VIROLOGY

189, 304-3 16 (1992)

The DNA Sequence of Equine Herpesvirus-l ELIZABETH A. R. TELFORD,’

MOIRA S. WATSON, KATHRYN MCBRIDE, AND ANDREW J. DAVISON

Institute of Virology, University

of Glasgow,

Received February

Church Street, Glasgow,

7, 1992; accepted

G 11 5/R, United Kingdom

March 27, 1992

The complete DNA sequence was determined of a pathogenic British isolate of equine herpesvirus-1, a respiratory virus which can cause abortion and neurological disease. The genome is 150,223 bp in size, has a base composition of 56.7% G + C, and contains 80 open reading frames likely to encode protein. Since four open reading frames are duplicated in the major inverted repeat, two are probably expressed as a spliced mRNA, and one may contain an internal transcriptional promoter, the genome is considered to contain 76 distinct genes. The genes are arranged collinearly with those in the genomes of the two previously sequenced alphaherpesviruses, varicella-zoster virus, and herpes simplex virus type-l, and comparisons of predicted amino acid sequences allowed the functions of many equine herpesvirus 1 proteins to be assigned. o 1992 Academic PWSS. 1~.

INTRODUCTION

laghan, 1990). Thus, the EHV-1 genome is similar in arrangement to the genomes of pseudorabies virus (PRV) and varicella-zoster virus (VZV) (Ben-Porat et a/., 1979; Dumas eta/., 1981; Davison, 1984; DeMarchi et a/., 1990). VZV and PRV virions contain genomes with predominantly a single orientation of U,, but approximately 5% of genomes contain U, in the alternative orientation (Davison, 1984; DeMarchi et al., 1990). U, is present in a single orientation in EHV-1 virions, and the alternative orientation has not been detected to date. EHV-1 is classified on biological grounds as a member of the Alphaherpesvirinae, a subfamily of the herpesviruses which is typified by herpes simplexvirus type-l (HSV-1) and also includes VZV and PRV (Matthews, 1982). Evidence of a genetic relationship between EHV-1 and HSV was first provided by DNA hybridization studies (Davison and Wilkie, 1983). The collinear arrangement of related regions indicated that the viruses may share a common gene arrangement, and this was subsequently borne out by the mapping of several EHV-1 genes (Allen and Coogle, 1988; Robertson and Whalley, 1988; Grundy et a/., 1989; Guo et a/., 1989; Whalley eta/., 1989; Audonnet eta/., 1990; Guo et a/., 1990; Yalamanchili et a/., 1990; Elton et a/., 1991; Flowers et a/., 1991; Robertson et a/., 1991; Whalley eta/., 1991; Holden eta/., 1992). In this paper, we report the complete DNA sequence of a pathogenic British isolate of EHV-1, interpret its genetic content, and discuss the genetic relationships between it and other herpesviruses. The analysis described was first presented in July 1991 at the XVI International Herpesvirus Workshop.

Equine herpesvirus-l (EHV-1) is a pathogen of major economic importance in horses, causing respiratory disease, abortion, and neurological disorders (reviewed by Bryans and Allen, 1989). Primary infection of the upper respiratory tract of young horses results in a febrile illness which lasts for 8 to 10 days. Immunologically experienced mares may be reinfected via the respiratory tract without disease becoming apparent, so that abortion usually occurs without warning. The neurological syndrome is associated with respiratory disease or abortion and can affect animals of either sex at any age, leading to incoordination, weakness, and posterior paralysis. The EHV-1 genome has been characterized as a linear double-stranded DNA molecule with an estimated base composition of 56 or 57% G + C (Darlington and Randall, 1963; Soehner eta/., 1965). Restriction endonuclease and electron microscopic analyses have shown that the DNA molecule is approximately 150 kbp in size and may be described as two covalently linked components, L and S (Whalley et a/., 1981; Henry et a/., 1981; Ruyechan et a/., 1982). The S component comprises of an unique sequence (U,) flanked by a large inverted repeat (IR, and TR,). The two orientations of U, are present in equimolar amounts in virion DNA. The L component consists of an unique sequence (U,) flanked by a small inverted repeat (IR, and TR,) (Chowdhury et a/., 1990; Yalamanchili and O’Cal-

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304

DNA SEQUENCE

MATERIALS

AND METHODS

Growth of EHV-1 Equine dermal cells (NBL-6) were obtained from ICN Flow and grown as monolayers at 37” in minimal essential medium containing Earle’s salts, 2 mM glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 100 U/ml mycostatin, 1% (vol/vol) nonessential amino acids, and 10% (vol/vol) fetal calf serum. Strain Ab4 of EHV-1 was kindly supplied at passage 7 by Dr. N. Edington (Royal Veterinary College, London, UK). This strain has been passaged only in equine cells and originated from a clinical case showing paresis. It manifests all of the recognized pathogenic characteristics of EHV-1: abortion, neurological signs, viremia, and respiratory disease (Pate1 et al., 1982). The virus was plaque purified three times in NBL-6 cells using medium containing 1% (wt/vol) low melting point agarose. An initial virus stock at passage 11 was prepared by infecting a 75cm’ flask of NBL-6 cells with the final plaque. A working stock at passage 12 was then obtained by infecting ten 175-cm* flasks of NBL-6 cells with the initial stock and incubating the cells at 37°C until they detached from the surface. The cells were pelleted and the medium was centrifuged at 15000 rpm for 2 hr in a GSA rotor operating in a Sorvall RC5B centrifuge. The virus pellet was resuspended in 5 ml of medium. The titer of the working stock, designated EHV-1 strain Ab4p, was 2 X 1O7 PFU/ml. Preparation

of EHV-1 DNA

NBL-6 cells in ten 175-cm2 flasks were infected at 0.1 PFU/cell and incubated at 37” until they detached from the surface. Virus was pelleted from the medium as described above and purified twice by centrifugation on 5-55% (wt/vol) sucrose gradients as described by Dumas eta/. (1980). Purified virus was resuspended in TE (10 mM Tris-HCI pH 7.5, 1 mM EDTA) and incubated in the presence of 20 Kg/ml proteinase K, 0.5% (wt/vol) sodium dodecyl sulfate for 1 hr at 37”. Released EHV-1 DNA was then extracted with 1 :l (vol/ vol) phenol:chloroform, ethanol precipitated, and resuspended in TE. The yield of DNA, as determined by spectrophotometry at 260 nm, was approximately 15 pg. Sequencing

