Journal of General Virology (1990), 71, 1793-1800. Printed in Great Britain

1793

The nucleotide sequence of an equine herpesvirus 4 gene homologue of the herpes simplex virus 1 glycoprotein H gene Lesley Nicolson, 1. Ann A. Cullinane 2 and David E. Onions 1 1Department of Veterinary Pathology, University of Glasgow Veterinary School, Bearsden Road, Bearsden, Glasgow G61 1QH, U.K. and Zlrish Equine Centre, Johnstown, Naas, County Kildare, Eire

The equine herpesvirus 4 (EHV-4) gene glycoprotein H (gH) gene homologue was localized by virtue of the conserved genomic position of this gene throughout members of the herpesvirus family. The gene maps immediately downstream of the thymidine kinase gene at approximately 0.49 to 0.51 map units within genomic fragment B a m H I C. The EHV-4 gH primary translation product is predicted to be a polypeptide of Mr 94 100, 855 amino acids long, which possesses features characteristic of a membrane glycoprotein, namely an N-terminal signal sequence, a large hydrophilic domain containing 11 putative N-linked glycosylation sites, a C-terminal transmembrane domain, and

a charged cytoplasmic tail. Comparison to other herpesvirus glycoproteins revealed identities of 85%, 26% and 32% with the gH counterparts of the alphaherpesviruses EHV-1, herpes simplex virus 1 and varicella-zoster virus, respectively, and of 17% and 18% with those of human cytomegalovirus, herpesvirus saimiri and Epstein-Barr virus. The EHV-4 gH exhibits features previously reported to be conserved throughout the gH polypeptides of herpesviruses of all three subgroups. A region of direct repeat elements and a possible origin of DNA replication are located immediately downstream of the gH gene.

Introduction

been implicated as targets for cell-mediated immunity through the recognition by equine lymphocytes of protein fractions containing major glycoproteins (Bridges et al., 1988). Electrophoretic analysis, by SDS-PAGE, of carbohydrate-containing envelope proteins of EHV-1 and EHV4 has indicated that virions possess at least 12 glycoprotein species, eight of which are highly abundant within the virus envelope (Turtinen & Allen, 1982). It is apparent from recent studies on the nature of these glycoprotein species that some may be specified by the same gene, differing from each other only in the degree of their post-translational processing (Meredith et al., 1989). Genomic map positions of the genes encoding the major glycoproteins of EHV-1, gp2, gpl0, gpl3, gpl4, gp17/18 and gp21/22, have been reported (Allen & Yeargan, 1987). Of these genes, three (gpl4, gpl3 and gp17/18) map at positions collinear with the herpes simplex virus type 1 (HSV-1) gB, gC and gE genes and analysis of EHV-1 sequence data has identified gpl4 and gpl 3 as gB and gC homologues respectively (Whalley et al., 1989; Allen & Coogle, 1988). Analysis of EHV-4 sequence data has revealed the EHV-4 gB and gC gene homologues are similarly collinear with the HSV-1 genes and that agE homologue is encoded within the Us region of the genome (Riggio et al., 1989; Nicolson & Onions,

Equine herpesviruses 1 and 4 (EHV-1 and EHV-4) are alphaherpesviruses responsible for significant economic loss within the equine industry. EHV-1 is associated with abortion, respiratory disease and neurological disease. EHV-4, previously classified as EHV-1 subtype 2, is primarily associated with respiratory disease but can also induce abortion (Allen & Bryans, 1986). Current vaccines against EHV-1 and EHV-4 require multiple administration and are of limited efficacy. The development of a more effective vaccine would be aided by a greater understanding of the host immune response to infection by these viruses and by determining which EHV-1 or -4-specific components are responsible for eliciting a protective immune response. Such studies have focused on the envelope glycoproteins since in herpesviruses these proteins are known to be important targets of humoral and cell-mediated immune responses (Carter et al., 1981 ; Spear, 1985; Rosenthal et al., 1987). Antibodies directed against EHV-1 and -4 glycoproteins are capable of virus neutralization (Allen & Bryans, 1986) and in the case of monoclonal antibodies against gpl3, gpl4 and gp17/18, can confer immunity to reinfection in a hamster model by passive immunization (Stokes et al., 1989). EHV-1 and -4 glycoproteins have 0000-9598 © 1990SGM

1794

L. Nicolson, A. A. Cullinane and D. E. Onions

1990; Cullinane et al., 1988). A candidate for an EHV-4 glycoprotein gene not yet mapped to the genome is a gH gene homologue. Glycoprotein H is one of only two glycoproteins, the other being gB, encoded throughout members of all three subgroups, alpha, beta and gamma, of the herpesvirus family (Baer et al., 1984; Gompels & Minson, 1986; McGeoch & Davison, 1986; Cranage et al., 1988; Gompels et al., 1988; Heineman et al., 1988; Robertson & Whalley, 1988). The gH proteins of varicella-zoster virus (VZV), HSV1, Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) are important in mediating virus infectivity since antibodies against gH can neutralize infectivity independent of complement (Buckmaster et al., 1984; Gompels & Minson, 1986; Montalvo & Grose, 1986; Keller et al., 1987; Cranage et al., 1988; Gretch et al., 1988; Oba & Hutt-Fletcher, 1988). The neutralizing effect of HSV-1 and EBV anti-gH antibody appears to occur at some stage after adsorption of virions to the host cell membrane (Fuller et al., 1989; Miller & HuttFletcher, 1988) and since anti-gH antibody also inhibits cell-to-cell spread of virus, it appears that gH may play a role in membrane fusion events of both the virion-host cell and intercellular type. HSV-1 gH is essential for viral replication (Desai et al., 1988). The herpesvirus gH gene is located within a block of conserved genes as indicated by sequencing studies and Southern hybridization analyses and maps immediately downstream of the thymidine kinase (TK) gene in those herpesviruses which encode this enzyme, including EHV-1. Having previously located the TK gene of EHV-

4 by virtue of collinearity of the EHV-4, EHV-1 and HSV-1 genomes (Nicolson et al., 1990), we sequenced downstream of this gene in order to search for an open reading frame (ORF) with the capacity to encode a gH homologue. Here we report the nucleotide sequence of the EHV-4 gH gene homologue, compare its predicted amino acid sequence to other herpesvirus gH-type proteins, and comment on sequence features downstream of the gene.

