Nucleotide sequence of the genes encoding the ... - Semantic Scholar
Journal of General Virology (1994), 75, 2029 2039. Printed in Great Britain
2029
Nucleotide sequence of the genes encoding the canine herpesvirus gB, gC and gD homologues Keith J. L i m b a c h , M . Paullin L i m b a c h , Darryl C o n t e and E n z o P a o l e t t i * Virogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy, N e w York 12180-8349, U.S.A.
The nucleotide sequence of the genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues was determined. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequences of CHV gB, gC and gD with the homologous sequences from other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. Alignment of the homologous gB,
gC and gD amino acid sequences indicates that most of the cysteine residues are conserved, suggesting that these glycoproteins possess similar tertiary structures. The nucleotide sequence of the open reading frame downstream from the CHV gC gene was also determined. The predicted amino acid sequence of this putative polypeptide appears to be homologous to a family of proteins encoded downstream from the gC gene in most, although not all, alphaherpesviruses.
Introduction
virulent PRV challenge (Brockmeier et al., 1993). Furthermore, vaccinia virus recombinants expressing PRV gII and gp50, or gII, gIII (gC) and gp50 have been shown to elicit a higher level of protection than recombinants expressing gII or gp50 alone, suggesting a potential synergistic effect with these glycoproteins (Riviere et al., 1992). The results of these studies indicate that an immune response against gB, gC a n d / o r gD can protect target species animals against a herpesvirus challenge. In this communication, we report the nucleotide sequence of the genes encoding the CHV gB, gC and gD homologues. This information will form a database upon which potential CHV vaccine candidates can be developed.
Canine herpesvirus (CHV) causes a fatal haemorrhagic disease in neonatal puppies and a self-limiting, usually subclinical, upper respiratory tract infection in adult dogs (Appel, 1987). Little is known about the genomic structure of CHV. Restriction enzyme maps of the CHV genome and nucleotide sequence of CHV genes have not been published. In particular, genes encoding immunologically pertinent proteins have not been characterized. During a herpesvirus infection, the majority of the immune response is directed against the viral envelope glycoproteins. These antigens have been shown to elicit both humoral and cellular immune responses. The contribution of the individual glycoproteins in eliciting a protective immune response, however, is not completely understood. Several reports have indicated that immunization with the herpesvirus gB, gC a n d / o r gD glycoproteins can induce a protective immune response. For example, immunization with purified bovine herpesvirus type 1 (BHV-1) gI (gB), gIII (gC) a n d / o r gIV (gD) has been shown to protect cattle against a BHV1/ Pasteurella haemolytica challenge (Babiuk et al., 1987). In addition, immunization with a fowlpox virus recombinant expressing Marek's disease virus (MDV) gB has been shown to protect chickens against a virulent MDV challenge (Nazarian et al., 1992). Immunization with NYVAC (highly attenuated vaccinia virus) recombinants expressing pseudorabies virus (PRV) gII (gB) or gp50 (gD) have also been shown to protect swine against a 0001-2025 © 1994 SGM
Methods Preparation of genomic CHV DNA. CHV (obtained from L. Carmichael, Cornell University, Ithaca, N.Y., U.S.A.) was propagated on Madin-Darbycaninekidney(MDCK)cells(ATCC CCL34). Viral DNA was isolated using standard methods (Tartaglia et al., 1990). DNA hybridization. CHV genomic DNA was digested with restriction enzymes,run on agarose gels and transferredto Gene-Screen membranes(New EnglandNuclear)under conditionsrecommendedby the manufacturers.Hybridizationswere performedat 44 °C, 53 °C or 59°C in 1M-NaC1, 1% SDS and 10% dextran sulphate. The hybridization probe included a 1800bp BamHI-XbaI fragment, containingan internal segmentof the felineherpesvirus(FHV) gB gene (Maeda et al., 1992), a 950 bp BamHI-EcoRI fragmentcontainingthe 3' end of the FHV gC gene (J.-C. Audonnet,unpublishedresults), and
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K. J. Limbach and others
a 970 bp BamHI-HindIIl fragmentcontainingthe 3' end of the FHV gD gene (J:C. Audonnet, unpublished results). Cloning and DNA sequencing. CHV genomic fragments were subcloned into pBluescript SK (Stratagene). Plasmid DNA was prepared and manipulatedusing standard techniques(Maniatis et al., 1982). Nucleotide sequencing was performed on both strands with double-stranded plasmid templates, using the modified T7 DNA polymerase, Sequenase Version 2.0 (U.S. BiochemicalCorporation), and standard protocols recommended by the manufacturer. M13 forward and reverse primers were used to obtain initial sequence; and custom primers, prepared with a Biosearch 8700 or an Applied Biosystems380B oligonucleotidesynthesizer,were used for subsequent reactions. DNA and amino acid sequence analyses. DNA and amino acid sequence analyses were performed with PC/GENE (IntelliGenetics), ALIGN Plus (Scientificand Educational Software) and IBI-Pustell (International Biotechnologies)softwarepackages.Homologysearches were conducted on the SWlSS-PROT (Release 20 and 23) (lntelliGenetics) databases, using the FASTA program (Pearson & Lipman, 1988).