305

OF EQUINE HERPESVIRUS-

of EHV-1 DNA

Cloning and sequencing were carried out as described by Davison (1991 a). Fifteen micrograms of EHV-1 DNA was sonicated and then precipitated using polyethylene glycol to give random fragments 4001500 bp in size. The sheared ends of the fragments were repaired, and 0.5 pg was ligated to 0.2 pg bacteriophage M 13mpl9 RFI DNA which had been linear-

ized using Smal and treated with calf intestinal phosphatase. Ligated DNA was transfected into Escherichia co/i XLl-Blue cells (Stratagene) and clones containing inserts were detected by growth as clear plaques on lawns of E. co/i XL1 -Blue cells in the presence of isopropylthio-P-o-galactoside and 5-bromo-4chloro-3-indolyl-/3-p-galactoside. A total of 5 138 single-stranded DNA templates were prepared and sequenced using the dideoxynucleotide chain termination technology (Bankier and Barrell, 1989; Messing and Bankier, 1989). Reaction products were labeled using [35S]dATP (SJ 264, Amersham International) and the Klenow fragment of E. co/i DNA polymerase I and separated on 6% polyacrylamide, 8 NI urea buffer gradient gels using Model S2 DNA sequencing apparatuses and double-fine sharkstooth combs (Bethesda Research Laboratories). Gels were dried directly on Whatman 3MM chromatography paper and exposed to Kodak XSl film for 5 days. Data handling

and analysis

Autoradiographs were read using a Summagraphics Digitizer and the database was compiled using version 1 .O of Staden’s sequence assembly program (SAP) running in a DEC MicroVax II computer. SAP incorporates programs from the Staden DB system (Staden, 1987). The database contained 69 contigs after all of the gel readings had been entered. The 64 contigs containing single gel readings were removed from the database on the assumption that they arose from contaminating equine cellular DNA rather than from EHV-1 DNA. These clones represented only 1.2% of the total; that is, the EHV-1 DNA was 98.8% pure. The remaining 5 EHV-1 contigs were then joined and ambiguous regions were resolved using a set of 26 oligonucleotide primers. The sequence was edited by reference to autoradiographs and analyzed using programs from the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin (Devereux et al., 1984). RESULTS AND DISCUSSION Genome organization Two alphaherpesvirus genomes have been sequenced previously, those of VZV (Davison and Scott, 1986) and HSV-1 (McGeoch et al., 1988). The EHV-1 genome sequence is thus the third to be reported. We decided to sequence EHV-1 strain Ab4 because it possesses the full range of pathogenic features displayed by EHV-1. Like Ab4, the plaque-purified clone Ab4p which was actually sequenced has been passaged only in equine cells. Moreover, Ab4p possesses the

306

TELFORD ET AL

pathogenic properties of Ab4 (J. S. Gibson, I. D. Slater, and H. J. Field, submitted for publication). The DNA sequence was obtained via direct ligation of EHV-1 virion DNA fragments into a sequencing vector, without preliminary subcloning into other vectors. Therefore, for the reasons discussed by Davison (199 1a), the finished sequence is as close to that of the majority population of Ab4p genomes as can be obtained using current technology. Owing to considerations of space, the sequence is not given in this paper. It has been deposited with the GenBank Data Library under Accession No. M86664. The DNA sequence was derived from approximately 1.2 X 1O6 nucleotides of data and about 97% was determined on both strands. Thus, each base pair was determined an average number of nine times. By virtue of the identity of IR, and TR,, the final database was 137,509 bp in size (i.e., the full genome length minus the length of TR,). The complete sequence was established by reference to clones containing the IR$U, or TR$U, junctions. It contains 150,223 bp and the sizes of its components are: U,, 1 12,870 bp; TR,/IR,, 32 bp; Us, 11,861 bp; and IRJTR,, 12,714 bp. The G + C content is 56.7% when averaged over the genome, but, as in VZV and HSV-1 (Davison and Scott, 1985; McGeoch et a/., 1986) is significantly higher in IRJTR, (67%) than U, (55%) or U, (52%). The genome ends are identical to those published for strains KyA (Yalamanchili and O’Callaghan, 1990) and Austrian IV (Chowdbury et a/., 1990). The HSV-1 and VZV genomes possess an unpaired nucleotide at the 3’ end of each DNA strand (Mocarski and Roizman, 1982; Davison, 1984), but a similar feature would not have been apparent from our sequence because the ends of random fragments were repaired prior to cloning. The first 87 bp at the left genome terminus are repeated in inverse orientation at nucleotides 1032 to 11 18. The intervening 945 bp contain, in addition to unique sequences, two sets of tandem reiterations of short sequence elements. A 17-bp element is repeated 13 times and once partially and an 18-bp element is reiterated 19 times and once partially (Table 1). This organization is similar to that reported for strains KyA (Yalamanchili and O’Callaghan, 1990) and Austrian IV (Chowdhury et al,, 1990) except that the copy numbers of reiterated elements differ and the 18-bp element is only 15 bp in size in Austrian IV. The EHV-1 genome contains three distinct candidates for origins of DNA replication. Two (ori, and ori,, Fig. 1) contain a 9-bp sequence identical to that recognized by the origin-binding protein encoded by HSV-1 gene UL9, adjacent to an A + T-rich palindrome. The genome contains two copies of oris in IR, and TR, at positions corresponding to or& of HSV-1 and VZV

(Stow, 1982; Stow and Davison, 1986). EHV-1 oris has been shown to contain c&acting sequences essential for DNA replication (Baumann et a/., 1989). EHV-1 ori, has been identified previously in strain HVS25A by Robertson et al. (1991) and is located between genes 39 and 40. It has not yet been shown to function as an origin. In contrast, HSV-1 or& is located between the genes corresponding to EHV-1 genes 30 and 31 (Gray and Kaerner, 1984) and, moreover, VZV lacks an oriL (Stow and Davison, 1986). A functional origin has been mapped recently near the left terminus of the PRV genome (Kuperschmidt et al., 1991). The structure of the L terminus of the PRV genome is similar to that of EHV1, comprising an 82-bp inverted repeat separated by 352 bp which contain potential UL9- and NFl -binding sites. Neither of these elements was shown in an in vitro assay to be essential for origin function, but their removal did have a detrimental effect (Kuperschmidt et a/., 1991). The ability of EHV-1 sequences at the left terminus of the genome to act as an origin has not yet been tested, but we note that a consensus ULS-binding site is present in the 945-bp sequence between the 87-bp inverted repeats. The EHV-1 genome contains a total of 20 sets of tandem reiterations: eight in U,, two in U,, and five each in TR, and IR, (Fig. 1, Table 1). Each contains several copies of a short sequence element plus a partial copy. Most are located in regions which are not predicted to encode proteins, but two are within the protein-coding regions for genes 24 and 71. The HSV1, VZV, and Epstein-Barr virus (EBV) counterparts of EHV-1 gene 24 also contain repeat sequences in approximately similar locations, but neither the reiterated element nor the encoded protein sequence is conserved. Nevertheless, this single example of related genes containing reiterations suggests that the repeated amino acid sequence has a role in protein structure.