Methods Construction of recombinant plasmids. An EHV-4 strain 1942 BamHI library in pUC9 was previously reported (Cullinane et al., 1988). Subfragments of EHV-4 BamHI C, generated by restriction endonuclease digestion, were cloned into a Bluescript M 13 + vector (Stratagene) for sequence analysis.

D N A sequencing. DNA sequencing was carried out by the Sanger dideoxynucleotide technique (Sanger et al., 1977) using denatured recombinant plasmid DNA as template and Bluescript-specific or " custom oligonucleotides as primers. Primer sequences annealed to single-stranded plasmid DNA were extended by the Klenow fragment (BCL sequencing grade) in the presence of dNTPs, ddNTPs (Pharmacia) and [35S]dATP (Amersham). Sequencing reaction products were electrophoresed through 0.6% acrylamide wedge gels using an LKB Macrophor apparatus. Gels were fixed in 10% acetic acid, dried, and exposed overnight to Amersham MP autoradiograph film. The sequence of 3.85 kbp of DNA was determined by analysis of overlapping sequence data (Fig. I b). Sequence analysis was performed using Beckman Microgenie software (Queen & Korn, 1984) which utilizes the algorithms of Gamier et al. (1978) and Hopp & Woods (1981) for prediction of secondary structure and hydropathic plot analysis. S (35 kb)

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Fig. 1. (a) BamHI restriction map of the EHV-4 genome (from Cullinane et al., 1988). The EHV-4 TK gene maps upstream of the rightward XhoI restriction site of BamHI fragment C at map position 0.48. (b) Sequencing strategy and localization of the EHV-4 gH gene.

Nucleotide sequence o f the E H V - 4 g H gene

Results and Discussion Analysis of E H V - 4 genome map units 0.49 to 0.52

The EHV-4 genome is composed of long (L) and short (S) components of 109 kbp and 35 kbp respectively, the latter bounded by a set of inverted repeats (CuUinane et al., 1988) (Fig. l a). The EHV-4 strain 1942 TK gene maps within a 2 kbp E c o R V / X h o I subfragment of the B a m H I C fragment in the long unique component of the genome at approximate map position 0.48 (Nicolson et al., 1990). The nucleotide sequence of 3-85 kbp of DNA immediately downstream of the TK gene, determined according to the strategy outlined in Fig. 1 (b), is detailed in Fig. 2. An ORF of 2565 bp located 170 bp downstream of the putative polyadenylation signal of the TK gene (Fig. 2) is predicted to encode a polypeptide possessing features characteristic of a membrane glycoprotein. Comparison of this putative EHV-4 glycoprotein to published herpesvirus glycoprotein sequences indicated similarity to the gH of HSV-1 and to its homologue within other alpha-, beta- and gammaherpesviruses. The gene is thus hereafter identified as the EHV-4 gH gene pending identification of its protein product and review of equine herpesvirus glycoprotein gene and gene product nomenclature. The DNA sequence upstream and downstream of this ORF was searched for putative gene control elements. Two potential TATA boxes, consensus sequence TATA(A)A(~0 (Corden et al., 1980), are located at positions 87 to 93 (TATATAA) and 157 to 162 (TATATT). The polyadenylation signal AATAAA (Proudfoot & Brownlee, 1976) is not specified within the 3' non-coding DNA proximal to the termination codon, TAA, at 2750 bp. However the minor polyadenylation signals ATTAAA and AGTAAA (Birnstiel et al., 1985) are specified within the extreme Y-terminal coding region of the gene. The sequence, TGTGTTGT (2770 to 2777) shares homology with the consensus element of McLauchlan et al. (1985), YGTGTTYY, often present 24 to 30 bp downstream of the polyadenylation signal element of HSV and mammalian mRNAs, and is located at an appropriate distance from the ATTAAA element at 2737 to 2742. However since consensus sequence elements may occur fortuitously within DNA, we cannot rule out the possibility that the EHV-4 gH transcript is terminated considerably farther downstream of the gene, and that it may encode a contiguous gene. The modified scanning hypothesis of translation (Kozak, 1984) proposes that the initiation codon of a transcript is the first ATG downstream of the mRNA start site which is accessible to the ribosomal translational apparatus. The ATG at 185 to 187 bp, within the