Results Identification and sequencing of the C H V gB gene
Hybridization of restriction enzyme-digested CHV genomic DNA with a radiolabelled probe containing the FHV gB, gC and gD genes (Maeda et at., 1992; J.C. Audonnet, unpublished results) identified one complementary sequence (data not shown). A 6 kb XbaI fragment containing this sequence was cloned into pBluescript SK and the nucleotide sequence of the hybridizing region was determined (Fig. 1). An open reading frame (ORF) starting at position 201 and ending at position 2840 was identified. The predicted translation product of this ORF is 879 amino acids long. Comparison of this amino acid sequence with the SWISS-PROT (Release 20) database revealed significant homology with the gB glycoprotein of numerous herpesviruses. Additional analyses revealed that the predicted CHV gene product was more similar to the gB glycoprotein of alphaherpesviruses, such as herpes simplex virus type 1 (HSV-1), than those of beta- or gammaherpesviruses, such as human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV) (Table 1). These results indicate that CHV should be classified as an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties (Carmichael et at., 1965; Roizman, 1982).
Analysis o f the C H V gB nucleotide sequence
The 5' and 3' non-coding regions of the CHV gB gene contain numerous R N A polymerase II regulatory sequence motifs, such as TATA box, CAAT box and
polyadenylation signal sequences (Corden et al., 1980; Proudfoot & Brownlee, 1976) (Fig. 1). The nucleotide sequence surrounding the initiation codon has been shown to affect the efficiency of translation initiation (Kozak, 1986). In particular, the sequence ( A / G ) N N A T G G has been found to be most efficient. Therefore, according to Kozak's rules, the nucleotide sequence surrounding the CHV gB initiation codon (AGTATGT) is favourable at position - 3 , but not at position + 4 (Fig. 1). The fact that the CHV gB gene does not follow Kozak's rules is not unusual. The FHV (Maeda et al., 1992), PRV (Robbins et al., 1987), varicella-zoster virus (VZV) (Keller et al., 1986), MDV (Ross et al., 1989) and HSV-1 (Bzik et al., 1984) gB genes also contain a pyrimidine at position + 4. Analysis of the predicted C H V gB amino acid sequence
The deduced amino acid sequence of the CHV gB homologue is presented in Fig. 1. Hydropathicity analysis of this amino acid sequence revealed the presence of two prominent hydrophobic peaks (data not shown). The first peak, located at the N terminus, may represent a potential signal sequence. N-terminal signal sequences initiate transport across the endoplasmic reticulum membrane and are critical for the proper posttranslational modification and targeting of glycoproteins (Blobel, 1980). Signal sequences vary in length from about 15 to 30 residues and usually consist of a basic Nterminal region, a central hydrophobic region and a short, relatively polar C-terminal region. In addition, the cleavage site usually conforms to the - 3, - 1 rule, where the residue at position - 1 is small (Ala, Ser, Gly, Cys, Thr