin per ml, 12% glycerol, 5 pg of salmon sperm DNA per ml and a cocktail of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 0.24 trypsin inhibitor units of aprotinin per ml). DNA-protein complexes were separated on 5% native polyacrylamide gels in 0.5x TBE at 4°C. In experiments involving antibody, the proteins were incubated with antibody for 30 minutes followed by incubation with the labeled probe for an additional 30 minutes. The competition experiments were performed as described above, with premixing of specific probe and competitor DNAS prior to the addition of proteins in DNA-binding buffer. The dried gels were dried for quantitation on a phosphor-imager (Molecular Dynamics, Sunnyvale, CA). Results Detection of MDV UL9 gene product. The GST-MDVUL9 fission protein was purified as described in Materials and Methods and as expected, a 43 kDa fusion protein was detected (data not Shown). Antiserum was produced by immunization of New Zealand white rabbits with purified GST-MDV UL9 fusion protein. To determine if MDV UL9 protein was expressed in the lytically infected cells, western blot analysis. was performed using MDV-infected nuclear extracts and GST-BK antiserum as a primary antibody. A 95 kDa protein was detected in both MDV-GA- and Mdl l-infected CEF cells but not in CEF cells. The Size of this polypeptide is consistent with the predicted Size of MDV UL9 protein (95 kDa) (Fig. 1). In Figure l of Chapter 2, three virus-specific proteins could be detected. Compared to the results of western blot analysis with anti-MDV UL9 antibody, the protein with an apparent molecular Size, 95 kDa, Should be the MDV UL9 protein while the other two proteins were likely the degradation products. Cloning and expression of MDV UL9 gene. In order to examine binding activity of MDV UL9 independent of other viral proteins, the MDV UL9 gene was cloned into a pBKCMV expression vector. Based on our previously determined DNA sequence of the MDV UL9 gene, PCR primers were designed with KpnI restriction sites engineered at their 5’-ends. The MDV UL9 gene was amplified by PCR using total cellular DNA isolated from MDV-GA-infected CEF cells (Fig. 2). A 84 85 Figure 1. Detection of MDV UL9 in MDV-infected cells. Western blot hybridization was performed as described in Material and Methods. Mock-infected CEF nuclear extract (CEF) was used as negative control Position of the putative MDV UL9 protein identified in extracts of CEF cells infected with MDV strain GAp14 (CEF/GAp l4) and Md11p89 (CEF/Mdl 1p89) is indicated by an arrow to the right. Protein Sizes were determined by comparison to standards of known size. a D k 5 H awn r EEEMO 6 8 Egoimo “.mo kDa 101 - 83 — " 50.6 — . 87 Figure 2. Cloning of MDV UL9 gene. Two primers for PCR reactions contain KpnI Sites at their 5’-ends. PCR reaction was performed as described in Material and Methods. PCR fiagment was digested with Kpnl and cloned into the Kpnl site of pBKCMV vector (Stratagene, Ja Jolla, CA. ). The schematic shows the orientation of MDV gene relative to MDV genome, the resultant fi'agment, the restriction enzyme used and the final ligation into pBKCMV. 88 m, m, m, MDV ATG fitOP I I MDV UL9 Gene PCR Fragment E—l Ligation MDV UL9 Gene _ Restriction Digestion g—i 89 Figure 3. PCR results of amplification of MDV UL9 gene from total cellular DNA isolated from CEF cells infected with MDV strain GA. Maker used was 1 Kbp ladder marker. PCR reactions were performed as described in Material and Methods. Lanes 2-4 were PCR reactions performed with total cellular DNA isolated from MDV-GA-infected CEF cells (CEF/GA). The positive control (CEF/Mdll) was PCR reactions performed with contour-clamped homogeneous electric field (CHEF) isolated Mdll DNA Negative controls were PCR reactions performed with total cellular DNA isolated from mock- infected CEF cells (CEF) and without DNA template (negative). 90 m + 91 2.6 Kbp fragment was amplified from total cellular DNA isolated from MDV-GA-infected CEF cells (Fig. 3, lanes 2, 3 and 4) but no amplification was detected when mock infected CEF cellular DNA used as the template (Fig. 3, lane 6). PCR fragments were purified and cloned into the Kpn I site of pBKCMV vector (Fig. 2). The wild type MDV UL9 gene construct was used to express the protein in vitro with the TNT system (Progema). An aliquot of the 3SS-labeled protein was run on an 10% SDS-polyacrylamide gel. A predominant 95 kDa polypeptide was present in the wild-type lane (Fig. 4A, lane 2) but not detected in the control reaction (Fig. 4A, lane 1). The 95 kDa protein was immunoprecipitated by antiserum against WV UL9 (Fig. 4B, lane 4), indicating that the MDV UL9 protein was expressed. The in-vilro expressed MDV UL9 protein will be utilized in EMSA Binding of MDV OBP to the HSV-1 UL9 binding site I. The high degree of Similarity between MDV UL9 and HSV-l UL9 implies that MDV UL9 may bind to HSV-1 UL9 binding site I. In order to test the binding activity of MDV UL9 gene product, electrophoretic mobility gel Shift assays (EMSA) were performed with MDV UL9 gene product expressed in vitro using a 26-mer oligonucleotide probe (HSV-1 UL9 site I DNA) which contains a 11-bp motif (CGTI‘CGCACTT) identified as an HSV- 1 UL9 binding site I (Fig. 5B). Following incubation of the plasmid programmed-lysate with HSV-1 UL9 Site I DNA probe, three protein-DNA complexes, which ran with electrophoretic mobilities indistinguishable from those of complexes 1, II and C’ identified in EMSA with the mock lysate, were detected (Fig. 6, lane 3). However, complexes I and II identified in EMSA with the programmed-lysate became much weaker when compared 92 Figure 4. SDS-polyacrylamide gel analysis and Immunoprecipitation of L- [”S]metthionine-labeled MDV UL9 gene product synthesized in vitro by using a reticulocyte lysate. MDV UL9 gene was expressed with TNT lysate system (Promega) as described in Material and Methods. An aliquot of 35S-labeled protein was an on an SDS- 10% polyacrylamide gel and visualized by autoradiography. (A) Lane 1, mock- programmed control; lane 2, full length product of MDV UL9 gene. (B) lane 1, mock programmed lysate; lane 2, full length product of MDV UL9 gene; lane 3, immunoprecipitation of fiill length MDV UL9 gene product by preimrmnie antisenun; lane 4, imrrnmoprecipitation of fiill length MDV UL9 gene product by anti-MDV UL9 antiserum; lane 5, immimoprecipitation of mock programmed lysate by anti-MDV UL9 antiserum; The synthetic MDV UL9 protein is shown by the arrow to the right. Molecular mass marker is shown to the left. l01 _ 83 — 50.6 — Control Synthetic MDV UL9 93 B. Antiserum Preimmune <— 95 kDa Synthetic MDV UL9 ' + Control 94 Figure 5. MDV origin and comparison of OBP binding sites of HSV-1 and MDV. (A) MDV serotype 2 origin. The potential OBP binding sites are indicated by the bar imder the sequence. The 9 bp sequence is conserved in the origins of HSV-1, VZV and EHV-1. (B) HSV-1 UL9 binding Site I. The 11 bp conserved sequence is indicated by the sequence within the square. (C) Comparison of HSV-1 UL9 binding Site I and the MDV UL9 binding site I. Oii 22 is indicated by the sequence above the bracket. 95 10 2o 30 MDV origin ACGCGTCAW CGAACCAATA 40 50 60 TAAGATTATA TATATAATAT ATTATTGGCG 70 80 9O CWCG‘TCCG CGCAATCGGG ll-bp element HSV-1 UL9 binding site I cccmotmcccacrfizorcccmr ll-bp element HSV-l UL9 binding site I GCGAA GT'I‘CGCA CGTCCCAAT MDV OBP binding site CGTCA GT'TCGCACC GAACCAAT l I Oii22 96 Figure 6. Binding of iii-vitro synthesized MDV UL9 gene product to HSV-1 UL9 site I DNA. EMSAS of mock lysates (control) or MDV UL9 gene product (synthetic MDV UL9) on HSV-1 UL9 site I DNA were performed as described in Material and Methods. Lane 1, EMSA was performed without addition of the proteins. See Fig. 5 for the sequence of site 1 DNA s: specific competitor. ns : nonspecific competitor. The complexes formed are indicated by letter to the left. 97 E Synthetic MDV UL9 compefitor -‘S'\S g, .15.:- 98 to those identified in EMSA with the mock lysate (Fig. 6, lane 2 and 3). An additional complex (M), which was not present in EMSA with the mock lysate, was detected in EMSA with the programmed-lysate (Fig 6, lane 3). This complex presumably resulted from the binding of synthetic MDV UL9 protein to the lone OBP binding Site on HSV-1 UL9 site I DNA The complex between synthetic IVfl)V UL9 protein and the HSV-1 UL9 site 1 DNA was specific (Fig. 6, lane 4 and 5), Since it could be inhibited by a specific competitor (nonlabeled probe) but not by a nonspecific competitor. Thus, MDV UL9 protein forms a specific complex with the HSV-l UL9 binding Site 1. Binding activities from MDV-infected extracts on HSV-1 UL9 binding site I. In order to test for origin-binding activity from MDV-infected cells, electrophoretic mobility gel shift assays (EMSA) were performed with the nuclear extracts prepared from MDV-GA- or Mdl l-infected CEF cells or uninfected CEF cells using the HSV-1 UL9 site I DNA probe. In EMSAS with MDV-GA- or Mdl l-infected extracts, three protein-DNA complexes (M, C and C’) were identified (Fig 7A, lane 3 and 7B, lane 3) and an additional complex (G) was occasionally seen in EMSA with MDV Mdl l-infected-cell extract (Fig 7B, lane 3). Formation of all three complexes (M, C and C’) identified in EMSAS with the infected extracts were specific (Fig. 7A, lane 4 and 5, Fig. 7B, lane 4 and 5), as they could be inhibited by a specific oligonucleotide competitor but not by a nonspecific competitor. In EMSAS with the mock extracts, two complexes with electrophoretic mobilities indistinguishable from those (C and C’) identified in EMSAS with the infected extracts were detected (Fig 7A, lane 2 and 7B, lane 2). Thus, the complex M, which was identified in EMSAS with the infected extracts and not present in EMSAS with the mock extracts 99 Figure 7. Detection of origin-binding activities from MDV-infected cells on HSV-1 UL9 site I DNA. (A) Binding of MDV-GA-infected nuclear extract proteins. EMSAS of uninfected extracts (CEF) or MDV-GA-infected extracts (CEF/GA) on HSV-1 UL9 Site 1 DNA were performed as described in Material and Methods. Lane 1, EMSA was performed without addition of the extracts. The complexes formed are indicated by the letters to the left. s: specific competitor. ns: nonspecific competitor. (B) Binding of MDV- GA-infected nuclear extract proteins. Legends are the same those in Fig. 7A @ CEF/GA @ CEF/Md11 Competitor - - SNS Competitor - - SNS m ...