1795

sequence CGATTATGT, is the first ATG downstream of the in-frame stop codon at 116 to 118 bp. Although the local sequence of this codon is only moderately homologous to Kozak's consensus sequence, CC(A)CCATGG, it does retain the purine at position - 3 , the most highly conserved feature of the 5' flanking sequence of the initiation codon of eukaryotic mRNAs. Assignment of this codon as the initiation codon of the gH gene is supported by the potential of the DNA immediately downstream to encode a functional signal peptide domain. The gene has a G + C content of 45 % which contrasts with the 67% G + C content of the HSV-1 gH (McGeoch & Davison, 1986) presumably reflecting a difference in the overall G + C content of the EHV-4 and HSV-1 genomes. Downstream of the gH gene a region of direct repeat elements and a putative origin of replication were identified. The region of repeats comprises 20 elements from 18 to 27 bp of sequence (GCG)I 3GCGGCGAGG(GCT)I 3. Since similar repeat elements in other herpesvirus genomes are unstable on cloning, the repeat region may be of greater size in the intact EHV-4 1942 genome. Specific repeat elements within HSV-I and VZV have been implicated in transcriptional regulation and identified as target sites for site-specific recombination (Davison & Scott, 1986). However, the functional significance, if any, of most of these isolated repeat elements (as distinct from components of the major terminal and internal repeats) is unknown. A possible EHV-4 UL origin of DNA replication (oriL) was identified through sequence similarity to the origins of replication of HSV-1 (Stow & McMonagle, 1983), HSV-2 (Lockshon & Galloway, 1986), VZV (Stow & Davison, 1986) and EHV-1 (Baumann et al., 1989). Alphaherpesvirus origins are characterized by the presence of a 9 bp conserved sequence element, CGTTCGCAC, proximal to an A/T-rich region. In EHV-4, the sequence CGTTCGCAC (3355 to 3363 bp) and its complement GTGCGAACG (3400 to 3408 bp) flank an A/T-rich region of size comparable to that of the EHV-1 ori s sequence (Baumann et al., 1989). The HSV and VZV origin sequences are palindromic implying a possible role for intrastrand base pairing in their function or regulation. The reported EHV-1 oris possesses fewer potential intrastrand base matches than those of HSV and VZV. Similarly the EHV-4 candidate sequence does not possess a perfect palindrome with only 21 potential base pairing events within each 54 base arm of a hypothetical cruciform structure (Fig. 3). An ORF of transcriptional sense right to left was localized downstream of the EHV-4 repeats and putative origin of replication. The predicted C-terminal region of the product of this gene shares identity with the

1796

L. Nicolson, A. A. Cullinane and D. E. Onions

~TTGT~TA~TT~TTTG~TAT~T~ATAG~G~TTGTGTGTTTCGTGTGTAAAcTT~GTTTCTAGTTTGGG~TATATAAGC~GTTGTGCTCTTAAATcATTTAGTA 120 CAGCGC~GCC~TACTCGAGGTATCCAGTGGTTGTATATTGGGAATAAATACTGCTG~TTATGT~CAACCGTATCTAAAAATAG~TATCTTAGTGGCCGCTACTATTGTGTCTGC240 M S O p y L K [ A ] L V A A T I V $ A GATTCCCGTTTG~C~CACCGGTTTCAACTT~C~ACCCCAACAAACAAAATTGCACTATGTGGGAAATGGTACCTGGGTA~CAAC~TA~TTr..AACGT~CCAGGTAT~GGAT 3 ~ ] p V W T T p V S T $ p p Q O T K L H Y V G N G T W V H N N T F N V T R Y D R ! ~CCATGG~cAGTTTAT~T~CAATTTATCCTCTACTACCTTTTTTGTTGCTATATCGGAGAGAAATTTT~G~ACGGTT~CTCCACTTGGAGCGTCCGTATTTTGGATTTTAAA 480 T M E p V y N N N L S S T T F F V A ] S E R N F R T V N T P L G A S V F W [ L K ~GCGCTCTT~TCCTCCCAAA~CC~CCCTGTATAGCT~TGTG~CAG~CCCGGT~CC~CGCG~CGTG~GTCAA~TCMCTGT~GTCTATTTTTTAAT~CAATTTGGAGCC S A L N p p K H 0 p C I A N V p E p G D p R G P C V N S T V S L F F N D N L E