Gene arrangement Potential protein-coding regions in the EHV-1 genome were identified first by searching for ATG-initiated open reading frames (ORFs) containing more than 99 codons. General features of gene layout in other alphaherpesvirus genomes were then used as additional criteria: ORFs are arranged compactly on both strands, do not overlap extensively, and either terminate near candidate polyadenylation sites (containing AATAAA or minor variants ATTAAA and AGTAAA) or are transcribed as members of 3’-coterminal families. The codon and base compositional biases of the sequence were not sufficiently pronounced to aid in the identification of protein-coding regions. The similarities of encoded protein sequences to those of other alpha-

DNA SEQUENCE

307

OF EQUINE HERPESVIRUSTABLE 1

REITERATEDELEMENTSIN THE EHV-1 STRAINAB~P GENOME Location (nucleotides) 105-339 383-728 44,221-44,471 44.974-45,161 45.197-45,275 73,870-74,17-l 108,963-l 09,022 112,349-l 12,476 113,091-l 13,349 (149,809-l 50,067)* 1 13,350-l 13,796 (149,362-149,808) 121,107-121,220 (141,938-142,051) 121,290-121,366 (141,792-141,868) 123,828-l 24,179 (138,979-l 39,330) 129,645-l 29,757 129,780-l 29,871

Element size (nucleotides)

Partial element size (nucleotldes)

17 18 63

14 4 62

33 12 32 18 13

23 7 20 6 11

15

4

12

3

7

2

(CCCAGCTCCGGCGACCCCGGCCCAG)3

25

2

(AGGCCGAGCGGGGGAGAGCGGTAGGGG)13 (ACGACCACAGCAGCA) 7 (ACCACCGCGGCTACC) 6

27 15 15

1 8 2

Protein-coding region -a Gene 24 Gene 24 Gene 24

Sequence

and copy number

(AGGCCACGCCCACTGGG) 13 (TGGGGCTCACTGCTATGC) 19 (CTCCTCCCACTCTXCACCCGCTCCCCCTCTGC CCCAATCCACTTCAAAGGCCGCCAGCGGCC) (CCCCCGCCGCGGCCCCGGCCAAATCTGCGGCGG)5 (GGCCAAGGACCA) 6 (GCGGGAGCGAGGGCTGCTGCGGCGGCGGCGCG)9 (GCTAGCGCTAACGCTAGG) 3 (TGCCCTAGCCCCC) 9 (GGAAGGGGAGGAGCA) (CCATCAACCCGC)

17

37)

(GGTGGTC) 16

Gene 7 1 Gene 71

a Not located in a predicted protein-coding ’ Inverse sequence in TRs.

3

region.

herpesviruses were also analyzed. Sequences between predicted protein-coding regions were then assessed for coding potential by comparing small ORFs with corresponding regions in the VZV genome. The arrangement of 80 ORFs likely to encode protein is shown in Fig. 1. ORFs 64, 65, 66, and 67 are duplicated in TR, and IRS, and ORFs 44 and 47 are homologous to the two exons of HSV-1 gene UL15, which is expressed as a spliced mRNA (Costa et a/., 1984; DoIan et al., 1991). HSV-1 gene UL26 is expressed as a full-length mRNA from an upstream promoter and as a 3’-coterminal truncated mRNA which is initiated internally (Liu and Roizman, 1991 a). The protein specified by the smaller overlapping gene (termed gene UL26.5) is identical to the carboxy-terminal portion of the gene UL26 protein. A similar pattern of expression is likely to pertain to EHV-1 and VZV, since initiation codons are located appropriately. The overlapping genes are termed 35 and 35.5 in EHV-1 and 33 and 33.5 in VZV (Table 2, Figure 1). Thus, the EHV-1 genome contains 76 distinct genes. The transcript layout of the EHV-1 genome, as predicted from the position of potential polyadenylation sites, is largely similar to those proposed for VZV (Davison and Scott, 1986) and HSV-1 (McGeoch et a/., 1988). Differences may reflect real divergence or lack of functionality of certain predicted polyadenylation sites.

Four sizeable regions of the EHV-1 genome appear not to encode protein. The first is approximately 1000 bp in size and is located at the left end. It contains TR, and sequences which may be involved in cleavage of genomes from replicated concatameric DNA (Chowdhury et al., 1990; Yalamanchili and O’Callaghan, 1990). The second is approximately 1500 bp in size and is located between genes 62 and 63. A noncoding region of approximately the same size is present at a corresponding position in the VZV genome. Comparison between these regions of the EHV-1 and VZV genomes at the levels of their DNA sequence and protein-coding potential revealed no clue as to function. The third region, approximately 2000 bp in size, is located between genes 63 and 64 and includes the lRL/ IRS junction. It contains three sets of tandem reiterations (Table 1). Comparison of this region with that published for EHV-1 strain KyA (Grundy et a/., 1989) revealed that the copy numbers of the repeated elements are different. The three elements are reiterated 9, 17, and 37 times in Ab4p and 23, 12, and 20 times in the KyA plasmid clones sequenced. Also, a 54-bp sequence which is reiterated 20 times in KyA is present as a single copy in Ab4p. The fourth region not encoding protein is located in IRJTR, between genes 64 and 65; it is approximately 2500 bp in size and contains oris.

308

TELFORD

29

75

32

31

30

57

68

69

70

90

63

58 59606162

71

3435.535

85

80

56

33

72

73

74 7576

67

66

36

3738

39

40

95

100

64

65

65

64

66

67

FIG. 1. Predicted EHV-1 gene arrangement. The genome is shaded, the thinner and thicker portions denoting the unique regions (U,, U,) and Inverted repeats (TR,, IR,. TR,, IR,), respectively. The scale is in kbp. Protein-coding regions are shown as open arrows with gene nomenclature below. ORF 35.5 is shown by a thinner arrow to reveal the region of overlap with ORF 35. ORFs 44 and 47, represented as filled arrows, are probably expressed as a spliced mRNA. Vertical arrows indicate candidate polyadenylation sites in the appropriate strand. The locations of reiterations (B) and candidate origins of DNA replication (0) are indicated above the genome.