—...»... 101 (Fig. 7A, lane 3 and Fig. 7B, lane 3), was a virus-specific complex It presumably resulted from the interaction between MDV UL9 protein present in the infected extract and HSV-1 UL9 Site I DNA The virus-specific complex (M) identified in EMSAS with the infected extracts and the MDV UL9-specific complex (M) identified in EMSA with the in-vitro expressed MDV UL9 were shown to migrate at the similar rates when the in-vitro translated MDV UL9 gene product was used in EMSA and run in parallel with the infected extracts (Fig. 8, lane 3 and 7). This result indicated that MDV-infected cells contained MDV UL9 which could bind to HSV-1 UL9 binding Site 1. Binding of MDV UL9 OBP to MDV serotype 2 origin. A serotype 2 MDV origin had been fimctionally identified by Camp et al (1991). serotype 2 MDV origin contains two ll-bp sequences each of which is highly similar to HSV-l UL9 binding site I (Fig. 5A). Thus, MDV serotype 2 origin contains two potential MDV OBP binding sites. These two ll-bp elements are present at the lefi (MDV UL9 binding Site I) and right (MDV UL9 binding Site II) arms of the palindrome sequence within the serotype 2 MDV origin. The sequence of MDV UL9 binding Site I is different from that of HSV-1 UL9 binding site I in two positions while that of MDV UL9 binding site II is different only in one position (Fig. 5). Using a 22-mer oligonucleotide (MDV UL9 site I DNA) probe containing MDV UL9 binding site I, no viral or cellular binding activities were detected in EMSAS with MDV GA-infected-cell nuclear extracts (data not shown). However, using a 26-mer oligonucleotide (MDV UL9 Site 11 DNA) probe containing MDV UL9 binding site 11, two virus-specific complexes (M and M’) were 102 Figure 8. Comparison of binding of MDV-GA-infected extract proteins and iii-vitro synthesized MDV UL9 gene product to HSV-1 UL9 site 1 DNA. EMSAS of uninfected extracts (CEF) or MDV-GA—infected extracts (CEF/GA) and mock lysates (control) or in- vitro synthesized MDV UL9 gene product (synthetic MDV UL9) on HSV-1 UL9 Site I DNA were performed as described in Material and Methods. Lane 1, EMSA was performed without addition of the proteins. 5: specific competitor. ns: nonspecific competitor. The complexes formed are indicated by the letters to the left. 103 i S"'""“°@ MDV UL9 CEF/GA Competitor ' ' 303" SNS pone-u.» 104 Figure 9. Detection of binding activities from MDV-GA-infected cells on MDV UL9 site II DNA. Lane 1-5, EMSAS were performed using HSV-l UL9 Site Iprobe; lane 6-10, EMSAS were performed using MDV UL9 site 1] probe. EMSAS of uninfected extracts (CEF) or MDV-GA-infected nuclear extracts (CEF/GA) on HSV-1 UL9 Site I DNA or WV UL9 Site 11 DNA were performed as described in Material and Methods. Lane 1 and 6, EMSAS were performed without addition of the extracts. 5: specific competitor. ns : nonspecific competitor. The complexes formed are indicated by the letters to the left. 105 HSV—1 UL9 MDVUL9 sitele site II DNA II @CEF/GA @CEF/GA .l _ - sNS ' ' SNS . ,..~"...~-~ "fifl- l 106 identified (Fig. 9, lane 8). These two complexes were specific and not present in EMSAS with the mock extracts (Fig. 9, lane 9 and 10). These two complexes (M and M’) probably resulted fiom the interaction between MDV UL9 present in the infected extract and MDV UL9 site 11 DNA The two complexes identified in EMSAS with MDV UL9 site 11 DNA probe migrated at rates Similar to those of two complexes identified in EMSA with HSV- 1 UL9 site I DNA probe (Fig. 9, lane 3 and 8). The slowly migrating virus-specific complex (M’) detected both in EMSAS with HSV-l UL9 site I DNA and MDV UL9 Site 11 DNA probes were occasionally seen and probably resulted from intra- and intermolecular interactions between WV UL9 bound-DNA Rabkin and Hanlon (1991) reported that HSV-l UL9, purified from baculovirus vector-infected insects cells, could form a high order of nucleocomplex with the plasmid containing HSV-1 oiis via inter- and intramolecular interactions. Compared to the results of EMSAS with HSV-l UL9 site I DNA probe, cellular binding activities were difi‘erent in EMSAS with MDV UL9 site 11 DNA probe (Fig. 9, lane 3 and 8). Super-EMSA were also performed to detect to the MDV UL9 protein present in the complex M. The complex M was reduced a little in presence of MDV UL9-specific antibody (data not Shown). When EMSA was performed with the in-vitro expressed MDV UL9 by using MDV UL9 Site 11 DNA probe, four specific-binding complexes not present in the control reaction were detected (Fig. 10, lane 3). The fastest migrating complex (M) migrated at a rate similar to that of complex M identified in EMSAS with MDV GA-infected nuclear extracts, while the other three complexes (H, H’ and H”) migrated at a slower rate (Fig. 10, lane 3 and 7). The three slowly migrating complexes detected in EMSA with the in- 107 Figure 10. Binding of iii-vitro synthesized MDV UL9 gene product to MDV UL9 site [1 DNA. Lane 1-5, EMSA were performed with mock lysate (control) or in-vitro synthesized MDV UL9 gene product (synthetic MDV UL9); lane 6-10, EMSAs were performed with uninfected extracts (CEF) or MDV-GA-infected nuclear extracts (CEF/GA). EMSAs of in-vitro synthesized MDV UL9 gene product or MDV-GA- infected nuclear extracts on MDV UL9 site 11 DNA were performed described in Material and Methods. Lane 1, EMSA was performed without addition of the proteins. 108 Synthetic MDV UL9 CEF/GA Competitor --SNS--SI\B ~01...” ZEII 109 vitro expressed MDV UL9 were also likely due to the intra- and intermolecular interactions between MDV UL9 bound-DNA The result of EMSA with the in-vitro expressed MDV UL9 suggested that MDV-infected cells contain MDV UL9 which can bind to MDV UL9 binding Site 11. Since MDV UL9 can be shown to bind to one of the potential OBP binding Sites, it is not surprising that MDV UL9 can bind to MDV serotype 2 origin. The result of EMSA with MDV-GA infected extracts using a 96-bp MDV serotype 2 origin as the probe Showed that two closely migrating complexes were present in EMSAs from infected extracts but not in EMSAs from mock extracts. (Fig. 11). Effect of single-base-pair mutation on the binding of MDV UL9 to MDV UL9 site II DNA. As mentioned above, no viral binding activities were detected in EMSA using MDV UL9 site I DNA probe. The sequence of MDV UL9 binding site I is different from that of MDV UL9 binding site II in one position (Fig. 12A). The last nucleotide of MDV UL9 binding Site I is a G while that of MDV UL9 binding Site II is a T. In order to investigate if nucleotide T within the MDV UL9 binding site H is important for the viral binding activities of MDV UL9 binding site II in vitro, a mutant MDV UL9 site H DNA was made and tested in competitive EMSAS. The ll-bp motif within the mutant MDV UL9 site 11 DNA was made to have the same sequence as that of 1 l-bp motif within MDV UL9 site I DNA; that is, nucleotide T within MDV UL9 site H DNA was mutated to nucleotide G (Fig. 12A). The results of competitive EMSAs analyzed in a phosphor 110 Figure 11. Detection of binding activities from MDV-GA-infected cells on MDV serotype 2 origin. EMSAs of uninfected extracts (CEF) or MDV-GA-infected extracts (CEF/GA) on MDV serotype 2 origin were performed as described in Material and Methods. Lane 1, EMSA was performed without addition of the extracts. ' 111 @ CEF/GA Competitor NSS QO Free probe 112 Figure 12. Mutation analysis of MDV UL9 binding site II. (A) The position of point mutation. The point nmtation is indicated by the letter within square. (B) Graph of the interpolated phosphor-imager analysis of competition analyses of the M complexes. Competitive EMSAs were performed with MDV-GA-infected extracts using MDV UL9 Site 11 DNA probe as described in Material and Methods. Each band was scanned with the phosphor-imager and results were compared (as a percentage) with those obtained without competitor (100%). The binding percentage was plotted against the molar ratio of unlabeled competitor. Each value represents the average of 2 to 3 experiments. Binding Percentage (%) 113 F-q MDV UL9 siteIIDNA GGACGGCG’I'I‘CGCACCTTGCGCCAAT Miriam MDV UL9 m 11 DNA GGACGGCGT'I‘CGCACCGTGCGCCAAT MDV UL9 site MututMDV UL9 MDV UL9 ‘ [IDNA “°“ SiteIIDNA — — siteIDNA 120 . 108 E / 96 E '\ ,/ 84 E A\-\ .