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GTTTTT~TGA~AAAAAATCTTTTGGAGTTTG~GTATTG~CGAC~CTACAT~C~ATGGACGTTT~GC~T~TAA~CTGTGGCTACGAAAGGC~CC~G~TTG~GTGGTTCT 720 F L M T K N L L E F E V L P D N y I T G W T F E R S K T V A T K G N P V G V V L CTCCCCTCCCCG~C~GTCCG~TGTAAAT~CAC~T~GAGAT~TGGCACCCCTAAACAG~CTTGAG~TTATAGACGAACATACTACGTTCGTGCTCGACCTGCAAAATTTTAC ~ 0 S p p R T S p D V N N T I R D D G T P K Q H L S I I D E H T T F V L D L Q N F T ~ACTTTAA~TTATAT~GCCCATTTGCTGCGGTGTGGCC~T~CAGCCTTTCATGCCGGAATTA~GT~TGGGGTGT~CAACTCAGG~TTGCGTACCTCGGC~TGGGTT 9~ K T L T y I S P F A A V W P I T A F H A G ] T V M G C D T T Q A I A Y l G N G F 1ATGGGTTTGCAAAT~GCTCGGT~AC~TC~CCG~TG~TGATTGTTGCACCAAATGACGTCCGTGCTCGGATAGTT~CCGCCTTCCCCC~CGTC~CTTGAGCCACC~GG 1080 M G l Q I S S V N N P p L E M I V A P N D V R A R I V N R L P P R R R L E P P G GC~ATATGCAGGA~CTATCTAC~GGTGTACGTACTCAGT~TGGAAATTTTTACTTGGGTCATGG~TGAGCAAGATTTCTAGGGAGGTTGCCGCGTACCCAG~GTTTGGACTA 1200 p y A G p I y K ~Y V l S D G N F Y L G H G M S K I S R E V A A Y P E E S E D Y CCGCTACCACTTATCG~TTG~C~C~TT~TACTCTGGCTATGTTGGCAG~TTTCTTCCGGT~G~GGATGT~GCTATTA~TTGTAT~G~T~TTGCGAGGCTGGCCGTAGC 1320 R y H L S L A N L D T L A M L A E L S S G K S K D V S Y Y L Y R I I A R L A V A ~CGTTTTCCCTTG~GAAGTTATA~GCCT~GT~CTATATGCTCCTTC~GGCCATCGACGT~ATATAAACCTCCGCCT~TTGTACCTCTAGTGATGAAGTA~GCCGCTGGGGG 1440 T F S l A E V I R L S D y M L l Q E A I D V D I N L R L I V P L V M K Y A A G G ~CGGCAGATAGCTCGTA~CAT~CTcG~CGTAGCTATG~CC~TTCGAGGTGGCTCAAGCC~TT~GAAGATAGTAG~TATAAATATCGAAAATG~TTGCG~AAACCTAT 1560 T A D S S y T S S D V A M D Q F E V A Q A Q I E K I V A D I N I E N E L R K P M GTAC~GCAC~GcT~TTATTGAAAAGCGTGTACGCTTATTCTAGAAAGCCG~TACCAAACG~GGT~GCTTTGCT~C~GGCT~TCACGGCTATGTATAAAG~GC~TT~GGACAG I ~ 0 y E H R S L L K S V y A Y S R K p L P N A V S F A N R L I T A M Y K E A ] K 0 R ~TTACGTGG~CTCTAC~TGCGAGAGGTGTTATTTTTTGCGGTTGGTGCTGCTG~GGTTCG~TGTTATCCTCACGGATGGGC~TCTCGGTTTA~TGCC~CAAA~TTCTTC 1 ~ 0 I T W N S T M R E V L F F A V G A A A G S H V ] L T D G P D L G L H A H K D S $ ~TGTTTCTATCTCTT~CCGC~CATACTCTTGTTGTGTACGG~CATGTGTACGGCGTCGCATGCCGTGTCCG~GGAGTAAAACTA~GG~GTTATGGCTGGCCTTATTGCCGGGGG 1920 M F L S L N R N 1L L L C T A M C T A S H A V S A G V K L E E V M A G L I A G G TGTACAATTTAGCCTCC~AG~GTATTTAGTC~TGTATGGCGTCTGCTC~TTTGACC~GGCCGAAG~G~ATGTGCTA~TCTACTGTCCGTTATCCCACCTCGCCTGTA~CC~ 2 ~ 0 V Q F S L L E V F S P C M A S A R F D L A E E E H V L D L L S V $ P P R L Y T D CTTA~CACTGGCTTG~GGAC~CGGAAC~CCATCCATTCATACGGACGGTCTGCT~CGGAATTTTAAACTCTCG~TCGCATAT~CTTT~TGCTGTTCGTGTATTTACTC~ 2160 L N T G L E D D G T T ] H S Y G R S A N G I L ~ S R I A Y N F D A V R V F T P E GTTGGCCTCATGCAGCACTAAACTAC~AAAA~TTTTGGTAGTGCTACCCTTAGCATCAAACCGAAGCTACGTTAT~CT~GTACTGCGC~AATATAGGTTT~cTTACTcTCTTGATGG 2280 L A S C S T K L p K V L V V L P L A S N R S Y V % T R T A P N I G L T Y S L 0 G GGTA~TATAGC~AGCCTATAGT~TCAGTTACAT~CTTATGGAAATTGTCAAGTTTC~GCTACAAT~GGTCAGTTTACTTG~CCATCCGGGC~CC~GTCGTGCGTATA V N ] A K p ] V I S y I T Y G N C O V S R A T I R S V Y L D H P G H T Q S C V

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Nucleotide sequence of the EHV-4 gH gene corresponding region of the polypeptides specified by gene UL21 of HSV-I and gene 38 of VZV (unpublished results). Neither the HSV-1 nor the VZV genome possesses repeat elements or a putative origin of replication in the intervening D N A between the coding regions of the gH gene and genes UL21 (HSV-1) or 38 (VZV).

hydrophobic amino acid residues within the extreme Nterminal region of the primary translation product. The hydrophobic region of the EHV-4 gH signal peptide is evident within the hydropathic plot shown in Fig. 4. Application of the eukaryotic weight matrix developed by von Heijne (1986) for the prediction of signal sequence cleavage sites indicates that Ala(19) is the most probable terminal residue of the signal peptide. The predicted Mr of gH after cleavage of the signal peptide is 92130.

The EHV-4 gH polypeptide The EHV-4 gH gene encodes a protein 855 amino acids in length with a predicted Mr of 94100. It remains to be determined whether the native, processed EHV-4 gH corresponds to any of the previously reported EHV-4 glycoproteins (Turtinen & Allen, 1982; Meredith et al., 1989; Bridges et al., 1988). The predicted polypeptide has the potential to exhibit the following structural features characteristic of membrane glycoproteins. (i) N-terminal signal sequence peptide Signal peptides are characterized by a stretch of G

4GCGTT CGCACT TGT TACAATAATTAT III1-11111 II I I I I I I I I I I t ~GCAAGCGT GGTTAAGGTTAT TAATA A

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Fig. 3. Potential intrastrand base pairing events within a putative EHV-4 origin of DNA replication. I

1797

(ii) Hydrophilic external domain In membrane glycoproteins this major domain projects from the virion envelope or infected cell plasma membrane and comprises the glycosylated portion of the polypeptide. It is therefore usual to find potential Nlinked glycosylation sites (N-X-S/T) within this domain. Residues 20 to 816 may constitute the external domain of EHV-4 gH. Within this region there are 11 putative Nlinked glycosylation sites, seven of which are located within the N-terminal half of the polypeptide and four in the C-terminal half. Due to its exposure on the virion or cell surface this part of the protein is likely to contain the major antigenic determinants of gH with respect to the host humoral immune response and, in this case, possibly one or more complement-independent neutralization epitopes as in HSV-1, VZV, EBV and HCMV.