DNA sequences have been reported for limited regions in the genomes of various EHV-1 strains. Most differ to some extent from the Ab4p sequence, as summarized in Table 3. Many differences probably represent genuine strain variation, but some, particularly those causing frameshifting, may have arisen either from mutations in plasmid subclones or from sequencing errors. We are confident that the Ab4p sequence is correct in these regions. It is clear that the hamsteradapted strain KyA lacks at least five genes (genes 1, 2, 73, 74, and 75). Gene functions The precise locations of EHV-1 ORFs and characteristics of their primary translation products are listed in

ET AL.

Table 2. In the absence of experimental data, it is assumed that translation starts at the first ATG in each ORF. Homologues of EHV-1 proteins in HSV-1 and VZV were identified using FastA with a word size of 2 (Pearson and Lipman, 1988) and are listed in Table 2. Scores in excess of 100 were considered to indicate significant homology. EHV-1 genes are closely collinear with their counterparts in HSV-1 and VZV. The properties or functions of EHV-1 proteins are predicted in Table 2 on the basis of present knowledge of HSV-1 gene function. The primary translation products of eight EHV-1 genes share features characteristic of class I membrane proteins: a hydrophobic signal sequence near the amino terminus for translation of the mRNA on membrane-bound ribosomes and a carboxy-terminal hydrophobic sequence followed by basic residues which acts as a transmembrane anchor. Six EHV-1 genes encoding such proteins have been mapped previously on the basis of positional collinearity and homology of their products to HSV-1 glycoproteins. They include genes encoding counterparts of HSV-1 gB (Whalley et al., 1989; Guo eT al., 1990) gC (Allen and Coogle, 1988; Guo et al., 1989), gD (Audonnet et al., 1990; Flowers et al., 1991; Whalley et a/., 1991; Whittaker et a/., 1992), gH (Robertson et a/., 199 l), gE, and gl (Audonnet et al., 1990; Elton et al., 1991). Two additional genes are likely to encode membrane glycoproteins. Gene 70 corresponds in location to HSV-1 gene US4, which encodes gG, but the encoded proteins lack significant homology (Table 2; FastA score = 24). The gene 70 protein is related, however, to HSV-2 gG (McGeoch et al., 1987; FastA score = 184) and its counterpart in PRV, gX (Rea et al., 1985; FastA score = 441). HSV-2 gG is larger (699 residues) than its HSV-1 counterpart (238 residues) and appears to comprise sequences homologous to HSV-1 gG plus an additional region near the amino terminus (McGeoch et a/., 1987). HSV-2 gG, PRV gX, and the EHV-1 gene 70 protein are related in the additional region. Thus, the similarity between the EHV-1, PRV, and HSV-2 proteins is most simply accounted for by evolution of their genes from an alphaherpesvirus gene which was present before HSV, PRV, and EHV-1 diverged, with loss of the near amino-terminal region from HSV-1 after its divergence from HSV-2, as proposed by McGeoch et al. (1987). The amino acid sequence of the glycoprotein predicted to be encoded by EHV-1 gene 71 is shown in Fig. 2. It possesses a single putative asparagine-linked glycosylation site. It also contains an extensive region encoding a high proportion of serine and threonine residues, part of which is encoded by two sets of tandem reiterations (Table 1). The region between residues 22

DNA SEQUENCE

309

OF EQUINE HERPESVIRUS-

TABLE 2 CHARACTERISTICSOFEHV-1 PROTEINS Gene

Star?

Stopb

Codons

M,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 35.5 36 37 38 39 40 41 42 43 44; 471 45 46 48 49 50 51 52 53 54 55

1,298 2,562 2,841 4,249 5,874 7,042 10,301 10,300 12,115 12,084 12,549 13,505 15,317 18,083 21,146 22,851 24,234 25,696 26,262 28,859 31,276 32,916 33,292 36,588 47,311 48,230 48,857 48,763 50,618 55,184 55,453 59,243 61,432 64,578 67,093 66,142 68,975 69,897 69,910 71,192 76,224 76,793 77,703 82,083 84,319 88,917 84,480 86,620 88,947 89,369 91,305 92,783 94,471 94,389 97,173 100,331

1,906 1,945 3,614 3,647 4,462 6,011 7,056 11,037 11,135 12,386 13,463 14,944 17,932 20,326 20,487 21,445 23,029 24,479 27,755 27,894 28,904 31,519 36,354 46,853 46,952 47,403 48,369 50,625 51,598 51,522 59,082 61,570 64,374 65,060 65,153 65,153 67,212 69,079 70,968 73,738 74,632 77,512 81,833 83,027 83,148 87,885 86,600 87,732 89,900 91,153 92,831 93,007 93,119 97,052 99,323 99,420

202 205 257 200 470 343 1081 245 326 100 304 479 871 747 219 468 401 405 497 321 790 465 1020 3421 119 275 162 620 326 1220 1209 775 980 160 646 329 587 272 352 848 530 239 1376 314 734

21,671 23,398 28,021 22,379 51,318 38,042 118,956 26,364 35,207 10,801 33,239 53,644 96,966 82,305 23,798 50,887 43,203 45,084 56,540 36,015 88,394 51,304 111,605 367,061 13,596 30,679 17,993 67,297 36,524 135,949 129,976 85,308 109,800 17,305 68,576 34,679 63,689 29,186 38,748 92,837 57,912 26,534 152,175 33,840 81,074

14(365) 15(153) 16(185) 17(876) 18(1053) 19(1992) 20(809) 21(1314) 22(2243) 23(114) 24(620) 25(231) 26(1499) 27(198) 28(3130) 29(3283) 30(1927) 31(1980) 32(158) 33(785) 33.5[86] 34(1138) 35(438) 36(604) 37(889) 38(777) 39(334) 40(3935) 41(956) 45/42(2222)

706 370 317 594 508 74 450 887 716 303

76,316 40,800 35,814 65,244 56,064 8,408 49218 97,270 77.769 33,852

43(1030) 44(739) 46(310) 47(787) 48(735) 491471 50(607) 51(1933) 52(898) 53(586)

VZV c0unterpaf-F

HSV-1 counterpart”

Properties

or functrons of HSV-1 proteinsd

1WI 2(163) 3(263) 4(436) 5(666) 6(1921) 7(453) 8(292) 9A( 113) 9(257) 1O(838) 1 l(397) 12(987)