or Gln) and the residue at position - 3 is not aromatic (Phe, His, Tyr or Trp), charged (Asp, Glu, Lys or Arg) or large and polar (Asn or Gln), and residues - 3 to + 1 are not Pro residues (von Heijne, 1986). Although analysis with PSIGNAL, a P C / G E N E program designed to detect eukaryotic signal sequences, did not identify the N terminus of CHV gB as a potential signal sequence, this region does have elements consistent with typical signal sequences, namely a hydrophobic core (residues 2 to 17) and a relatively polar C-terminal region (Fig. 1). That PSIGNAL does not detect a signal sequence in the N-terminal region of CHV gB is not unique. This algorithm also fails to detect a signal sequence in the Nterminal region of the VZV gB homologue. The second, very broad, hydrophobic peak is located near the C terminus, with predicted membrane-spanning segments between amino acid residues 725 and 741, and 747 and 771 (using the method of Klein et al., 1985), and probably functions as a membrane anchor region. It has been hypothesized that the transmembrane domain of HSV-1 gB, as well as other gB homologues, traverses the
Sequence of CH V gB, gC and gD homologues
2031
TTTTCTGGATTTCAGCTATGTCCTTCGGGAGTTTATAT~CTTAT~GAAAACTGTCCTTTGGTA~CAGTTTTACAAAGCGGTGTAAATTGCGAAATTGGACC~CTA~CTGTM~
120
T~CGACAGTGATATTTTTTCTCTTCTTTATAcCGTTCTTCAAAAATT--GGGCTCCTGGTGTT~TATAGAAATTT~T~GTATGTTTTcATTGTATCTATATATTTTTTTTATTATTTATA 240 M
F
S
L
Y
L
Y
Z
F
F
I
]
Y
13
CTTTAAT~TAT~TGAT~C~C~CACCGGAAAGTACTATT~TC~TTAAAT~TCA~TTTATC~CACCT~CCTACTTCG~T~TATTCGTGAAATTTTACGTG~TCCC~ 360 T L ] I C D P T T P E S T ] N P L N H H N L S T P K P T S D D I R E I L R E $ 0 53 TT~ATCTGATGATACATC~CATTTTACATGTGCCCACCACCATCGG~TC~CATTGGTGCGTTTG~GCCACCTA~GCATGTCCT~TATA~ACTTGGTAAJBJ~ATTTTA~A~G I E S D D T S T F Y N C P P P S G S T L V R L E P P R A C P N Y K L G K N F T E
480 93
G~TTGCTGT~TATTT~GGAAAATATTT~TCCTTAT~TTTAAAGCT~TATATACTACAAAAATATTATTATCACCACTGTATGGTCTGGAAGCA~TATGCAGT~TTACT~TA G I A V I F K E N I S P Y K F K A N ] Y Y K N I I I T T V W S G S T Y A V I T N
600 133
GATATACAGATCGTGTACCTATAGGTGTTCC~GAAATTACA~GTT~TT~TA~GGTATGTGTTTATCA/~L/AAG~T~TTATATTCG~T~TTATG~TTA~CGCATTT~TA F
T
A
F
D
720 I~
AGGATG~GA~AGA~GTT~ATTT~G~cTTC~GTTT~TACACCAG~TCc~GTGGATGG~ATA~AGTT~TAc~TA~TTG~GGGTTCTG~TTTTATCATT K D E D P R E V H l K P S K F N T P G S R G W H T V N D T Y T K I G G S
G
F
Y
H
~0 213
CTGGAACAT~TGT~TTGTATAGTTG~GTT~TG~CA~TC~GT~TAT~CATAT~TT~ATTTGCTATCT~CCGGG~TAT~TT~ATATGTC~TTTTTTTGGATTACGAG S G T S V M C I V E E V D A R S V Y P Y D S F A I S T G D I I H N S P F F G l
R
960 253
ATGGTG~T~ATACTG~TATATTAGTTATT~cT~TA~TTTC~TAG~GGTTATTATCCTAT~cTTAGATA~TA~C~A~AGCTT~GTGCA~CA~TTT~TA~G~TTTTT D G A H T E Y I S Y S T D R F Q Q I E G Y Y P ] D l D T R L Q l G A P V S R N
F
1080 293
TAA~AA~A~AA~AcGTTAC~GTTGCTTGG~TTGGGTT~TTcGTG~GTGT~TACTTTGGCT~TGGCGTGAAATT~GAAATTATTCGTGAT~G~A~GGGATCTTACA L T T Q H V T V A ~ N W V P K I R E V C T l A K W R E I D E I I R D E Y K G S
Y
1200 333
~TTTA~AGCAAAAT~TATCTGC~ATTTATTT~T~TACTACT~T~TGATATTGATCGTGT~TT~GT~TTGTGC~GTG~GC~A(A~GCTATT~T~GAT~T R F T A K S I S A T F I S D T T Q F D I 0 R V K L S D C A K R E A I E A