\ 1' ' l] 72 E ”Mm“- ..i i J . . E i E 43 9 E. T I \\ O 36 E \\:r l\; E é\* l — a 24 E \z 12 :— 0 C l l l J l l 1 5 10 15 25 50 Fold of competitor l 14 imager (Molecular Dynamics, Sunnyvale, CA) are shown in Fig. 12B. Since complex M is the major viral complex, the results were derived from quantitation of complex M The wild type MDV UL9 site 11 DNA gave the predicted reduction in binding whereas the mutant MDV UL9 Site II DNA had much less competition ability than that of wild type MDV UL9 site 11 DNA MDV UL9 site I DNA has the least ability to compete and was comparable to that of mutant MDV UL9 site 11 DNA This result suggested that the last nucleotide T of MDV UL9 binding site II is essential for the viral binding activities of MDV UL9 binding Site II in vitro. Binding activities from MDV-infected cells on MDV UL9 site I DNA. Our previous studies showed that no viral or cellular binding activities were detected in EMSAs with MDV GA-infected extracts using MDV UL9 site I DNA probe (data not shown). Since the sequence differences between MDV UL9 site I DNA and HSV-1 UL9 site I DNA are primarily within the flanking sequences (Fig. 5C), we investigated the efi‘ects mutations within the flanking sequence may have on the viral binding activities. To investigate the effects of mutations, five 26-mer oligonucleotides each of which contained point mutations within MDV UL9 site I DNA were synthesized and tested in competitive EMSAs by using HSV-1 UL9 site I DNA probe. The mutant oligonucleotides were designed by mutating wild type MDV UL9 Site I DNA to become more HSV-l UL9 site I DNA-like. Figure 13A shows the positions of mutations. The results of assays are shown in Fig 13B. HSV-1 UL9 site I DNA (competitor 1) gave the predicted reduction in binding. While all competitors had the efl‘ects on the binding, competitor 3 containing the 115 Figure 13. Mutation analysis of MDV UL9 binding site I. Competitive EMSAs were performed with MDV-GA-infected extracts using HSV-1 UL9 Site I DNA probe as described in Material and Methods. (A) Sequences of the point mutants. Competitor 1 indicates the sequence of HSV-1 UL9 site I DNA with the ll-bp UL9 recognition sequence indicated by the bracket on top of the sequence. Competitor 2 indicates the sequence of wild type MDV UL9 Site I DNA Competitors 3-7 indicate the sequences of a series of point mutants. The point mutations are indicated by the letters within squares. (B) Graphic display of the phosphor-imager analysis of competitive analyses of the M complexes. Each band was scanned with the phosphor-imager and results were compared (as a percentage) with those obtained without competitor (100%). The bars indicate binding in the presence of indicated amounts of excess competitor expressed as percentage of binding with no competitor. 116 l- l Competitor 1 GCGAA 'I'I'CGCA CGTCCCAAT Competitor 2 CAGCG'ITCGCACCGCGAACCAA Competitor 3 CGTCAGCG'ITCGCACEPGAACCAAT Competitor 4 CGTCAGCGTI'CGCACCGCEFCAAT Competitor 5 CGTCAGCGTI’CGCACEFqEFCAAT Competitor 6 @GCGNCGCAWCAAT Competitor 7 @GCGNCGCAWCAAT I Competitor 1 I Competitor 2 Competitor 3 E Competitor 4 I Competitor 5 [:1 Competitor 6 Binding Percentage (%) Competitor 7 80 60 40 20 Fold of Competitor 117 perfect HSV-1 ll-bp element within the context of MDV UL9 Site I DNA had the most profound efl‘ect. Although competitors 5, 6 and 7 contained the perfect HSV-1 ll-bp element and the flanking sequence was more HSV-1 UL9 Site I DNA-like, they had much less competitive abilities than competitor 3 and had competition abilities comparable to competitor 2 and competitor 4 both of which contained the MDV UL9 binding Site I. This result suggested that the last two nucleotides within the ll-bp motif of HSV-1 UL9 Site I DNA are essential for the binding activity of MDV UL9 protein in vitro. This result was consistent with the result of previous section in which the last nucleotide (T) of MDV UL9 binding Site II was important for the binding activitiy of MDV UL9 in vitro. The results of competitive EMSAS also indicated that mutations changing the flanking sequences to become more HSV-1 UL9 site I DNA-like had a detrimental effect on the MDV OBP binding. Discussion Virus-specific OBPs have been identified and characterized extensively in HSV-1 and other systems, including large T antigens of simian virus 40 (SV40) and polyomavirus (Borowiec et al, 1990; Gaudray et al, 1981). In SV40, large T antigen plays an essential role in initiation of viral DNA replication. It is required to initiate the protein-DNA interactions which result in maturation of the replication machinery and subsequent unwinding of the origin. The precise role of HSV-1 OBP in HSV-1 DNA replication has not been established but it has been suggested that it plays a role in initiation of HSV-1 DNA replication similar to the role of SV 40 large T antigen. Less is known about the requirements for MDV DNA replication. In our previous studies, we determined the sequence of MDV gene homolog to the HSV-1 UL9 gene. Based on predicted amino acid sequences, HSV-l UL9 and MDV UL9 share 49% identity and 66% similarity while those of VZV gene 51 (VZV UL9 homolog) and MDV UL9 Share 46% identity and 63% Similarity. MDV UL9 also Shares numerous motifs with both HSV-1 UL9 and VZV gene 51. Conserved motifs include several helicase moieties and a leucine zipper in the N—terminus, a C-terminal pseudo-leucine zipper, and a putative helix-turn-helix structure. Our previous data suggested that MDV UL9 may have a fimction Similar to that of HSV-1 UL9. In this report, we detected a MDV UL9 protein in infected-cell nuclear extracts, with an apparent size consistent with the predicted size for MDV UL9 (95 kDa) Futhermore, a predominant 95 kDa protein was expressed in the in-vitro coupled transcription- translation programmed with MDV UL9 expression plasmid. Alignment of HSV-1 UL9, 118 119 VZV gene 51 and MDV UL9 protein sequences suggests that MDV UL9 may have biochemical activities Similar to HSV-1 UL9, including the origin-binding activity. Electrophoretic mobility gel shift assays (EMSAs) Showed that synthetic MDV UL9 not only bormd to HSV-1 UL9 binding Site I but also to MDV UL9 binding site H. In addition, MDV UL9 binding site H- and HSV-1 UL9 binding site I-binding activities were present in the infected-cell nuclear extracts. This activity generated two complexes in which the faster migrating complex migrating at a rate Similar to the fastest migrating complex identified in EMSA with the in-vitro expressed MDV UL9. The slower migrating complex probably resulted from inter- and intramolecular interaction between MDV UL9 site H DNA-bound MDV UL9 protein. The EMSAS indicated that WV UL9 gene encodes an origin-binding protein. Although viral binding activities could be detected for MDV UL9 binding Site H, no viral binding activities were detected on MDV UL9 Site I DNA By using a mutant oligonucleotide mutated from MDV UL9 site H DNA as the competitor, competitive EMSA indicated that nucleotide T in the last position of MDV UL9 binding Site H was essential for the viral binding activities of MDV UL9 bindng Site H in vitro. Elias er al. (1990) reported that the 11-bp sequence (CGTTCGCACT'I‘) was required for high- aflinity binding of HSV-1 UL9. However, Hazuda et al. (1990) refined the UL9 recognition sequence to 10 bp (CGTTCGCACT), suggesting that nucleotide T in the last position of ll-bp motif was not important. Compared to our results, nucleotide T in the last position is essential for the binding. This difference may be because the sequence in the 11-bp element (CGTTCGCACCT) within MDV UL9 site H DNA is different from 120 that within HSV-l UL9 site I DNA in one position, or that the spatial arrangement of conserved motifs within C-terminal domain of MDV UL9 is difl‘erent fi'om that of C- terminal of HSV-1 UL9 (data not shown). The sequence of 11-bp element (CGTTCGCACCG) within MDV UL9 Site I DNA is different fiom that within HSV-1 UL9 site 1 DNA (CGTTCGCACTT) in two positions. By using five mutant MDV UL9 Site I DNA as competitors, which were designed by imitation of wild type MDV UL9 site I DNA to become more HSV-1 UL9 site I DNA- like, competitive EMSAs indicated that the last two nucleotides (TT) of ll-bp element within HSV-1 UL9 Site 1 DNA was important for the binding activities of MDV UL9 protein in vitro. No binding has yet been demonstrated for HSV-1 UL9 binding site H1 in vitro but HSV-1 UL9 site IH is required for viral replication in viva. This is also probably true for MDV as well Although MDV UL9 was shown not to bind to MDV UL9 binding Site I in vitro, it is likely to be important for the viral replication in viva. Several explanations can be ofl‘ered to account for the inabilities to determine the binding on MDV UL9 Site I DNA in vitro. First, it is possible that binding of the MDV UL9 protein to MDV UL9 binding site I needs the cooperation fiom the MDV UL9 binding Site H-bound MDV UL9 proteins. Ifthis is the case, it is not surprising that MDV UL9 protein can not bind to MDV UL9 site I DNA containing the lone MDV UL9 binding Site I in EMSAs. DNase I footprinting on the whole serotype 2 MDV origin should solve the question. Second, it is postulated that MDV UL9 actually binds to MDV UL9 binding site I in an in viva environment but that other cellular or viral factors need to bind to the site in order to 121 change the overall structure making the site more accessible for MDV UL9 binding. Thus, the binding of MDV UL9 to MDV UL9 binding site I which may occur in in viva, can not be detected under the current conditions used for in-vitra DNA-binding studies. Third, MDV UL9 binding Site I may provide sites for other factors to act upon as the replicon is formed. Fourth, certain nucleotides within MDV UL9 binding Site I may be important for maintaining secondary structure of the origin for the recruitment of replication machinery. Acknowledgements We thank Jean Wilms—Robertson, Carolyn Cook, and Ronald Southwick for excellent technical assistance. We also thank Dr. Richard Schwartz for suggestions for electrophoretic mobility gel shift assays. 