UL55(181) UL54(677) UL53(544) UL52(1912) UL51(369) UL50(299) UL49A[38] UL49(281) UL48(531) UL47(280) UL46(502) UL45[46] UL44(108) UL43[45] UL42(101) UL41(706) UL40(899) UL39(1600) UL38(762) UL37(1733) UL36(2325) UL35[72] UL34(565) UL33(254) UL32(1393) UL31(852) UL30(3432) UL29(2852) UL28(1804) UL27(2020) UL26(790) UL26.5(215) UL25(1255) UL24(380) UL23(590) UL22(454) UL21(482) UL20(133) UL19(4074) UL18(724) UL15(2016) UL17(1005) UL16(491) UL14(211) UL13(638) UL12(793) UL11[43] ULlO(493) UL9(1587) UL8j532) UL7j376)

Transcriptional activator Membrane glycoprotein DNA helicase/primase complex Deoxyuridine triphosphatase Possrble transmembrane protein Tegument protein Tegument protern Tegument protein Virion protern Membrane glycoprotein (gC) Multiply hydrophobic protein DNA polymerase processivity factor Host shutoff virion protern Ribonucleotrde reductase (RR2) Ribonucleotrde reductase (RRl) Capsid protern Tegument protern Capsid protern Possible vlnon protein Probable vlnon protern DNA polymerase ssDNA binding protean Probable vlnon protein Membrane glycoprotern Protease Capsrd assembly Virion protern

(gB)

protein

Thymidine klnase Membrane glycoproteln

(gH)

Multiply hydrophobic protein Major capsid protean Capsid protein Possible DNA packaging protein

Vlnon protein klnase Deoxyribonuclease Mynstylated won protein Multiply hydrophobic protein Onbinding protein DNA hellcaseiprimase complex

TELFORD

310

ET AL.

TABLE 2-Continued Gene

Start”

stop*

Codons

n/r,

56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

102,390 102,374 105,069 106,415 107,115 108,143 108,802 111,984 118,590 121,367 122,861 125,193 126,274 126,410 127,680 129,096 131,432 132,898 134,405 136,054 136,782

100,129 105,019 105,746 105,876 106,477 107,205 108,146 110,386 114,127 122,248 123,571 124,375 125,018 127,558 128,915 131,489 132,790 134,172 136,057 136,446 137,441

753 881 225 179 212 312 218 532 1487 293 236 272 418 382 411 797 452 424 550 130 219

83,988 99,448 24,286 19,451 23,681 34,776 24,424 58,627 154,859 32,115 25,032 30,101 46,786 42,541 45,267 80,338 51,097 46,390 61,180 14,854 22,417

VZV counterpaiY 54(1665) 55(2877) 56(586) 57[24] 58(532) 59(755) 60(151) 61(259) 62(1687) 63(323) 64( 146)

66(522)

67(228) 68(220) 65[74]

HSV-1 counterpartC UL6(1065) UL5(2321) UL4(376) UL3(432) UL2(757) ULl(lO9) IEl lO(l40) lEl75(1486) USl(lO3) US10(129)

Properties

or functions

of HSV-1 proteins“

Possible virion protein DNA helicase/primase complex

Uracil-DNA glycosylase Membrane glycoprotein (gL) Transcriptional activator Transcriptional activator In vitro host-range factor Vinon protein

US2(301) US3(418) US4[25] US5[24] USS[Sl] US7(177) US8(196) -

Protein kinase Membrane glycoprotein Membrane glycoprotein Membrane glycoprotein Membrane glycoprotein Membrane glycoprotein

US9(1 10)

Tegument

(gG) (gD) (gl) (gE)

protein

a Location of first base or its complement in first ATG, except for ORF 44, where first base in exon is given. * Location of third base or its complement in stop codon, except for ORF 47, where last base in exon is given. ’ Positional counterparts are listed regardless of the degree of amino acid sequence conservation. Genes lacking positional counterparts are indicated by hyphens. FastA scores greater than 100 are shown in parentheses and those less than 100 are shown in square brackets. d Gene functions are derived from McGeoch (1989) and Davison (1991 b), with additional data for the following HSV-1 genes: UL53, Debroy et al. (1985) Ramaswamy and Holland (1992); UL49, Elliott and Meredith (1992); UL47, McLean et al. (1990); UL45, H. Marsden (personal communication); UL42, Gottlieb et a/. (1990); UL35, M. Davison, A. Davison, and F. Rixon, unpublished data; UL26. Liu and Roizman (1991 b), Preston et al. (1992); UL26.5, Rixon et al. (1988) Liu and Roizman (199la); UL15, Davison (1992); UL13, Cunningham et al. (1992); ULl, Hutchinson et al. (1992)

and 465 is composed of 879/o serine and threonine residues and is likely to be heavily 0-glycosylated. Thus although the predicted Mr of the gene 71 protein is 80,338 (Table 2) the apparent n/l, on SDS-PAGE is likely to be very much greater. EHV-1 gene 71 corresponds in position to HSV-1 gene US5, which encodes a putative HSV-1 glycoprotein containing a single potential asparagine-linked glycosylation site (McGeoch et al,, 1985). The 92 residue US5 protein is, however, much smaller than the EHV-1 gene 71 protein and no similarity was detected between the amino acid sequences. Interestingly, HSV-2 gG (the counterpart of the EHV-1 gene 70 protein) is heavily 0-glycosylated (Serafini-Cessi et al., 1985) and has an extended region containing a high proportion of serine and threonine residues which is presumed to contain most of the glycosylation sites (McGeoch et a/., 1987). It has been proposed that HSV-2 gene US4, which encodes gG, is related to HSV-2 genes US6 and US7 and that these genes have evolved by duplication and divergence (McGeoch, 1990). Comparison of the encoded proteins from HSV-1, HSV-2 and PRV revealed that amino acids in a region containing several

cysteine residues are conserved. The distinctive pattern of cysteine residues characteristic of these proteins is observed in the EHV-1 gene 70, but not the gene 71, protein. In addition to class I membrane glycoproteins described above, EHV-1 encodes several other glycosylated proteins associated with the virion. These include the gene 13 (Whittaker et al., 1991), gene 28 (G. Whittaker and D. Meredith, personal communication), and gene 52 (D. Meredith, personal communication) proteins. EHV-1 gene 12 is the counterpart of HSV-1 UL48 and VZV gene 10. The HSV-1 UL48 protein is a structural component of the virion which also acts as a transactivator of HSV-1 immediate early (IE) promoters (Campbell et a/., 1984) by virtue of an acidic domain at the carboxy terminus (Triezenberg et a/., 1988). Functional comparisons of this protein with its VZV counterpart have shown that the latter lacks an acidic tail (Dalrymple et a/., 1985) and does not transactivate (McKee et a/., 1990). The EHV-1 gene 12 protein also lacks a carboxy-terminal acidic domain and therefore also may not act as a transactivator. In contrast to