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Fig. 4. Hydropathicplot of the EHV-4 gH protein (Hopp & Woods, 1981).Positive values indicate a local hydrophilicenvironment, negative values indicate a local hydrophobicenvironment. Fig. 2. The nucleotidesequenceof the EHV-4gH gene and flanking DNA. Sequenceelementswith homologyto consensus sequences associated with promoterfunction and polyadenylationsignal function are underlined: TATA box homologuesoccur at positions87 to 93 (TATATAA)and 157 to 162 (TATATT); putative polyadenylationsignals downstream of the TK and gH genes occur at 9 to 14 (AATAAA) and 45 to 54 (TTGTGTGTTT) and at 2737 to 2742 (ATTAAA) and 2770 to 2777 (TGTGTTGT) respectively.The predicted amino acid sequenceof the primary translation product of the gH gene is indicated. Direct repeat elements are lettered in order of increasing size. The conservedori sequence element CGTTCGCAC and its complement are boxed.

1798

L. Nicolson, A. A. Cullinane and D. E. Onions

(iii) Hydrophobic transmembrane domain A characteristic feature of membrane glycoproteins is the existence of a stretch of around 20 or more hydrophobic amino acids towards the C terminus. This has been hypothesized to constitute the membranespanning portion of the glycoprotein and may consist of one or more m-helical loops traversing the virion envelope or infected cell membrane. Amino acids 817 to 836 of the EHV-4 gH are enriched in hydrophobic residues (Fig. 4) and are predicted to constitute the gH transmembrane domain.

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(iv) Cytoplasmic domain The proposed cytoplasmic domain of EHV-4 gH stretches from amino acids 837 to 855 and is negatively charged• A proposed function of this domain is to anchor the transmembrane domain within the lipid bilayer and thus to fix the C-terminal end of the glycoprotein to the membrane. With the exception of pseudorabies virus gH which is only 686 amino acids in length, the gHs of alphaherpesviruses tend to be larger than their beta- and gammaherpesvirus counterparts, ranging from 838 (HSV-1) to 882 (BHV-1) as compared to 706 to 743 for the EBV, HVS and HCMV gHs. At 855 amino acids in length the EHV-4 gH is of comparable size to most of the alphaherpesvirus gHs. Identities between the gH glycoproteins of EHV-4 and other herpesviruses are 859/0, 32~, 26~, 18~ and 17~ with the gHs of EHV-1 (J. M. Whalley, G. R. Robertson, N. A. Scott & J. M. Miller, personal communication), VZV, HSV-1, HCMV and HVS respectively. Dot matrix comparisons of EHV-4 gH are presented in Fig. 5. The C-terminal region of the protein is clearly conserved to a greater degree than the N-terminal region. A comparison of the amino acid sequence of the gH proteins of alpha-, beta- and gammaherpesviruses by Gompels et al. (1988) and Cranage et al. (1988) indicated this greater diversity of sequence in the N-terminal region of the protein and highlighted several features of the gH protein conserved throughout the herpesvirus family: firstly, an unusually short cytoplasmic domain of 14 or 15 amino acids in alphaherpesviruses and of seven or eight amino acids in beta- and gammaherpesviruses, secondly, four conserved cysteine residues at similar positions relative to the putative transmembrane domain and within conserved local sequence, and thirdly, a conserved glycosylation site sequence NGTV 13 to 18 amino acids N-terminal to the transmembrane domain. EHV-4 gH exhibits all the above features as shown in an alignment of the EHV-4, HSV-1 and VZV gHs (Fig. 6): the proposed cytoplasmic domain is under 20 amino acids in length, the four conserved cysteines are present at positions 556, 591,663 and 716, and the C-terminal

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glycosylation site is located within the sequence NGTV (amino acids 796 to 799) which is positioned 19 amino acids N-terminal to the putative EHV-4 transmembrane domain. The Cys residues at 737 and 740 in the EHV-4 gH occur at sites of cysteine conservation throughout most herpesvirus gHs, with the exception of HSV-1. The strong conservation of cysteine residues between the

Nucleotide sequence o f the E H V - 4 g H gene

EHV-4 HSV'1 VZV

NSQPYLKIAILVAATIVSAIPVWTTP-VSTSPPQQTKL . . . . . . . HYVGNGTWVHNNTFNVTRYDRITNEPVYNNNLSRTT. . . . . . . . FFVAISEEMFR NGN~LwFV~VI~LG~AWGQV~wT~-~EQT~WFLDGLG~DR~YwRDTNTGRLWL~NT~K~RGFLAppDELNLTTASLPLLRUYEERFCFVLV~T NFALVLAVV|LPLWTTANKSYVTPTPATRSIGHNSALLREYSDRNNSLKLEAF.YPTGF . . . . DEELIKSLHWGNDRKNVFLV-IVKVNPT

EHV-4

T~NTP.L~A~VFW|LK~AL..NPPKH°PC~A~V~E~G~RGPCv~STvsLFF~°NLE~FL~TKNLLEFEVL~N~I~GWTFERsK~VAT[~PVGV~LsP TAEFPR~P6~LLYIPKTYLLGRP~NA~LPAPTTVEPTAQPPP~APL~GLLHNPAA~LLR~RA~VTFSA~P~..EALT~P~G~VA~A~HPSGPR~P