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0
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0
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1320 3~
A~AAAA~TAT~T~T~ATATT~CAGGA~TTGGAAACATA~TTGG~TAGAGGGG~TTTATTATAG~ATT~A~CC~TGATTAGT~T~G~TAGCAAAATTGTATA Y K K K Y N K T H I ~ T G E l E T Y l A R G G F ] I A F R P M I S N E L A K
l
Y
1440 413
N
1560 453
I
TA~TGAGTTAGT~GAT~T~T~GTA~GGTTGATTTG~TCTcTTTT~T~CATCTGT~GAGGGGGGGCTAC`/~`A-~G~T~AGTA~GG/~AAATA]q~J~CAAAACGT~TA I
N
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G A
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R
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E
N
K
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S
K
R
TTG~GGTGGTATTGA~TGT~T~TTC~C~T~TT~GAC~CTTCATCTGTT~ATTTTGCTATGCTTCAGTTTGCCTATGATCATATTC~T~ACATGTT~TGAAATGCTTA1680 ] E G G I E M V N N S T I ] K T T S S V H F A H L O F A Y D H ] O S H V N E M L ~93
GTAGAATT~AA~TG~AT~TGT~T~TTCAAAAT~GAGA~CCCTTTG~T~GTTATG~TT~TCC~CTAGTGTGGCTTCGGTTGCTATGGAT~pd~p~GAG~TTCAGCAC 1800 S
R
I
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A
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533
GAATG~TAGGGGATGTT~TTG~AGTTA~TC~GTGT~TATATCAGGTTCTAGTGTTTTTATT~TT~CA~GCGTGTT~TAGGGT~C~CTA~ATGTTACAGT~GTc~T~TTA R H l G D V l A V T Q C V N [ S G S S V F [ 0 N S N R V L G S T T T C Y S R P L
1920 5~
TAT~ATTTAAAGCACTAG~TC~T~CTATATT~G~CTTGGGGAAAAT~T~TATTAGTAG~C~GCT~TT~CcATGTACAGCT~CCAT~GATATT ] S F K A L E N S T N Y ] E G 0 L G E N N E l l V E R K L I E P C T
20~0 613
A
0
N
R
H
V
K
S
R
Y
TTAAATTTGGTGCAGATTATGTATATTTTGAAAACTATGcATATGTT~GAAAGG~ACCTCTT~TGAAATT~TGAT~AGTGCATATGTAGATCTT~TATTACATTACTTGAGGATC F K F G A D Y V Y F E N Y A Y V R K V P l N E I E N I S A Y V D l N I T l l E D
2160 653
GTG~TTTTTACCACTAGAGGTATATACTCGAG~AGAGTTA~GATACAG~C~ATTG~CTATAGT~GATTC~GTAG~TC~CTACAT~CACTT~GTTTTATGATATTGA~A R E F l P l E V Y T R A E L E 0 T G l l D Y S E [ ~ R R N Q l H A l K F Y D I 0
2280 693
GTGTTGTAAAAGTTGAT~T~TGTTGT~TTATGAG~G~ATTGC~TTTTTTCC~G~CT~G~TGT~G~GCGGGATTTGGAAAA~TTGTTTTGGGTGCTGC~G~TGTTA S V V K V D N N V V I N R G I A N F F O G L G 0 V G A G F G K V V l G A A N A
V
2~00 ~3
TTGCAACTGTTTCTGGAGTGTCCTCGTTTCTT~T~cCCATTTGGG~CG~TAG~GT~G~TTGCT~TTTTAGCTG~CTAT~TGCAGCGTTTTTGGCTTATA~TATGT~TCT~AC [ A T V S G V S S F l N N P F G A l A V G L l ] l A G l F A A F L A Y R Y V S K
2520 7~
TTAAGTCA~T~CAATG~G~ACTATACCCAGT~TA~TTT~G~GTGTT~TGGT~TTCTG~T~TAGTGATGGAG~GAAAAT~TGAT~AT~TG2~ l K S N P M K A l Y P V T T K N l K E S V K N G N S G N N S D G E E
N
D
0
N
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813
AAG~AG~TTC~C~GCT~G~TGATT~TATATGTCTCTAGTTTCTGCTATG~AGCAGG~CAT~GCTATT~TAGTG~CCTGCCCTTcTAGCAAGT~ACA E E K L O 0 A K E N I K Y N S L V S A N E Q 0 E H K A [ K K N S G P A l
L
A
S
H
2760 853
TTACAAAc~TATCTCTT~CATCGTGGTc~AAAA~ACAAA~GTTTGAAAAA~GT~d~TGAAAGT~GTTT~TAJU~TTT~TATTA~GTAAAATTTTCTGACTCTGC~ [ T N l S l K H R G P K Y K R l K N V N E N E $ K V *
2~0 879
~ACTTT~T~TAT~TAT~TTTTAGAJ~ATTTTA~T~ATTTTATTATCTTTTAT~CCTC~CTATTTAT~G~T~T~TG~ATTT~TG~GGTGCCTGTATAT~CTACT~
3000