122 Summary and Conclusion In many of the DNA replication systems that have been studied in vitro, the first step in initiation is the recognition of a specific DNA sequence at the origin of DNA replication by an origin-binding protein (OBP). The OBP is required to initiate the protein- DNA interactions which result in the formation of mature replication complexes and subsequent destabilization of the origin. Using transient replication assays, seven genes were identified, which are both necessary and suflicient for origin-dependent DNA replication (Wu et al, 1988). HSV-1 UL9 gene, one of these seven essential genes, encodes an origin-Specific binding protein. It binds to specific sequence elements within the viral DNA replication origin. The biochemical activities of HSV-1 UL9 protein suggests that it may play a role in initiation of viral DNA replication Similar to SV40 large T antigen (Bruckner et al., 1991; Kofl‘ et al., 1991; Rabkin and Hanlon, 1991; Fierer and Challberg, 1992; Boehmer et al., 1993; Dodson and Lehman, 1993). An origin of serotype 2 MDV DNA replication has been fimctionally identified using transient replication assays (Camp et al, 1991). The serotype 2 MDV replication origin is located in the inverted repeats flanking the unique long region (Camp et al, 1991), suggesting that there are at least two copies of the origin. The origin of serotype 2 MDV DNA replication is located within a 90-bp region. It contains an imperfect palindrome with 30 bp of alternating AT sequence located at the center. The structure and sequence of serotype 2 MDV DNA replication origin is very Similar to the HSV-1 orig, and oris, VZV origin, and equine herpes virus type 1 (EHV-1) origin (Camp et al, 1991). In addition, the serotype 2 MDV DNA replication origin contains a 9-bp motif which is ‘ located both at the left and right arms of the palindrome sequence and this 9-bp motif is 123 124 highly conserved among alphaherpesviruses (Camp at al., 1991). The 9-bp motif is a subset of a 11-bp motif that is recognized by the HSV-1 origin binding protein (UL9). The presence of two copies of the 9-bp motif suggests that there are two potential binding sites for the MDV origin-binding protein Based on gene colinearity between MDV and other alphaherpesviruses, as well as origin Similarities, it seems likely that MDV encodes a UL9 homolo g which is critical for initiation of DNA replication. Conservation of potential UL9 binding sites within the MDV core origin of replication suggests that MDV UL9 may be highly Similar to the HSV-1 UL9 protein. To analyze this possibility, western blot analyses of MDV-infected and uninfected cell extracts were performed using a polyclonal anti-HSV-l UL9 protein antibody. A protein with an apparent molecular Size similar to HSV-l UL9 protein was specifically detected in extracts fi'om CEF infected with MDV, strain Mdll but not detected in mock-infected CEF. Based on gene colinearity and random sequencing analysis of BamHI G fragment of MDV, strain GA genome, it was likely that the major portion of the putative MDV UL9 gene was located within BamHI C fragment of MDV, strain GA genome. Terminal sequencing of five internal EcaRI subclones and comparison of deduced amino acid sequences with HSV-1 UL9 revealed that one internal EcaRI subclone with a 3.1 kb insert contains one end which is very Similar to the N-terminal portion of HSV-1 UL9. Using this end as a starting point, the whole putative MDV UL9 gene was identified. The MDV UL9 gene is located within BamHI G and C fragment of MDV, strain GA genome. It is transcribed fiom right to left relative to MDV genome. The orientation and location are 125 similar to HSV-1 UL9. The upstream region of MDV UL9 gene contains several transcription factor binding motifs, including CAAT boxes, GC boxes and four putative TATA boxes. The MDV UL9 gene encodes an 841 amino acid polypeptide which is Similar to HSV—1 UL9 (851 amino acids) and VZV gene 51 (835 amino acids). The predicted amino acid sequences of HSV-1 and MDV UL9 share 49% identity and 66% Similarity while those of VZV gene 51 and MDV UL9 share 46% identity and 63% similarity. Alignment of MDV UL9, HSV-l UL9, and VZV gate 51 protein predicted amino acid sequences revealed that MDV UL9 is highly similar to HSV-1 UL9 and VZV gene 51. Based on the predicted amino acid sequences, many features which are conserved within HSV-1 UL9 and VZV gene 51 can be found within MDV UL9. For example, all putative helicase motifs found within the N-terminal portion of HSV-1 and VZV UL9 proteins are also present in MDV UL9. Furthermore, the spatial arrangement of these motifs is conserved between the three origin binding proteins. It has been shown that the conserved helicase motifs are important for the helicase activity of HSV-1 UL9 (Martinez et al., 1992). Conservation of these motifs in MDV UL9 suggests that, like HSV-1 UL9, MDV UL9 possesses an intrinsic helicase activity within the N-terminal portion. Also within the N-terminal domain of MDV UL9, a leucine zipper region can be found, which is essential for dirnerization and cooperativity of HSV-1 UL9 (Elias et al. , 1992; Hazuda et al.,1992) The C-terminal domain of MDV UL9 contains two structural motifs in common with the C-terminal domains of HSV-1 UL9 and VZV gene 51, a pseudo-leucine zipper (not clearly defined in VZV UL9) and a helix-tum-helix motif The pseudo-leucine zipper 126 was shown to be important for DNA-binding activity of HSV-1 UL9 (Deb and Deb, 1991; Arbuckle and Stow, 1993; Martin et al., 1994). However, importance of the helix-tum- helix motif for the DNA-binding activity remains to be elucidated. In addition to these two recognizable structural motifs, a highly conserved region is formd within the very C- terminal end of MDV UL9. This sequence, termed the VZV homology region, is highly similar to corresponding sequences in HSV-l and VZV UL9 proteins. The VZV homology region has been shown to be critical in maintaining the DNA-binding activity of HSV-1 UL9 (Deb and Deb, 1991; Arbuckle and Stow, 1993; Martin et al., 1994). In addition to conservation of known structural/firnctional motifs, there are three highly conserved regions in MDV UL9, HSV-1 UL9, and VZV gene 51 which do not show clear homology to previously identified functional domains. The high Similarity between deduced amino acid sequences of MDV UL9, HSV-1 UL9 and VZV gene 51 suggests that MDV UL9 gene encodes an origin-binding protein which possesses the Similar biochemical activities to HSV-1 UL9 and may play a role in initiation of MDV viral DNA replication. To detect the MDV UL9 transcript, four DNA probes isolated fi'om the MDV UL9 gene were used in northern blot analysis. With the EcaRl-A val probe fiom the N- terminal portion of MDV UL9, 4.4 and 2.1 kb transcripts were detected whereas with the EcoRI subfi‘agment from BamHI C which contains the partial N-terminal domain and the upstream region, 4.4, 2.6 and 2,1 kb transcripts were detected. With the BglII-BamHI probe from the C-terminal domain, 4.4 and 2.0 kb transcripts were detected whereas with the EcaRl-BamHI probe which is approximately centrally located in BamHI C, 4.4 kb 127 transcript was detected. Based on gene colinearity, it is suggested that the 4.4 kb transcript may represent the primary MDV UL9 transcript and the 2.1 kb transcript may represent the MDV UL10 homolog transcript. In order to detect the MDV UL9 protein in the MDV-infected cells, western blot analyses were performed with a polyclonal antibody against the MDV UL9 protein raised fi'om MDV UL9-GST fusion protein. A protein with an apparent molecular size corresponding to the predicted size (95 kd) was detected in the infected nuclear extracts but not in uninfected nuclear extracts. To examine the binding activity of MDV UL9 independent of other viral protein, the MDV UL9 gene was amplified by PCR from the total cellular DNA isolated fi'om MDV, strain GA-infected cells. The resulting product was cloned into the pBKCMV vector. The MDV UL9 protein was expressed with the coupled transcription-translation system (Promega). A 95 kd protein could be expressed in the coupled transcription- translation lysate and was immunoprecipitated by the polyclonal antibody against the MDV UL9 protein. The in vitro expressed MDV UL9 protein was used in electrophoretic mobility gel shift assays (EMSA) using HSV-1 UL9 site I DNA probe. A MDV UL9- specific complex (M) could be detected in EMSAS with in vitro expressed MDV UL9 but not detected in EMSAS with the control lysates. Consistent with this result, a similar virus- specific complex (M) was also detected in EMSAs with MDV GA or Mdl l-infected nuclear extracts. These results demonstrated that MDV UL9 protein can bind to HSV-l UL9 site I DNA. 128 The serotype 2 MDV origin contains two potential OBP binding Sites. To detect if MDV UL9 can bind to the OBP binding sites within the serotype 2 MDV origin, EMSAs were performed with in vitro expressed MDV UL9 protein or MDV-infected nuclear extracts using MDV UL9 Site H DNA probe. The results of EMSAs showed that in vitro expressed MDV UL9 protein can form a complex (M) with MDV UL9 site H DNA migrating at a rate similar to a complex resulting fiom MDV GA-infected nuclear extracts. Besides the complex M, high order complexes were also detected in EMSAs with in-vitra expressed MDV UL9 protein or MDV GA-infected nuclear extracts. Using the entire serotype 2 MDV origin as a probe, two virus-specific complexes could be detected in EMSAs with MDV GA-infected nuclear extracts. The results of EMSAs demonstrated that MDV UL9 can bind to the serotype 2 MDV origin Although WV UL9 was Shown to bind to one of the potential OBP binding Sites (MDV UL9 binding Site H) within the serotype 2 MDV origin, MDV UL9 was not detected to bind to the MDV UL9 binding Site I. The sequence of WV UL9 binding site I is different from that of MDV UL9 binding site H in one position. The last nucleotide of MDV UL9binding SiteIisa Gwhilethat ofMDV UL9 binding siteHisa T. To examine the importance of the last nucleotide in MDV UL9 binding site H, a mutant MDV UL9 site H DNA was synthesized and tested in competitive EMSAs. The ll-bp motif within the rmrtant MDV UL9 site H DNA has the same sequence as MDV UL9 binding site I. The results of competitive EMSAS Showed that the last nucleotide (T) of MDV UL9 binding Site H is essential for the binding of MDV UL9 to MDV UL9 site H DNA in vitro and the flanking sequence may have a minor effect on the binding activities. 