DNA SEQUENCE

311

OF EQUINE HERPESVIRUS-

TABLE 3 COMPARISONOF THE EHV-1 STRAINAEI~P DNA SEQUENCEWITH PREVIOUSLYPUBLISHEDEHV-1 SEQUENCES

Reference Chowdhury

EHV-1 strain

et a/. (1990)

Yalamanchili

and O’Callaghan

Yalamanchill

et a/. (1990)

Austrian

(1990)

Ab4p sequence (nucleotides)

IV

l-1,189

KYA

l-2,864

KYA

2,674+4,858

Guo et al. (1989)

KyT43 1

21,ll

Allen and Coogle (1988)

KyT43 1

21,210-22,974

Whalley et al. (1989)

HVS25A

60,482-64,766

Guo et al. (1990) Robertson and Whalley (1988)

KyT431 HVS25A

61,133p64,483 68,815-71,959

Robertson

HVS25A

70,918%74,381

et a/. (1991)

l-22,974

Grundy et a/. (1989)

KYA

112,360-l

19,577

Holden et al (1992)

KYA

120,538-l

22,533

DIfferencesa 4C(l) 398 D (3) 653 D (72) 803 D (1) 839 C (1) 941 D (1) 1,115C(1) 89 C (1) 271 D (68) 653 D (72) 980 C (1) 1,203 C (1) 1,242 C (1) 1,350 D (1283) 2,702 C (1) 2,702 C (1) 3,583 D (1) 22,279 c (1) 22,387 C (1) 22,279 c (1) 22,387 C (1) 62,206 D (1) 62,234 I (1) 64,650 D (1) 64,668 D (1) 61,199 c (1) 68,826 I (1) 69,528 D (1) 69,531 I (1) 72,760 C (1) 73,953 D (224) 112,477 I (182) 112,530 I (5) 112,54OC(l) 112,741 I (1) 112,937 I (1026) 112,952 c (1) 113,010 c (35) 113,045 I (21) 113,275 D (75) 1 13,593 D (204) 114,891 C (1) 1 15,399 c (1) 116,795 C (1) 117,518 C (1) 117,880 C (1) 118,374 C (1) 1 18,400 C (1) 1 18,802 C (1) 119,254 c (1) 120,568 D (2) 120,631 D (1) 120,727 C (1) 120,759 c (1) 120,802 C (1) 120,935 c (1) 120,958 C (1) 121,221 I (7) 121,367 I (250)

Effect on coding potential* None None None None None None None None None None None None None ORFs 1 and 2 deleted None None Frameshift (3) Silent (16) Silent (16) Silent (16) Silent (16) Frameshift (33) Frameshift (33) Frameshift (34) Frameshlft (34) None Frameshift (36) Frameshift (37) Frameshift (37) Silent (39) None None None None None None None None None None None R to G (64) F to L (64) Silent (64) I to P (64) Silent (64) T to A (64) A to V (64) None None None None None None None None None None None

TELFORD

312

ET AL.

TABLE 3-Continued

Reference

EHV-1 strain

Ab4p sequence (nucleotides)

Baumann et al. (1989)

KYA

120,693-l

Whalley et al. (1991)

HVS25A

131,428-132,836

Flowers et a/. (1991)

KYA

130,922-137,011

Elton et a/. (1991)

Abl

132,305-137,011

Audonnet

KYD

130,609-137,011

et al. (1990)

20,898

Differences’

Effect on coding potentia?

120,727 C (1) 120,759 c (1) 120,802 C (1)

None None None

132,225 C (1) 132,341 C (1) 132,549 c (1) 132,719 D (2) 132,81 1 D (3,859) 136,119C(2) 136,532 C (1) 130,624 D (1) 130,708 D (1) 130,865 C (2) 130,901 c (1) 131,035 c (1) 131,121 C (1) 131,262 D (1) 134,398 C (2) 134,935 I (6) 135,035 c (2) 136,087 I (1) 136,109 D (1) 136,121 D (1) 136,166 D (1) 136,168 D (1) 136,170 D (2) 136,345 C (1) 136,922 D (1) 136,938 C (2)

Q to L (72) L to V (72) A to V (72) Frameshift (72) ORFs 73, 74 and 75 deleted KR to NG (75) None Frameshift (7 1) Frameshift (71) NATO KP (71) Silent (71) VtoE(71) D to H (71) Frameshift (71) None Insertion of QP (74) A to R (74) Frameshift (75) Frameshift (75) Frameshift (75) Frameshift (75) Frameshift (75) Frameshift (75) Introduces stop codon (75) Frameshift (76) A to R (76)

a The first number is the location in the Ab4p sequence; I, D. and C indicate an insertion, deletion, or change, with respect to the Ab4p sequence, with the number of nucleotides affected in parentheses. * The affected gene is shown in parentheses. Amino acid changes are indicated in single letter code, with the Ab4p residue shown first. c Austrian IV has a deletion of 3 bp at nucleotide 398 of the Ab4p sequence (i.e., the last 3 bp of an 18.bp reiterated element). There are 15 copies of the element in Austrian IV. and 3 bp are also deleted at every 16th nucleotide from 401-650.

HSV-1, EHV-1 expresses a single gene (gene 64) under IE conditions which encodes a major transcriptional regulatory protein (Gray eta/., 1987; Smith et a/., 1992). EHV-1 gene 10 is clearly homologous to an 87 codon ORF located between VZV genes 8 and 9 (Fig. 3). We have denoted this VZV gene as gene 9A in Table 2, since it is predicted to be 3’-coterminal with gene 9 but is not translated as a truncated form of another VZV protein (as is proposed for VZV genes 33 and 33.5). The primary translation products of VZV gene 9A and EHV-1 gene 10 have hydrophobic domains near the amino and carboxy termini suggesting a transmembrane location, but lack potential asparagine-linked glycosylation sites. A 91 codon ORF located between HSV-1 genes UL49 and UL50, designated UL49A in Table 2, is positionally equivalent to VZV gene 9A

(Barker and Roizman, 1992) and EHV-1 gene 10. Small ORFs are also located in appropriate regions of the HSV-2, EBV and human cytomegalovirus (HCMV) genomes; the amino acid sequences of the encoded proteins, like that specified by HSV-1 UL49A, are not convincingly related to the EHV-1 gene 10 and VZV gene 9A proteins but have strikingly similar hydrophobicity profiles (B. Barnett, A. Dolan, E. Telford, A. Davison, and D. McGeoch, unpublished data). Genome evolution The gene layouts of the three sequenced alphaherpesviruses are very similar, but EHV-1 proteins are in general more closely related in amino acid sequence to their VZV than to their HSV-1 counterparts (Table 2).