HSV-1 VZV

1799

THE~.~VG.LV|FPK.YLLSPY~FKAEHRAPFPAGRFGFL~HPVTPD~SFF~S~FAP~LTT~LVAFT~FPPNPL~w~L~RAETAA~ERPFG~SLLP

EHV-4 HRV-1 VZV

~RTSPDV~NT~R~GTP~HLS~IDEHTTFVLDL~NFTKTL.TY1~FAAVw~TAFHA~1TVNGC~TT~A~AYLGNGF~GL~VNN~LE~VA~N~ ~RP~GARRH~TTELD~T~L~NASTTWLATR~LL-~GRYvYFSP~A~TWpvG~WTTGELVLGCDAALVRA~YGREF~GLV~DR~EVNwPAG AR~T-~KNT~L~KA~FATV~ALARHTFFsAEA~TNSTLR~HVPLFG~VUP~RYWATG~vLLT~SGI~E~Gv~FN~sL~G~P~EL~VVP~T

EHV-4 HSV'I VZV

VKLNAvTS~TTWFQLNP-P~PGPSYRVYLL~RGLD~N~-~KHAT~|CA-YPEE~L~YRY~L~A~TEALRNTTKADQ~|NEE~YY~AAR|ATS

VRAR%~NRLPPRRRLEP~PGPYAG~YKvY~LSDGNFYLGHG~S~SREvAAY~EE~LDYR~HLSLANL~TLA~LAELS~GK~KDV~YYLYR~ARLAvA

~TL~RV~DPADENPP~AL~PP~PR~RVFVLG$LTRA~NGSALDALRRVGGYPEEGTR~A~FLSRAYAEFFSG~AGAEQGPRPPLF~--URLTGLLAT~

EHV-4 HSV-1 VZV

GFAFvNAAHANGAvCLSDLLGFLA•SRALAGLAARGAAG•AADSVFFNVSvL•PTARL-•LEARLQHLVAE•LEREQSLALHALGY-•QLAF-vL••PSA

T FRLAEVI RLSDYNLLQEAIDVD 1NLRL l VPLVNKYAAGGTADSSYTSSOVANOQFEVAOAQ I EK-- IVAO I NI EN.ELRI(PNYENRSLLKSVYAYSRKp

EHV-4 HSV-1 VZV

l P " NAVSFANRLi TANYKEA[ KORI TWNSTHREVLFFAVGAAAGSHV%l TOGPDl GLHAHKO$SNFLSL NRNi l l LCTAN;TASHAVSAGVKLEEVI4AGL YDAVAPS-AANL| DALYAEFLGGRVLTT-~VHRALFYA. . . . . . SAVLRQPFLA-GVPS" --AVQRERA- RRSLL | ASALCTSDVAAATNADLRTALARA DHVNALSLARRVI MSI YKGLLVKQNL- NATERQALFFA. . . . . . SN1LL-NFRE- GLEN'-" RSRVLOG-RTTLLLMTSMCTAAHATQAALN|QEGLAYL

ENV-4 HSV-1 VZV

IAGGVQFSLLEVFSPCMASARFDLAEEEHVLDLLSV | PPRLYTDLNTGLEDDGTT[ HRYGRSANGI LNSRI AYNFDAVRVFTPELAR.;STRLPKVL..DHQKTL FWLPDHFSPCAASLRFDLDESVF[ LDALAQATRSETPVEVLAQQTHGLAST. . . . . . . . . LTRWAHYNALl - RAFVPEARHRCGGQSANVEPR| NPSKHNFTI PNVYSPCNGRLRTDLTEEi HVNNLLSA | PTRPGLNEVLNTQLDESE| FDAAFKTMN| FTTWTAKDLHi LHTHVPEVFT. CQDAAARNGEYV

E,V-, HSV'I VZV

VVL,.S,.,VI,.A.IOL ,,SL°OV, I,K, LV'P I THNASYVVTHSPLPRGI GYICLTGVDVRRPLFLTYLTA- TCEGSTRD1ESKRLVRTQNORDLGLVGAVFNRYTPAGEVMSVLLVDTONTO001AAG L I LPAVOGHSYVITRNI(PORGLVYSLADVOVYNPI SVVYLSRDTCVSEHGV| ETVALPHPGNLICECLYCGSVFLItYLTTGA|NO| I ] IDSICDTEItOLAAN

EHV'4 HSV-1 VZV

EN•T•PAFNPKLYTPS••ALL•FPNGT•TL•SAFASYSAFK•PSTYLWA••GGLLLA•L•LY•••K•LCGG•••N•YSLLLNRE* PT~GAPS~F~SD~P~TALLLFP~GT~LLAFDT~PVAA~APG~LAA~ALGVVN~TAALAG~L~VLRTS~PFF~RRE* GNRT~F~DN~GDDSKA~LLF~NGTV~TLLGFERR~A~R~SG~YLGASLGGAFLAVVGFG~IG~L~GNSRLREYNK~LT*

I FALRENGRTTE¥ FLLOE IVDVOYQLKFLN¥ I LHR I GAGAHPNT |S G ~ L

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iv,s,,,,o,;ovs.,

wo

855 838 841

Fig. 6. Alignment of the predicted primary translation products of the EHV-4, HSV-1 (McGeoch & Davison, 1986) and VZV (Keller et al., 1987) gH genes. Conserved cysteines are asterisked and putative N-linked glycosylation sites are underlined.