129 Our previous studies showed that no viral or celhilar binding activities were detected in EMSAs with MDV GA-infected extracts using MDV UL9 site I DNA probe (data not shown). Since the sequence difl‘erences between MDV UL9 Site I DNA and HSV-1 UL9 Site I DNA are primarily within the flanking sequences, the efl‘ects mutations within the flanking sequence may have on the viral binding activities were investigated. To investigate the effects of imitations, five 26-mer oligonucleotides each of which contained point mutations within MDV UL9 site 1 DNA were synthesized and tested in competitive EMSA using HSV-1 UL9 Site 1 DNA probe. The mutant oligonucleotides were designed by mutating wild type MDV UL9 site 1 DNA to become more HSV-l UL9 Site I DNA- like. The results of competitive EMSAs demonstrated that the last two nucleotides within the 11-bp motifs of HSV-1 UL9 site I DNA are essential for the binding activity of MDV UL9 protein in vitro. Future Research Directions Less is known about the mechanisms of MDV DNA replication compared to HSV- 1. Identification of MDV UL9 gene adds important information to studies of MDV DNA replication, particularly regarding the initiation of MDV DNA replication. Alignment of the predicted amino acid sequences suggests that MDV UL9 may have the Similar biochemical activities to HSV-1 UL9. However, the real fimctions of MDV UL9 need to be investigated. Many fimctions need to be examined and future research should focus on structural and fimctional analysis. Structure-function studies of HSV-1 UL9 have been intensive. The N-terminal domain of HSV-1 UL9 encodes the helicase activity and responsible for cooperativity. Within the N-terminal domain of HSV-1 UL9, the conserved helicase motifs have been Shown to be essential for the helicase activity as well as for viral replication and the leucine zipper region is essential for the cooperativity. MDV UL9 also contains the same conserved motifs within the N-terminal domain. The importance of helicase conserved motifs within the N-terminal domain of MDV UL9 could be investigated by point mutations or deletion of each helicase conserved motif The wild type or mutant WV UL9 protein could be purified from the MDV UL9-expressing recombinant baculovirus- infected insect cells. Purified wild type or mutant MDV UL9 proteins could be tested for in vitro helicase assays. Although MDV UL9 protein has been shown to bind to one of two OBP binding Sites, it was not detected to bind MDV UL9 site I DNA containing the lone MDV UL9 binding Site I located on the left arm of palindrome sequence. It is possible that binding of 130 131 the MDV UL9 protein to MDV UL9 binding site I requires the cooperation from MDV UL9 proteins bound to MDV UL9 binding Site H. This cooperativity could be examined by DNase I footprinting of the serotype 2 MDV origin. If MDV UL9 protein did cooperatively bind to the two OBP binding Sites, the importance of leucine zipper region within the N-terminal portion of MDV UL9 for cooperativity Should be investigated by insertion nnrtation of the leucine zipper region. The cooperativity could be addressed by DNase I footprinting with wild type or mutant MDV UL9 protein purified from recombinant baculovirus-infected insect cells. The C-terminal domain of HSV-1 UL9 encodes the DNA-binding domain. A pseudoleucine zipper region and a VZV homolog region have been Shown to be important for the DNA binding activity whereas the importance of helix-tum-helix motif is not clear. The C-terminal domain of MDV UL9 protein contains all conserved motifs except that the spatial arrangements of leucine zipper and helix-tum-helix motif are different. The boundaries of C-terminal domain Should be determined by EMSAs with in vitro coupled transcription-translation expressed wild type or deletion mutant MDV UL9 proteins. The importance of leucine zipper could be addressed by insertion irmtation of wild type DNA- binding domain using EMSAs while the VZV homolog region can be examined by the deletion. In addition to the conserved motifs mentioned above, three small regions are also conserved within HSV-1 UL9 and MDV UL9. The conserved regions H and HI are located within the N-terminal domain while the conserved region I is located within the C- terminal domain. The importance of these three regions could be investigated by the 132 deletion mutation. Conserved region H and HI will be tested for cooperativity and conserved region III will be tested for DNA binding activity. Studies on the structure-frmction of MDV UL9 will give insight to possible mechanisms for initiation of MDV DNA replication and may help elucidate mechanisms in other related alphaherpesviruses. List of references Adams, A (1987). Replication of latent Epstein-Barr virus genomes in Raji cells. J. Virol 61, 1743-1746. Adldinger, H. K, Cahiek, B. W. (1973). Pathogenesis of Marek’s disease: early distribution of virus and viral antigens in infected chickens. J. Natl Cancer Inst. 50, 1287- 1298. Albrecht, M., Darai, G., and Flugel, R M. (1985). Analysis of the genomic termini of tupaia herpesvirus DNA by restriction mapping and nucleotide sequencing. J. Virol 56, 466-474. Ambinder, R F., Mullen, M., Chang, Y.-N., Hayward, G. S., and Hayward, D. S. (1991). Functional domains of Espstein-Barr virus nuclear antigen EBNA-l. J. Virol 65, 1466- 1478. Anders, D. G., Kacica, M. A, Pari, G., and Puntuiieri, S. M. (1992). Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. J. Virol 66, 3373-3384. Anders, D. G., and Puntuiieri, S. M. (1991). Multicomponent origin of cytomegalovirus lytic-phase DNA replication. J. Virol 65, 931-937. apRhys, C. M. J., Ciufo, D. M., O’Neill, E. A, Kelly, T. J., and Hayward, G. S. (1989). Overlapping octamer and TAATGARAT motifs in the VF65-response elements in herpes simplex virus immediate-early promoters represent independent binding Sites for cellular nuclear factor HI. J. Virol 63, 2798-2812. Arbuckle, M. 1., and Stow, N. D. (1993). A mutation analysis of the DNA-binding domain of the herpes Simplex virus type 1 UL9 protein. J. Gen. Virol 74, 1349-1355. Bankier, A T., Dietrich, W., Baer, R, Barrel], B. G., Colbere-Garapin, F., Fleckenstein, B., and Bodemer, W. (1985). Terminal repetitive sequences in herpesviruses Simiri virion DNA J. Virol 55, 133-139. Baradaran, K, Dabrowski, C. E., Schaffer, P. A (1994). Transcriptional analysis of the region of the herpes Simplex virus type 1 genome containing the UL8, UL9, and UL10 genes and identification of a novel delayed-early gene product, OBPC. J. Virol 68, 4251- 4261. Baumann, R P., Yalamanchili, R R, and O’Callaghan. (1989). Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication. J. Virol 63, 1275-1283. 133 134 Beasley, J. N., Patterson, L. T., and McWade, D. H. (1970). Transmission of Marek’s disease by poultry house dust and chicken dander. Am J. Vet. Res. 31, 339-344. Ben-Porat, T ., Kaplan, A S., Stehn, B., and Rubenstein, A S. (1982). Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA Cell 31, 89-97. Biggs, P. M. (1968). Marek’s disease- current state of knowledge. Curr. Top. Microbial. Imnnmol 43, 92-125. Boehmer, P. E., Craigie, M. C., Stow, N. D., and Lehman, I. R (1994). Association of origin binding protein and single strand DNA-binding protein, ICP8, during herpes simplex virus type 1 DNA replication in viva. J. Biol Chem. 269, 29329-29334. Boehmer, P.E., Dodson, M. S., and Lehman, I. R (1993). The herpes Simplex virus type- 1 origin binding protein. DNA helicase activity. J. Biol Chem 268, 1220-1225. Boehmer, P. E., and Lehman, I. R. (1993). Physical interaction between the herpes simplex virus type 1 origin-binding protein and single-stranded DNA-binding protein ICP8. Proc. Natl Acad. Sci. 90, 8444-8448. Borowiec, J. A, Dean, R B., Bullock, P. A, and Hurwitz, J. (1990). Binding-rmwinding- how T antigen engages the SV 40 origin of DNA replication. Cell 60, 181- 184. Bradley, G., Hayashi, M., Lancz, G., Tanaka, A, and Nonoyama, M. (1989). Structure of the Marek’s disease virus BamHI-H gene family: Genes of putative importance for tumor induction. J. Virol 63, 2534-2542. Bruckner, R C., Crute, J. J., Dodson, M. S., and Lehman, I. R (1991). The herpes simplex virus 1 origin binding protein: A DNA helicase. J. Biol Chem. 266, 2669-2674. Brunovskis, P., and Velicer, L. F. (1992). Genetic organization of the Marek's disease virus unique short region and identification of Us-encoded polypeptides. In "Proceedings 19th World Poultry Congress" Vol 1, pp. 74-78. Ponsen & Looijen, Wageningen, The Netherland. Buckmaster, A E., Scott S. D., Sanderson, M J. S., Boursnell, M. E. G., Ross, N. L. J., and Binns, M. M. (1988). Gene sequence and mapping data from Marek's disease virus and herpesvirus of turkeys : Implications for herpesvirus classification. J. Gen. Virol. 62, 2033-2042. 135 Bulow, V. V., and Biggs, P. M. (1975a). Differentiation between strains of Marek’s disease virus and turkey herpesvirus by irnnnmofluorecence assays. Avian Pathol 4, 133- 146. Bulow, V. V., and Biggs, P. M. (1975b). Precipitating antigens associated with Marek’s disease viruses and a herpesvirus of turkeys. Avian Pathol 4, 147- 162. Bzik, D. J., and Preston, C. M. (1986). Analysis of DNA sequences which regulate the transcription of hereps simplex virus immediate early gene 3: DNA sequences required for enhancer-like activity and response to trans-activation by a virion polypeptide. Nucleic Acids Res. 14, 929-943. Calnek, B. K, and Witter, R L. (1991). Marek’s disease, p342-385. In B. W. Calnek, H. J. Barnes, C. W. Beard, W. M. Reid, and H. W. Yoder (ed.), Disease of poultry. Iowa State University press, Ames, Iowa. Calnek B. W. (1985). Marek’s disease-model for herpesvirus oncology. CRC Critical Reviews in Microbiology 12, 293-320. Cahiek, B. W., Schat, K A, Ross, L. J. N., Shek, W. R, and Chen, C. L. H (1984). Further characterization of Marek’s disease virus-infected lymphocytes 1. in vivo infection. Intl J. Cancer 33, 389-398. Calnek, B. W. (1980). Marek’s disease and lymphoma, p104-143. In F. Rapp.