DNA SEQUENCE

1

OF EQUINE HERPESVIRUS-

MGFIYARKLLLC"AVSIYAIGSTTTTETTTSSSSTSGSGQSTSSGTTNSS

51

101

PTSTSTETTTTTPTASTTTPTTTTAAPTTAATTTAVTTAATTAT ...... .... ...... .

151

201

................. .... . ... ... ...... .... TMTTTAATTTAATTTAATTTAATTSSATTSSATTMTTTAATTTAATTTAATTT

251

.. ... AATTTAATTTGSPTSGSTSTTGASTSTPSASTATSATPTSTSTSAAATTS

301

351 401

TESDSTDSSTVPTTGTESITESSSTTEASTNLGSSTYESTFALETPDGNT

451

TSGNTTPSPSPRTPSFADTQQTPDNGVSTQHTTINDHTTINDHTTANAQKHAGHHR

501

GRAGGRRGSPQGGSHTTPHPDRLTPSPDDTYDDDTNHPNGRNNSIEWPQ

551

LPPDRPIIELGVATLRKNFMEASCTVETNSGLAIFWKIGN

601

: : THTRLMRNGVPVYALVSTLRVPWLNVIPLTKITCAACPTNLVAGDGVDLN

651

S;TTKSTTIP;PGQQRTHIFFSAKGDRA"&TSEL"SQPTITWS"GSDRL

701

RNDGFSQTWYGIQPGVCGILRSEVRIHRTTWRFGSTSKDYLCEVSASDSK

751

TSDYK"LPNAHSTSNFAL"AATTLT"TILCLLCCLYCMLTRPRAS"Y

***

: :: : FIG. 2. Predicted ammo acid sequence of the EHV-1 gene 71 protein. Hydrophobic amino- and carboxy-terminal domains are underlined and cysterne residues are indicated by colons. A single potential asparagrne-linked glycosylation site is denoted by asterisks. The domain which is rich in serine and threonine residues is delimited by angle brackets, and regrons encoded by two sets of tandem reiteratrons are indicated by dots. Residue numbers are given on the left.

According to the criteria used in constructing Table 2, only three EHV-1 genes (genes 1, 67 and 75) lack positional and sequence counterparts in VZV or HSV-1. The gene 1 protein, however, has a very similar hydrophobicity profile to the gene 2 protein, including a carboxy-terminal hydrophobic domain suggestive of an association with membranes. The two proteins are also similar in size and share local sequence similarities. Genes 1 and 2 are encoded on opposing DNA strands and thus it is likely that they have arisen by gene duplication and inversion events. VZV gene 1 occupies an equivalent position to EHV-1 gene 2 and the VZV protein shares a similar hydrophobicity profile and a weak sequence relationship with the EHV-1 gene 1 and 2 proteins. EHV-1 gene 67 specifies a 272 residue protein in IRJTR,. Gene 75 encodes a small protein and was first reported for EHV-1 strain Abl by Elton et a/. (1991). A homologous coding region is present in the corresponding region of the genome of EHV-4 strain

313

1942, but is divided by a frameshift mutation (Cullinane et al., 1988). Four EHV-1 genes (genes 15, 51, 59, and 72) lack amino acid sequence homologues in VZV and HSV-1 but have positional counterparts in VZV or HSV-1 to which they are probably related evolutionarily. EHV-1 gene 15 corresponds in position to HSV-1 UL45 and each encodes a protein which possesses a hydrophobic amino-terminal domain. The corresponding region of the VZV genome contains a larger unrelated gene (gene 13) on the opposing strand which encodes thymidylate synthase (Thompson et a/., 1987). EHV-1 gene 51 corresponds in position to VZV gene 49 and HSV-1 gene ULll; each encoded protein contains a potential myristylation site at the amino terminus. EHV1 gene 59 corresponds in position to VZV gene 57, and the encoded proteins are markedly hydrophilic. EHV-1 gene 72 corresponds in position to HSV-1 US6 (which specifies gD) but the encoded proteins are not significantly related by FastA (FastA = 6 1). The gene 72 protein, however, shares extensive homology with PRV gp50 (Petrovskis et a/., 1986a; FastA = 283) and the six cysteine residues conserved in HSV-1 gD and PRV gp50 (McGeoch, 1990) are also present in the EHV-1 protein. The gene layout in the S component of alphaherpesviruses varies to a much greater extent than that in the L component (Davison and McGeoch, 1986). Figure 4 shows a comparison of the S components of EHV-1, PRV, HSV-1, and VZV. The S component ranges widely in size from 19,871 bp in VZV to 37,289 bp in EHV-1 and contains from 10 genes in VZV to 17 in EHV-1. Six genes are conserved in all four viruses: genes 64 (major transcriptional activator), 65 (in vitro host-range factor), 69 (protein kinase), 73 (gl), 74 (gE), and 76 (tegument protein). The relative positions of some genes differ. For example, EHV-1 gene 76 is located at the same end of Us as its counterparts in PRV and HSV-1 but at the opposite end and in the opposite orientation from its VZV counterpart. Also EHV-1 gene 66, like its VZV counterpart, is present as two copies in IR,/TR,, but the corresponding HSV-1 gene is located as a single copy in Us and no counterpart has been identified in

EW-1gene10 vzv gene cO"Se"S"S

9R

EHV-1 gene 10 "Z" gene 9A COilSeIlS"S

1

**************

50

MLSTRFVTLAILACLLVVLGLARGAGGDPGVKQRIDV~EEERRD~ MGSITASFILITMQILFFCEDSSGEPNFAE..........RNWHAS -------T----------L-------G-~--------------R---

******************Lb** 51 CSGHGFPITTPSTAAILFYVSLLAVGVAVACQAYRAVLRIVTLEMLQHLH CSARGVYIDGSMITTLFFYASLLGVCVALISLAYHACFRLFTRSVLRSTW CS--G--I---------FY-SLL-"-"~----*~-*--R--T---L----

100

FIG. 3. Alignment of the predicted amino acrd sequences of the EHV-1 gene 10 and VZV gene 9A proterns. Hydrophobrc regions are indicated by asterrsks.