E H V - 4 a n d HSV-1 g H s and, indeed, t h r o u g h o u t the alpha-, beta- a n d g a m m a h e r p e s v i r u s g H s i n v e s t i g a t e d i m p l i e s some degree o f c o n s e r v a t i o n o f the s e c o n d a r y a n d t e r t i a r y structure o f these p r o t e i n s p r e s u m a b l y i n v o l v i n g d i s u l p h i d e b o n d i n g ( G o m p e l s et al., 1988). T h e p o t e n t i a l o f g H to elicit a n t i b o d y w h i c h c a n neutralize viral i n f e c t i v i t y in the a b s e n c e o f c o m p l e m e n t suggests t h a t the g l y c o p r o t e i n m i g h t be a useful constituent of a vaccine preparation. The investigation o f the a n t i g e n i c c h a r a c t e r i s t i c s o f the E H V - 4 g H a n d o f o t h e r glycoproteins specified by E H V - 4 should hopefully lead to a g r e a t e r u n d e r s t a n d i n g o f t h e i r i n d i v i d u a l a n d collective c o n t r i b u t i o n to the elicitation o f p r o t e c t i v e i m m u n i t y in infected horses. This should assist in the d e v e l o p m e n t o f v a c c i n e s w h i c h elicit a m o r e effective p r o t e c t i v e i m m u n e response t h a n the short-lived, a p p a r ently m i s d i r e c t e d response elicited by n a t u r a l E H V - l a n d -4 infection. We are grateful for the help of Mrs M. Riddell, Mr A. May and Mr J. Fuller in the preparation of this manuscript. We thank the Equine Virology Research Foundation for their generous funding.

References ALLEN, G. P. & BRYANS, J. T. (1986). Molecular

epizootiology,

pathogenesis and prophylaxis of equine herpesvirus-1 infections. In Progress in Veterinary Microbiology and Immunology, vol. 2, pp. 78144. Edited by R. Pandey. Basel: S. Karger. ALLEN, G. P. & COOGLE,L. D. (1988). Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gpl3) with homology to herpes simplex virus glycoprotein C. Journal of Virology 62, 2850-2858. ALLEN,G. P. & YEARGAN,M. R. (1987). Use of 2 gtl I and monoclonal antibodies to map the genes for the 6 major glycoproteins of equine herpesvirus 1. Journal of Virology 61, 2454-2461. BAER,R., BANKIER,A. T., BIGGIN, M. D., DEININGER,P. L., FARRELL, P. J., GIBSON,T. J., HATFULL,G., HUDSON,G. S., SATCHWELL,S. C., S~GUIN, C., TUFFNELL, P. S. & BARRELL, B. G. (1984). DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature, London 310, 207 211. BAUMANN,R. P., YALAMANCHILI,V. R. R. & O'CALLAGHAN,D. J. (1989). Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication. Journal of Virology63, 1275-1283. BIRNSTIEL,M. L., BUSSLINGER,M. & STRUB,K. (1985). Transcription termination and 3' processing: the end is in site! Cell 41, 349-359. BRIDGES,C. G., LEDGER,N. & EDINGTON,N. (1988). The characterisation of equine herpes virus-l-infected cell polypeptidesrecognised by equine lymphocytes. Immunology 63, 193 198. BUCKIVr_ASTER,E. A., GOMPELS,U. & MINSON,A. (1984). Characterization and physical mapping of an HSV-1 glycoprotein of approximately 115 x l03 molecular weight. Virology 139, 408-413. CARTER, V. C., SC/4-AFFER, P. A. & TEVETI-1IA, S. S. (1981). The involvement of herpes simplex virus type 1 glycoproteins in cellmediated immunity. Journal of Immunology 126, 1655-1660. CORDEN, J., WASYLYK, }]., BUCHWALDER,A., SASSONE-CORsI,P., KEDINGER, C. & CHAMBON, P. (1980). Promoter sequences of eukaryotic protein-coding genes. Science 209, 1406-1414. CRANAGE, M. P., SMITH, G. L., BELL, S. E., HART, H., BROWN, C., BANKIER,A. T., TOMLINSON,P., BARRELL,B. G. & MINSON,A. C.