(ed.), Oncogenic herpesvirus, Vol 1. CRC press, Boca Raton, Florida. Calnek, B. W. (1972). Effects of passive antibody on early pathogenesis of Marek’s disease. Infect. Immun. 6, 193-198. Calnek, B. W., Adldinger, H. K, and Kahn, D. E. (1970). Feather follicle epithelium: a source of enveloped and infectious cell-free herpesvirus from Marek’s disease. Avian Dis. 14, 219-133. Cahiek, B. W., Ubertini, T., and Adldinger, H. K (1970). Viral antigen, virus particle, and infectivity of tissues from chickens with Marek’s disease. J. Natl Cancer Inst. 45, 341- 347. Cahiek, B. W., and Hitchner, S. B. (1969). Localization of viral antigen in chickens infected with Marek’s disease herpesvirus. J. Natl Cancer Inst. 43, 935-949. Camp, H. S., Silva, R F., and Coussens, P. M. (1993). Defective Marek’s disease virus DNA contains a gene encoding a potential nuclear DNA binding protein and a HSV a-like sequence. Virology 196, 484-495. 136 Camp, H. S., Coussens, P. M., and Silva, R. F. (1991). Cloning, sequencing, and fimctional analysis of a Marek’s disease virus origin of DNA replication. J. Virol 65, 6320-6324. Cebrian, J., Kaschka-Dierish, C., Berthelot, N., and Sheldrick, P. (1982). Inverted repeat nucleotide sequences in the genomes of MDV and herpes virus of turkeys. Proc. Natl Sci. USA 79, 555-558. Chomczynsk, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidnium thiocyanate-phenol-chloroform extraction. Anal Biochem. 162, 156- 159. Challberg, M D., and Kelly, T. J. (1989). Animal virus DNA replication. Annu. Rev. Biochem. 58, 671-717. Chen, D., Stabell E. C., and Olivo, P. D. (1995). Varicella-Zoster virus gene 51 complements a herpes simplex virus type 1 UL9 null mutant. J. Virol 69, 4515-4518. Chen, D., and Olivo, P. D. (1994). Expression of the varicella-zoster virus origin-binding protein and analysis of its site-specific DNA-binding properties. J. Virol 68, 3841-3849. Chittenden, T., Lupton, S., and Levine, A (1989). Functional limits of oriP, the Epstein- Barr virus plasmid origin of replication. J. Virol 63, 3016-3025. Chou, J., and Roizman, B. (1985). 'The isomerization of herpes simplex virus type 1 genome: identification of the cis-acting and recombination sites within the domain of the a sequence. Cell 41, 803-811. Churchill, A E., Payne, L. N., and Chubb, R. C. (1969). Immunization against Marek’s disease using a live attenuated virus. Nature 221, 744-747. Churchill, A E., and Biggs, P. M. (1968). Herpes-type virus isolated in cell culture from tumors of chickens with Marek’s disease. H. Studies in vivo. J. Nat. Cancer Inst. 41, 951- 956. Confer, A W. and Adldinger, H. K (1980). Cell-mediated imnnmity in Marek’s disease: cytotoxic responses in resistant and susceptible chickens and relation to disease. Am. J. Vet. Res. 41, 307-312. Comitryman, J., and Miller, G. (1985). Activation of expression of latent Espatein-Barr herpesvirus after gene transfer with a small cloned subfragrnent of heterogeneous viral DNA Proc. Natl Acad. Sci. USA, 81, 7632-7636. Cox, M. A, Leahy, J., and Hardwick, J. M. (1990). An enhancer within the divergent promoter of Epstein-Barr virus responds synergistically to the R and Z transactivators. J. Virol 64, 313-321. 137 Dabrowski, C. E., Carmillo, P., and Shaffer, P. A (1994). Cellular protein interaction with herpes Simplex virus type 1 oriS. Mol Cell Biol 14, 2545-2555. Dabrowski, C. E., and Schafl‘er, P. A (1991). Herpes Simplex virus type 1 origin-binding protein : oriS-binding properties and efl‘ects of celhrlar proteins. J. Virol 65, 3140-3150. Dambaugh, T., Heller, M., Zraab-Traub, N., King, W., Chermg, A, Beisel, C., Hummel, M., van Santen, V., Fennewald, S., and Kiefl‘, E. (1980). DNAS of Epstein-Barr virus and herpesvirus papio, p85-90. In A Nahmias, W. Dondle, and R. Schinazi (ed.), The human herep svirus. Elsevier, New York Davison, A J. (1984). Structure of the genome termini of varicella-zoster virus. J. Gen. Virol 65, 1969-1977. Deiss, L. P., Chou, J ., Frenkel, N. (1986). Functional domain within the a sequence involved in the cleavage-packaging of herpes Simplex virus DNA J. Virol 59, 605-618. Deiss, L. P., and Frenkel, N. (1986). Herpes Simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence. J. Virol 57, 933-941. Deb, S., and Deb, S. P. (1991). A 269-amino-acid segment with pseudo-leucine zipper and a helix-tum-helix motif codes for the sequence-specific DNA-binding domain of herpes simplex virus type 1 origin-binding protein. J. Virol 65, 2829-2838. Deb, S., and Deb, S. P. (1989). Analysis of Ori-S sequence of HSV-1 : identification of one ftmctional DNA binding domain. Nucleic Acids Res. 17, 2733-27 52. Deb, S., and Doelberg, M. (1988). A 67-base-pair segment from the ori-S region of herpes simplex virus type 1 encodes origin function. J. Virol 62, 2516-2519. Dodson, M. S., and Lehman, I. R. (1993). The herpes simplex virus type I origin protein. DNA-dependent nucleoside triphosphate activity. J. Biol Chem 268, 1213-1219. Elias, P., Gustafsson. C. M., Hammarsten, O., and Stow, N. D. (1992). Structural elements required for the cooperative binding of the hereps Simplex virus origin binding protein to oriS reside in the N-terminal part of the protein. J. Biol Chem 17424-17429. Elias, P., Gustafsson, C. M., and Hammarsten, O. (1990). The origin binding protein of herpes Simplex virus 1 binds cooperatively to the viral origin of replication oriS. J. Biol Chem 265, 17167-17173. 138 Elias, P., and Lehman, I. R (1988). Interaction of origin binding protein with an origin of replication of herpes Simplex virus 1. Proc. Natl Acad. Sci 85, 2959-2963. Elias, P. O’Donell, M. E., Mocarski, E. S., and Lehman, I. R (1986). A DNA binding protein specific for an origin of replication of herpes Simplex type 1. Proc. Natl Acad. Sci USA 83, 6322-6326. Fierer D. S. and Challberg, M. D. (1992). Purification and characterization of UL9, the herpes Simplex virus type 1 origin-binding protein. J. Virol 66, 3986-3995. Fierer, D. S., and Challberg, M. D. (1995). The stoichiometry of binding of the herpes Simplex virus type 1 origin binding protein, UL9 to oriS. J. Biol Chem 270, 7330-7334. Fixrnan, E. D., Hayward, G. S., and Hayward, S. D. (1995). Replication of Epstein-Barr virus oriLyt: Lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays. J. Virol 69, 2998-3006. Fixman, E. D., Hayward, G. S., and Hayward, S. D. (1992). Trans-acting requirements for replication of Epstein-Barr virus oriLyt. J. Virol. 66, 5030-5039. Epstein, M A, and Achong, B. G. (1979). The Epstein-Barr virus. Spring-Verlag, Berlin. Frappier, L. and O’Donnell (1991). Proc, Natl Acad. Sci. USA 88, 10875-10879. Friedmann, A, Shlomai, J., and Becker, Y. (1977). Electron microscopy of herpes simplex virus DNA molecules isolated from infected cells by centrifugation in CsCl density gradient. J. Gen. Virol 34, 507-522. Fukuchi, K, Sudo, M., Lee, Y.-S., Tanaka, A, And Nonoyama M. (1984). Structure of Marek's disease virus DNA: Detailed restriction enzyme map. J. Virol 51, 102-109. Fukuchi, K, Tanaka, A, Schierman, L. W., Witter, R L., and Nonoyama, M. (1985). The structure of Marek’s disease virus DNA: the presence of unique expansion in nonpathogenic viral DNA Proc. Nat. Acad. Sci. 82, 751-754. Gahn, T. A, and Schildkraut C. L. (1989). The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication. Cell 58, 527-535. 139 Gaudray, P., Tyndall, C., Kamen, R, and Cuzin, F. (1981). The high aflinity binding Site on polymoa virus DNA for the viral large-T protein. Nucleic Acids Res. 9, 5697-5710. Glaubiger, C., Nazaiian, K, and Velicer, L. F. (1983). Marek's disease herpesvirus. IV Molecular characterization of Marek's disease herpesvirus A antigen. J. Virol 45, 1228- 1234. Grufl‘at, H., Manet, E., Rigolet, A, and Sergeant, A (1990). The enhancer factor R of Epstein-Barr virus (EBV) is a sequence-specific DNA binding protein. Nucleic acids Res. 18, 6835-6843. Gussander, E., and Adams, A (1984). Electron microscopic evidence for replication of circular Epstein-Barr virus genomes in latently infected Raji cells. J. Virol 52, 549-556. Gustafsson, C. M., Falkenberg, M., Simonsson, S., Valadi, H., and Elias, P. (1995). The DNA ligands influence the interactions between the hereps Simplex virus 1 origin binding protein and the single strand DNA-binding protein, ICP8. J. Biol Chem 270, 19028- 19034. Hammerschmidt, W., and Sugden, B. (1988). Identification and characterization of oriLyt, a lytic origin of DNA replication of Epstein-Barr virus. Cell 55, 427-433. Hamper, B., Tanaka, A, Nonoyama, M., and Derge, J. (1974). Replication of the resident repressed Epstein-Barr virus genomes during the early S phase (S-l period) of non- producer Raji cells. Proc. Natl Acad. Sci. USA 71, 631-633. Hamzeh, F. M., Lietman, P. S., Gibson, W., and Hayward, G. S. (1990). Identification of the lytic origin of DNA replication in human cytomegalovirus by a novel approach utilizing Ganciclovi-induced chain termination. J. Virol 64, 6184-6195. Harrison, S., Fisenne, K, and Hearing, J. (1994). Sequence requirement of the Epstein- Barr virus latent origin of DNA replication. J. Virol 68, 1913-1925. Hazuda, D. 1., Perry, H C., and Mcclements, W. L. (1992). Cooperative interactions between replication origin-binding molecules of herpes simplex virus origin-binding protein are mediated via the amino terminus of the protein. J. Biol Chem 267, 14309- 14315. Hazuda, D. 1, Perry, H C., Naylor, A M., and Mcclements, W. L. (1991). Characterization of the herpes simplex virus origin binding protein interaction with oriS. J. Biol Chem 266, 4621-24626. 