314

TELFORD

EHV-1

PRV

HSV-1

vzv

FIG. 4. Comparison of the organization of genes in the S components of EHV-1, PRV, HSV-1, and VZV. The S components are shaded, the thinner and thicker portions denote Us and IRflR,, respectively. Protein-coding regions are represented as open arrows and the EHV-1 gene nomenclature is applied to PRV, HSV-1, and VZV in order to indicate gene counterparts (Table 2). HSV-1 genes US1 1 and US1 2, located at the right end of Us, lack counterparts in EHV-1 and are labeled A and B, respectively. Data for HSV-1 are from McGeoch et al. (1988), VZV is from Davison and Scott (1986), and PRV from van Zijl et al. (1990), Rea et al. (1985), Petrovskis and Post, (1987), Petrovskis et al. (1986a, 1986b), Vlcek et al. (1990), Zhang and Leader (1990), and Zhang er a/. (1990).

PRV. Davison and McGeoch (1986) concluded that differences in gene layout between the S components of HSV-1 and VZV have resulted from expansion and contraction of IR,/TR, during evolution. They also noted that adjacent protein-coding regions in Us imposed evolutionary constraints on the positions of the IRS/U, and U,/TR, boundaries. The gene arrangements in PRV and in EHV-1, where gene 76 terminates 68 bp from the UJTR, junction and gene 68 terminates in IRS, are in accord with these conclusions. EHV-1 is also related, albeit much more distantly, to EBV, a gammaherpesvirus, and HCMV, a betaherpesvirus. The pattern of relationships is very similar to those reported for VZV and HSV-1, where up to 40 genes in U, are conserved in EBV and HCMV U, in several rearranged blocks of genes (Davison and Taylor, 1987; Chee et al., 1990). In contrast, the S component of alphaherpesviruses probably evolved more recently, since all the genes therein lack counterparts in the beta- and gammaherpesviruses. Unlike previous projects concerned with sequencing entire herpesvirus genomes, the priority of obtaining a sequence as close as possible to that of a natural virulent strain of EHV-1 had a major effect on decisions concerning choice of virus isolate and experimental approach. Therefore, the complete DNA sequence of EHV-1 strain Ab4p is certain to prove an important basis for advancing the knowledge of the molecular pathogenesis of this important equine virus. Moreover,

ET AL.

it will also provide a useful comparative dataset for studying related genes in other herpesviruses.

ACKNOWLEDGMENTS This work was funded by the Equine Virology Research Foundation. We are grateful to John Subak-Sharpe for his continued interest, to Duncan McGeoch for helpful discussions, to Duncan McGeoch and Walter Plowright for critical reading of the manuscript, and to Helen Johnson and Mary-Jo McLaren for technical assistance in the early stages of the work. We also thank Bill Bonass and Debra Elton for generous provision of unpublished sequence data from EHV-1 strain Abl. A.J.D. is a member of the MRC Virology Unit in the Institute of Virology.

REFERENCES ALLEN, G. P., and COOGLE, L. D. (1988). Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gpl3) with homology to herpes simplex glycoprotein C. J. Viral. 62,2850-2858. AUDONNET, J-C., WINSLOW, J., ALLEN, G. P., and PAOLE~I, E. (1990). Equine herpes virus type 1 unique short fragment encodes glycoproteins with homology to herpes simplex virus type 1 gD, gl and gE. 1. Gen. Viral. 71, 2969-2978. BANKIER, A. T., and BARRELL, B. G. (1989). Sequencing singlestranded DNA using the chain termination method. In “Nucleic Acids Sequencing: A Practical Approach” (C. J. Howe and E. S. Ward, Eds.), pp. 37-78. IRL Press, Oxford. BARKER,D. E., and ROIZMAN, B. (1992). The unique sequence of the herpes simplex 1 L component contains an additional translated open reading frame designated U, 49.5.1. Virol. 66, 562-566. BAUMANN, R. P., RAMANA, V., YALAMANCHILI, R., and O’CALLAGHAN, D. J. (1989). Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication. J. Viral. 63, 1275-l 283. BEN-P• RAT, T., RIXON, F. J., and BLANKENSHIP,M. L. (1979). Analysis of the structure of the genome of pseudorabies virus. Virology 95, 285-294. BRYANS, J. T., and ALLEN, G. P. (1989). Herpesviral diseases of the horse. In “Herpesviral Disease of Cattle, Horses and Pigs” (G. Wittmann, Ed.). Kluwer Academic Publishers, Nor-well, MA. CAMPBELL, M. E. M., PALFREYMAN,J. W., and PRESTON,C. M. (1984). Identification of herpes simplex virus DNA sequences which encode a transacting polypeptide responsible for stimulation of IE transcription. 1. MO/. Biol. 180, l-1 9. CHEE, M. S., BANKIER, A. T., BECK, S., BOHNI, R., BROWN, C. M., CERNY, R., HORSNELL, T., HUTCHISON, C. A., Ill, KOUZARIDES,T., MARTIGNE~I, J. A., PREDDIE, E., SATCHWELL,S. C., TOMLINSON, P., WESTON, K. M., and BARRELL.8. G. (1990). Analysis of the proteincoding content of the sequence of human cytomegalovirus strain AD1 69. Cur. Top. Microbial. Immunol. 154, 125-l 69. CHOWDHURY, S. I., BUHK, H-J., LUDWIG, H., and HAMMERSCHMIDT, W. (1990). Genomic termini of equine herpesvirus type 1. J. Viol. 64, 873-880. COSTA, R. H., DRAPER, K. G., KELLY, T. J., and WAGNER, E. K. (1984). An unusual spliced herpes simplex virus type 1 transcript with sequence homology to Epstein-Barr virus DNA. J. Viral. 54, 3 17328. CULLINANE, A. A., RIXON, F. J., and DAVIDSON, A. J. (1988). Characterization of the genome of equine herpesvirus 1 subtype 2. J. Gen. Virol. 69, 1575-l 590. CUNNINGHAM, C., DAVISON, A. J., DOLAN, A., FRAME, M. C., MCGEOCH, D. J., MEREDITH, D. M., Moss, H. W. M., and ORR, A. C. (1992). The UL13 virion protein of herpes simplex virus type 1 is phosphorylated by a novel virus-induced protein klnase. J. Gen. Viral. 73, 303-311.

DNA SEQUENCE

OF EQUINE HERPESVIRUS-

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