1800

L. Nicolson, A. A. Cullinane and D. E. Onions

(1988). Identification and expression of a human cytomegalovirus glycoprotein with homology to the Epstein-Barr virus BXLF2 product, varicella-zoster virus gpllI, and herpes simplex virus type 1 glycoprotein H. Journal of Virology 62, 1416-1422. CULLINANE,A. A., RIXON,F. J. & DAVISON,A. J. (1988). Characterization of the genome of equine herpesvirus 1 subtype 2. Journal of General Virology 69, 1575-1590. DAVISON,A. J. & SCOTT,J. E. (1986). The complete DNA sequence of variceUa-zoster virus. Journal of General Virology 67, 1759-1816. DESAI, P. J., SCHAFFER,P. A. & MINSON, A. C. (1988). Excretion of non-infectious virus particles lacking glycoprotein H by a temperature-sensitive mutant of herpes simplex virus type 1 : evidence that gH is essential for virion infectivity. Journalof General Virology 69, 1147-1156. FULLER, A. O., SANTOS, R. E. 8/. SPEAR, P. G. (1989). Neutralizing antibodies specific for glycoprotein H of herpes simplex virus permit viral attachment to cells but prevent penetration. Journal of Virology 63, 3435 3443. GARNIER,J., OSGUTHORPE,D. J. & ROBSON,B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. Journalof Molecular Biology 120, 97-120. GOMPELS, U. & MINSON, A. (1986). The properties and sequence of glycoprotein H of herpes simplex virus type 1. Virology 153, 230-247. GOMPELS, U. A., CRAXTON, M. A. & HONESS, R. W. (1988). Conservation of glycoprotein H (gH) in herpesviruses: nucleotide sequence of the gH gene from herpesvirus saimiri. Journalof General Virology 69, 2819-2829. GRETCH, D. R., KARl, B., RASMUSSEN,L., GEHRZ, R. C. & STINSKI, M. F. (1988). Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus. Journal of Virology 62, 875-881. HEINEMAN,T., GONG, M., SAMPLE,J. & KIEFF, E. (1988). Identification of the Epstein-Barr virus gp85 gene. Journal of Virology 62, 1101-1107. HOpp, T. P. & WOODS, K. R. (1981). Prediction of protein antigenic determinants from amino acid sequences. Proceedingsof the National Academy of Sciences, U.S.A. 78, 3824~3828. KELLER, P. M., DAVlSON,A. J., LOWE, R. S., RIEMEN,M. W. & ELLIS, R. W. (1987). Identification and sequence of the gene encoding gpIII, a major glycoprotein of varicella-zoster virus. Virology 157, 526-533. KOZAK, M. (1984). Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Research 12, 857-872. LOCKSHON,D. & GALLOWAY,D. A. (1986). Cloning and characterization of oriEL, a large palindromic DNA replication origin of herpes simplex virus type 2. Journal of Virology 58, 513-521. MCGEOCH,D. J. & DAVISON,A. J. (1986). DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genome of varicella-zoster virus and Epstein-Barr virus. Nucleic Acids Research 14, 42814292. MCLAUCHLAN,J., GAFFNEY,D., WHITTON, J. L. & CLEMENTS,J. B. (1985). The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini. Nucleic Acids Research 13, 1347-1368. MEREDITH, D. M., STOCKS,J.-M., WHI'FFAKER,G. R., HALLIBURTON, I. W., SNOWDEN,B. W. ~ KILLINGTON,R. A. (1989). Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies. Journal of General Virology 70, 1161 1172. MILLER, N. & HUTT-FLETCHER,L. M. (1988). A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of EpsteinBarr virus. Journal of Virology 62, 2366-2372.

MONTALVO, E. A. & GROSE, C. (1986). Neutralization epitope of varicella-zoster virus on native viral glycoprotein gpll8 (VZV glycoprotein gpIII). Virology 149, 230-241. NICOLSON,L. & ONIONS,D. E. (1990). The nucleotide sequence of the equine herpesvirus 4 (EHV-4) gC gene homologue. Virology (in press). NICOLSON, L., CULLINANE, A. A. & ONIONS, D. E. (1990). The nucleotide sequence of the equine herpesvirus 4 thymidine kinase gene. Journal of General Virology 71, 1801-1805. OBA, D. E. & HUTT-FLETCHER,L. M. (1988). Induction of antibodies to the Epstein-Barr virus glycoprotein gp85 with a synthetic peptide corresponding to a sequence in the BXLF2 open reading frame. Journal of Virology 62, 1108-1114. PROUDFOOT,N. J. & BROWNLEE,G. G. (1976). 3' Non-coding region sequences in eukaryotic messenger RNA. Nature, London 2,63, 211214. QUEEN, C. & KORN, L. J. (1984). A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Research 12, 581-599. RIGGIO, M. P., CULLINANE, A. A. • ONIONS, D. E. (1989). Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4. Journal of Virology 63, 1123-1133. ROBERTSON,G. R. 8¢ WHALLEY,J. M. (1988). Evolution of the herpes thymidine kinase gene: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cellencoded thymidylate kinase. Nucleic Acids Research 16, 1130311317. ROSENTHAL,K. L., SMILEY,J. R., SOUTH,S. & JOHNSON,D. C. (1987). Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognised by murine virus-specific cytotoxic T lymphocytes. Journal of Virology 61, 2438 2447. SANGER,F., NICKELS, S. 8¢ COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. SPEAR, P. G. (1985). Glycoproteins specified by herpes simplex viruses. In The Herpesviruses, vol. 3, pp. 315-356. Edited by B. Roizman. New York: Plenum Press. STOKES,A., ALLEN, G. P., PULLEN,L. A. & MURRAY,P. K. (1989). A hamster model of equine herpesvirus type 1 (EHV-1) infection; passive protection by monoclonal antibodies to EHV-1 glycoproteins 13, 14 and 17/18. Journal of General Virology 70, 1173-1183. STOW, N. D. & DAVISON,A. J. (1986). Identification of a varicellazoster virus origin of DNA replication and its activation by herpes simplex virus type 1 gene products. Journal of General ViJ "!ogy 67, 1613-1623. STOW, N. D. & MCMONAGLE,E. C. (1983). Characterization of the TRs/IR s origin of DNA replication of herpes simplex virus type 1. Virology 130, 427438. TURTINEN,L. W. 8/. ALLEN,G. P. (1982). Identification of the envelope surface glycoproteins of equine herpesvirus type 1. Journalof General Virology 63, 481485. YON HEIJNE, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 14, 4683~4690. WHALLEY,J. M., ROBERTSON,G. R., SCOTT, N. A., HUDSON, G. C., BELL, C. W. & WOODWORTH, L. M. (1989). Identification and nucleotide sequence of a gene in equine herpesvirus 1 analogous to the herpes simplex virus gene encoding the major envelope glycoprotein gB. Journal of General Virology 70, 383-394; corrigendum 3513.

(Received 29 March 1990; Accepted 24 April 1990)