140 Henle, W., Diehl, V., Kohn, G., Hansen, H. Z., and Henle, G. (1967). Herpes-type virus and chromosome maker in normal leukocytes after growth with irradiated Burkitt cells. Science 157, 1064-1065. Hernandez, T. R, Dutch, R E., Lehman, I. R, Gustafsson. C., and Elias, P. (1991). Mutations in a herpes simplex virus type 1 origin that inhibit interaction with origin- binding protein also inhibit DNA replication. J. Virol 65, 1649-1652. Hirai, K (1988). Molecular biology of Marek’s disease virus, p21-42. In S. Kato, T. Horiuchi, T. Mikami, and K Hirai (ed.), Advances in Marek’s disease research, Osaka, Japan. Hirai, K, Ikuta, K, and Kato, S. (1979). Comparative studies on Marek’s disease virus and herpesvirus of turkey DNAS. J. Gen. Virol 45, 119-131. Hirsch, 1., Cabral, G., Patterson, M., and Biswal, N. (1977). Studies on the intracellular replicating DNA of herpes simplex virus type 1. Virology 81, 48-61. Hsieh, D.-J., Camiolo, S. M., and Yates, J. L. (1993). Constitutive binding of EBNAl protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection. EMBO J. 12, 4933-4944. Hudson, L. and Payne, L. N. (1973). Analysis of T and B cells of lymphoma in chickens. Nature 241, 52-56. Igarashi, T., Takahashi, M, Donovan, J., Jessip, J., Smith, M., Hirai, K, Tanaka, A, and Nomoyama, M. (1987). Restriction enzyme map of herpesvirus of turkey DNA and its colinear relationship with Marek’s disease DNA. Virology 157, 351-358. Inoue N., Harada, S., Honma, T., Kitamura, T., and Yanag, K (1991). The domains of Epstein-Barr virus nuclear antigen 1 essential for binding to oriP region has a sequence fitted for the hypothetical basic-helix-loop-helix structure. Virology 182, 84-93. Iskenderian, A. C., Huang, L., Reilly, A, Stenberg, R M., and Anders, D. G. (1996). Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes. J. Virol 70, 383- 392. Jacob, R J., Morse, L. S., and Rozrnan, B. (1979). Anatomy of herpes simplex virus DNA XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA J. Virol 29, 448-457. Jakowsln', R M., Fredrickson, T. N., Luginbuhl, R E., and Helmboldt, C. F. (1969). Early changes in bursa of Fabricius from Marek’s disease. Avian Dis. 13, 215-222. 141 Kaaden, O. R, Scholtz, A, BenZeev, A, and Becker, Y. (1977). Isolation of Marek’s disease virus DNA from infected cells by electrophoresis on polyacrylamide gels. Arch Virol 54, 74-84. Kaemer, H. C., and Maichle, I. B., Ott, A, and Schroder, C. H. (1979). Origin of two different classes of defective HSV-1 Angelotti DNA Nucleic Acids Res. 6, 1467- 1478. Kato, S., and Hirai, K (1985). Marek’s disease virus. Adv. Virus Res. 30, 225-277. Kofl; A, Schwedes, J. F., and Tegtmeyer, P. (1991). Herpes Simplex virus origin-binding protein (UL9) loops and distorts the viral replication origin. J. Virol 65, 3284-3292. Kofl; A, and Tegtmeyer, P. (1988). Characterization of major recognition sequence for a herpes simplex virus type 1 origin-binding protein. J. Virol 62, 4096-4103. Kozak, M. (1989). The scanning model for translation : an update. J. Cell Biol 108, 229- 241. Kowalski, D., and Eddy, M. J. (1989). The DNA unwinding element: a novel, cis-acting component that facilitates opening of Escherichia Cali replication origin. EMBO J. 8, 4335-4344. LaFemina, R L., and Hayward, G. S. (1983). Replicative forms of human cytomegalovirus DNA with joined termini are found in permissively infected human cells but not in nonpermissive Balb/c-3T3 mouse cells. J. Gen. Virol 64, 373-389. Laine A, and Frappier, L. (1995). Identification of Epstein-Barr virus nuclear antigen 1 protein domains that direct interaction at a distance between DNA-bound proteins. J. Biol Chem 270, 30914-30918. Lee, S. S.-K, Dong, Q., Wang, T. S.-F., and Lehman, I. R (1995). Interaction‘of herpes Simplex virus type 1 origin-binding protein with DNA polymerase or. Proc. Natl Acad. Sci USA 92, 7882-7886. Lee, L. F., Kiefl‘, E. D., Bachenheimer, S. L., Roizman, B., Spear, P. G., Burmester, B. R, and Nazerian, K (1971). Size and composition of Marek’s disease virus deoxynucleotide acid. J. Viral 7, 289-294. Liberman, P. M., Hardwick, J. M., Sample, J., Hayward, G. S., and Hayward, S. D. (1990). The Zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocyte binds to both AP-l and ZRE sites in target promoter and enhancer regions. J. Virol 64, 1143-1155. 142 Lilierman, P. M., Hardwick, J. M., and Hayward, S. D. (1989). Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type- specific manner by two independent Signal regions. J. Virol 63, 3040-3050. Lindahl, T., Adams, A, Bjursell, G., Bomkamm, G. W., Kascha-Dierich, C., and Jehn, U. (1976). Covalently closed circular duplex DNA of Esptein-Barr virus in a human lymphoid cell line. J. Mol Biol 102, 511-530. Liptak, L. M., Uprichard, S. L., and Knipe, D. M. (1996). Fimctional order of assembly of herpes Simplex virus DNA replication proteins into prereplicative Site structures. J. Virol 70,1759-1767. Lockshon, D., and Galloway, D. A (1988). Sequence and structural requirements of a herpes Simplex viral DNA replication origin. Mol Cell Biol 8, 4018-4027. Lupton, S., and Levine, A J. (1985). Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Esptein-Barr virus-derived plasmids in human cells. Mol Cell Biol 5, 2533-2542. Marks, J. R, and Spector, D. H (1988). Replication of the murine cytomegalovirus genome: structure and role of the termini in the generation and cleavage of concatemates. Virology 162, 98-107. Marek, J. (1907). Multiple nervenentzundung (polyneuritis) bei Huhnem. Dtsch. tierarztl. Aschr. 15, 417-421. Martin, D. W., and Deb, S. (1994). Cloning and expression of an equine herpesvirus 1 origin-binding protein. J. Virol 68, 3674-3681. Martin D. W., Munoz, R M., Oliver, D., Subler, M. A, and Deb, S. (1994). Analysis of the DNA-binding domain of the HSV-1 origin-binding protein. Virology 198, 71-80. Martin, D. W., Deb, S. P., Klauer, J. S., and Deb, S. (1991). Analysis of the herpes Simplex virus type 1 oris sequence: Mapping of functional domain. J. Virol 65, 4359- 4369. Martinez, R, Shao, L., and Weller, S. (1992). The conserved helicase motifs of the herpes Simplex virus type 1 origin-binding protein UL9 are important for function. J. Virol 66, 6735-6746. 143 Matsuo, T., Heller, M., Petti, L., O’Shiro, E., and Kiefl; E. (1984). Persistence of the entire Epstein-Barr virus genome integrated into human lymphocyte DNA Science 226, 1322-1325. Mcgeoch, D. M., Dalrymple, M. A, Dolan, A, Mcnab, D., Perry, L. J., Taylor, R, and Challberg, M (1988). Structure of herpes Simplex virus type 1 genes required for replication of virus DNA J. Virol 62, 444-453. Mclean, (1994). The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein. J. Gen. Virol 75, 2699-2706. Middleton, T., and Sugden (1992). J. Virol. 66, 489-495. Mocarski, E. S., and Roizman, B. (1982). Herpesvirus-dependent amplification and inversion of cell-associated viral thymidine kinase gene flanked by viral a sequence and linked to an origin of viral DNA replication. Proc. Natl Acad. Sci. USA 79, 5626-5634. Mocarski, E. S., Post, L. E., and Roizman, B. (1980). Molecular engineering of the herpes Simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions. Cell, 22, 243-255. Morgan, R W., Cantello, J. L., Claessens, J. A J., and Sondermeyer, P. (1991). Inhibition of Marek’s disease virus DNA transfection by a sequence containing an alphaherpesvirus origin of replication and flanking transcription regulatory elements. Avian Dis. 35,70-81. Morse, LS, Buchman, TG, and Roizman, B. (1977). Anatomy of herpes simplex virus DNA IX. Apparent exclusion of some parental DNA arrangements in the generation of intertypic (HSV-l x HSV- l) recombinants. J. Virol. 24, 231-248. Nazerian, K (1973). Marek’s disease: A neoplastic disease of chickens caused by a herpesvirus. Adv. Cancer Res. 17, 279-315. Nazerian, K (1971). Further studies on the replication of Marek’s disease virus in the chicken and in cell culture. J. Natl Cancer Inst. 47, 207-217. Nazerian, K, and Witter, R L. (1970). Cell-free transmission and in viva replication of Marek’s disease virus (MDV). J. Virol 5, 388-397. Nazerian, K, Solomon, J. J., Witter, R L., and Burmester, B. R (1968). Studies on the etiology of Marek’s disease. H. Finding of a herpesvirus in cell culture. Proc. Soc. Exp. Biol Med. 127, 177-182. 144 Ono, M., Katsuragi-Iwanaga, R, Kitazawa, T., Kamiya, N., Horimoto, T., Niikura, M., Kai, C., Hirai, K, and Mikami, T. (1992). The restriction endonuclease map of Marek’s disease virus (MDV) serotype 2 and collinear relationship among three serotype of MDV. Virology 191, 459-463. Olivo, P. D., Nelson, N. J., and Challberg, M. D. (1988). Herpes simplex virus DNA replication : The UL9 gene encodes an origin-binding protein. Proc. Natl Acad. Sci 85, 414-5418. Pari, G. S., and Anders, D. G. (1993). Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dep endent DNA replication. J. Viol 67, 6979-6988. Pari, G. S., Kacica, M. A, and Anders, D. G. (1993). Open reading fi'ame UL44, IRSl/TRSl, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis. J. Virol 67, 2575-2582. Payne, L. N., Frazier, J. A, and Powell, P. C. (1976). Pathogenesis of Marek’s disease. Int. Rev. Exp. Pathol 16, 59-154. Payne, L. N., and Rennie, M. (1976). Sequential changes in the numbers of B and T lymphocytes and other leukocytes in the blood in Marek’s disease. Int. J. Cancer 18, 510- 520. Payne, L. N., and Rennie, M. (1973). Pathogenesis of Marek’s disease in chicks with and without maternal antibody. J. Natl Cancer Inst. 51,1559. Polvino-Bodnar, M., Kiso, J., and Schafl‘er, P. A (1988). Mutation analysis of Epstein- Barr virus nuclear antigen 1 (EBNA-l). Nuclear Acids Res. 16, 3415-3435. Polvino-Bodnar, M., Orberg, P. K, and Schafl’er, P. A (1987). Herpes simplex virus type 1 oriL is not required for virus replication or for the establishment and reactivation of latent infection in mice. J. Virol 61, 3528-3535. Preston, C. M., Frame, M. C., and Campbell, M. E. M. (1988). A complex formed between cell components and an HSV structural polypeptide binds to a viral immediate early gene regulatory DNA sequence. Cell 52, 425-434. Preston, C. M., Cordingley, M. G., and Stow, N. D. (1984). Analysis of DNA sequences which regulate the transcription of a hereps simplex virus immediate-early gene. J. Virol 50, 708-444. 145 Preston C. M., and Tannahill, D. (1984). Effects of orientation and position on the activity of a hereps simplex virus irmnediate early gene far-upstream region. Virology 137, 439- 444. Rabkin, S. D., and Hanlon, B. (1991). Nucleoprotein complex formed between herpes simplex virus UL9 protein and the origin of DNA replication: inter- and intramolecular interactions. Proc. Natl Acad. Sci USA 88, 10946-10950. Rabkin, S. D., and Hanlon, B. (1990). Herpes simplex virus DNA synthesis at a preformed replication fork in vitro. J. Virol 64, 4957-4967. Rawiins, D. R, Miiman, G., Hayward, S. D., and Hayward, G. S. (1985). Sequence- specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA- 1) to clustered sites in the plasmid maintenance region. Cell 42, 859-868. Reisman, D., and Sugden, B. (1986). Transactivation of an Epstein-Barr virus viral transcriptional enhancer by the Ep stein-Barr viral nuclear antigen 1. Mol Cell. Biol 6, 383 8-3 846. Reisman, D., Yates, J., and Sugden, B. (1985). A putative origin of replication of plads derived from Epstein-Barr virus is composed of two cis-acting component. Mol Cell Biol 5, 1822-1832. Ripalti, A, Boccimi, M. C., Campanini, F., and Landini, M. P. (1995). Cytomegalovirus virus-mediated induction of antisense mRNA expression of UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene. J. Virol 69, 2047-2057. Roizman, B. (1992). The family Herpesviridae : An update. Arch. Virol 132, 425-449. Roizman, B. (1991). Herpesviridae: A brief introduction, p841-847. In B. N. Field, D. M. Knipe, R M. Chanock, J. L. Mehiick, B. Roizman, and R E. Shope (ed.), Virology. Raven press, New York. Roizman, B. and Sears, A E. (1991). Herpes Simplex virus and their replication, p850- 895. In B. N. Field, D. M. Knipe, R M. Chanock, J. L. Melnick, B. Roizman, and R E. Shape (ed. ), Virology. Raven press, New York Ross, L. J., Sanderson, M., Scott, S. D., Binns, M. M., Doel, T., Milne, B. (1989). Nucleotide sequence and characterization of the Marek’s disease virus homologue of glycoprotein B of the herpes Simplex virus. J. Gen. Virol 70, 1789-1805. Ross, L.J.N., and Miline, B., and Biggs, P. M. (1983). Restriction endonuclease analysis of Marek’s disease virus DNA and homology between strains. J. Gen. Virol 64, 2785- 2790. 146 Sambrook, J., Frisstsch, E. F., and Maiatis, T. (1989). "Molecular cloning : A Laboratory Manual ," 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Sato, H, Takimoto, T., Tanaka, S., Tanaka, J., and Raab-Traub, N. (1990). Concatemeiic replication of Epstein-Barr virus: structure of the termini in virus-producer and newly transformed cell lines. J. Virol 64, 5295-5300. Schat, K A (1985). Characteristics of the virus, p77-112. In L. N. Payne (ed.), Marek’s disease. Martinus Nijhofl; Boston. Sharma, J. M. and Coulson, B. D. (1977). Cell-mediated cytotoxic response to cells bearing Marek’s disease tumor-associated surface antigen in chickens infected with Marek’s disease virus. J. Natl Cancer Inst. 58, 1647-1651. Shek, W. R, Calnek, B. W., Schat, K A, and Chen, C. L. H (1983). Characterization of Marek’s disease virus-infected lymphocytes: discrimination between cytolytically and latently infected cells. J. Natl Cancer Inst. 70, 485-491. Silva, R F., and Barnett, J. C. (1991). Restriction endonuclease analysis of Marek’s disease virus DNA: Differentiation of viral strains and determination of passage history. Avian Dis. 35, 487-495. Skaliter, R, Makhov, A M, Griffith, J. D., and Lehman, 1. R (1996). Rolling circle DNA replication by extracts of herpes Simplex virus type l-infected human cells. J. Virol 70, 1132-1 136. Skaliter, R, and Lehman, I. R (1994). Rolling circle DNA replication in vitro by a complex of herpes Simplex virus type l-encoded enzymes. Proc. Natl Acad. Sci. USA 91, 10665-10669. Smith, J. A, and Pari, G. S. (1995). Expression of human cytomegalovirus UL36 and UL37 genes is required for viral DNA replication. J. Virol 69, 1925-1931. Smith, G. D., Zelink, V., and Ross, L. J. N. (1995). Gene organization in herpesvirus of turkeys: Identification of a novel open reading frame in the long unique region and a truncated homologue pp38 in the internal repeat. Virology 207, 205-216. Solomon, J. J., Witter, R L., Nazerian, K, and Burmester, B. R (1968). Studies on the etiology of Marek’s disease. I. Propagation of the agent in cell culture. Proc. Soc. Exp. Biol Med. 127, 173-177. Spencer, J. L, and Cahiek, B. W. (1970). Marek’s disease: application of imnnmofluoresence of detection of antigen and antibody. Am J. Vet. Res. 31, 345-358. 147 Spaete, R R, and Frenkel, N. (1982). The herpes simplex virus amplicon: a new eucaryotic defective-virus cloning amplifying vector. Cell 30, 295-304. Stabell, E. C., and Olivo, P. (1993). A tnmcated herpes Simplex virus origin binding protein which contains the carboxyl terminal origin binding domain binds to the origin of replication but does not alter its conformation. Nucleic Acids Res. 21, 5203-5211. Stillman, B. (1989). Initiation of eucaryotic DNA replication in vitro. Annu. Rev. Cell Biol 5, 197-245. Stinski, M. F. (1991). Cytomegalovirus and its replication, p929-950. In B. N. Field, D. M. Knipe, R M. Chanock, J. L. Melnick, B. Roizman, and R E. Shope (ed.), Virology. Raven press, New York Stow, N. D., Weir, H. M., and Stow, E. (1992). Analysis of the binding sites for the varicella-zoster virus gene 51 product within the viral origin of DNA replication. Virology 177, 570-577. Stow N. D., and Davison, J. (1986). Identification of a varicella-zoster virus origin of DNA replication and its activation by hereps simplex virus type 1 gene product. J. Gen. Virol 67, 1613-1623. Stow, N. D, (1984). Mutagenesis of a herpes simplex virus origin of DNA replication and its effect on viral interference. J. Gen. Virol 66, 31 Stow, N. D., and McMonagle, E. C. (1983). Characterization of the TRS/IRS origin of DNA replication of herpes Simplex virus type 1. Virology 130, 427-438. Stow, N. D. (1982). Localization of an origin DNA replication within the TRS/IRS repeated region of the herpes simplex virus type 1 genome. EMBO J. 1, 863-867. Su, W., Middleton, T., Sugden, B., and Echols, H. (1991). Proc. Natl Acad. Sci USA 88, 10870-10874. Summers, I-I., Barwell, J. A, Pfuetzner, R A, Edwards, A M., and Frappier, L. (1996). Cooperative assembly of EBNAl on the Epstein-Barr virus latent origin of replication. J. Virol 70, 1228-1231. Tamashiro, J. C., and Spector, D. H. (1986). Terminal structure and heterogeneity in human cytomegalovirus strain Ad169. J. Virol 59, 591-604. 148 Varnniza, S. L., Smiley, J. R (1985). Signals for site-specific cleavage of herpes Simplex virus DNA: maturation involves two separate cleavage events at sites distal to the recognition sites. Cell 41, 792-802. Vlaniy, D. A, and Frenkel, N. (1981). Replication of herpes Simplex virus DNA: localization of replication signals within defective virus genomes. Proc. Natl Acad. Sci. 78, 742-746. Weir, H M., and Stow, N. D. (1990). Two binding sites for the herpes Simplex virus type 1 UL9 protein are required for efficient activity of the oris replication origin. J. Gen. Virol 71, 1379-1385. Weir, H. M., Calder, J. M., and Stow, N. D. (1989). Binding of herpes Simplex virus type 1 UL9 gene product to an origin of viral DNA replication. Nucleic Acids Res. 17, 1409- 1425. Weller, S. K, Spadaro, A, Schafl‘er, J. E., Murray, A W., Maxam, A M., and Schafl‘er, P. A (1985). Cloning, sequencing, and fimctional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis. Mol Cell Biol 5, 930-942. Wells, R D. (1988). Unusual DNA structures. J. Biol Chem 263, 1095-1098. Wilson, M. R, and Coussens, P. M. (1991). Purification and characterization of infectious Marek’s disease virus genomes using pulsed field electrophoresis. Virology 185, 67 3-680. Witter, R L., Burgoyne, G. H, and Solomon, J. J. (1969). Evidence for a herpesvirus as an etiologic agent of Marek’s disease. Avian Dis. 13, 171-184. Wong, S. W., and Schafi‘er, P. A (1991). Elements in the transcriptiomal regulatory region flanking hereps simplex virus type 1 oriS stimulate origin function. J. Virol 65, 2601-2611. Wu C. A, Nelson, N. J., Mcgeoch, D. J., and Challberg, M. D. (1988). Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis. J. Virol 62, 43 5-443. Wysokenski, D. A, and Yates, J. L. (1989). Multiple EBNAl-binding sites are required to form an EBNAl-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus. J. Viol 63, 2657-2666. 149 Yates, J. L., and Camiolo, S. M. (1988a). , p197-205. In B. Stillman and T. Kelly (eds), Cancer cells Vol 6: Eucaryotic DNA replication. Cold Spring Harbor Laboratory press, Cold Spring Harbor, N. Y. Yates, J. L., and Camiolo, S. M. (1988b). Dissection of DNA replication and enhancer activation fimctions of Epstein-Barr virus nuclear antigen 1. Cancer cells 6, 197-205. Yates, J., Warren, N., and Sugden, B. (1985). Stable replication of plasmids derived fi'om Epstein-Barr virus in a variety of mammalian cells. Nature 313, 812-815. Yates, J. L., Warren, N., Reisman, D., and Sugden, B. (1984)., A cis-acting element from the Esptein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc. Natl Acad. Sci 81, 3806-3810. Webster, C. B., Chen, D., Horgan, M., and Olivo, P. D. (1995). The varicella-zoster virus origin-binding protein can substitute for the herpes simplex virus origin-binding protein in a transient origin-dependent DNA replication assay in insect cells. Virol 206, 65 5-660. zur Hansen, H. (1981). Oncogenic herpesvirus, p. 747-795. In J. Tooze (ed.), DNA tumor viruses. Cold Spring Harber Laboratory, Cold Spring Harber, N. Y. zur Hansen, H, O’Neill, F., and Freese, U. (1978). Persisting oncogenic herpesvirus induced by the tumor promoter TPA Nature 272, 373-375.