Fig. l. Nucleotide sequence and predicted amino acid sequence of the CHV gB homologue. The putative cleavage site, transmembrane regions, potential glycosylation sites, TATA box, CAAT box and polyadenylation signal sequences are underlined. Nucleotides and predicted amino acid residues are numbered to the right of the sequence.
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K. J. Limbach and others
Table 1. Homology between the predicted amino acid sequences o f 10 herpesvirus gB glycoproteins* FHV CHV FHV EHV-1 PRV BHV-1 VZV MDV HSV-1 HCMV
EHV-1 PRV BHV-1 VZV MDV
78
. . .
61 57 . .
61 59 52 . .
.
59 58 52 63 -
.
. .
.
52 48 45 48 46 49
. .
.
55 51 47 52 52
HSV-1 HCMV EBV
.
.
.
50 48 44 50 48 48 48
29 27 27 29 28 30 30 28
27 27 26 29 28 28 29 29 32
* Values were obtained usingthe ALIGN Plus program and are expressedas percentageidentity. The entire gB amino acid sequence was used. The alignment parameters were: mismatch penalty = 2, open gap penalty = 4, extended gap penalty = 1. References: FHV (Maeda et al., 1992), EHV-1 (Whalleyet al., 1989),PRV (Robbins et al., 1987), BHV-I (Whitbecket al., 1988), VZV (Kelleret al., 1986),MDV (Ross et al, 1989),HSV-1 (Bziket al., 1984),HCMV (Kouzarides et al., 1987)and EBV (Pellett et al., 1985).
membrane three times (Pellett et al., 1985). As hydropathicity analysis of the C-terminal region of CHV gB reveals the presence of at least two membrane-spanning regions, it is possible that CHV gB and HSV-1 gB have similar transmembrane structures. Alignment of the CHV gB amino acid sequence with similar sequences from other herpesviruses revealed extensive homology throughout the entire sequence, with the exception of the N terminus, a region surrounding the putative cleavage site (see below) and a region near the C terminus (data not shown). This alignment also revealed that most of the cysteine residues are conserved. For example, CHV gB contains 11 cysteine residues, 10 of which are conserved in all alpha-, beta- and gammaherpesviruses. In fact, the only cysteine residue in CHV gB that is not conserved is found near the N terminus and may be located in the putative signal sequence. These results suggest that the gB glycoproteins may have similar tertiary structures. N-linked oligosaccharides can be added to Asn residues which have the sequence Asn-X-Ser/Thr, where X is not a Pro residue (Bause, 1983). Alignment of the CHV gB amino acid sequence with other gB sequences revealed that potential N-linked glycosylation sites are relatively well conserved (data not shown). It is not known whether glycosylation at these sites is structurally or functionally significant. The gB glycoprotein o f most herpesviruses is cleaved internally during maturation, with the subsequent peptides being held together by disulphide bonds. The VZV gB homologue (gpII), for example, is cleaved between Arg and Ser residues, resulting in two glycoproteins of approximately 60K (Keller et al., 1986). The gB glycoproteins o f FHV (Maeda et al., 1992), equine herpesvirus type 1 (EHV-1) (Whalley et al., 1989), PRV (Robbins et al., 1987), BHV-1 (Whitbeck et al., 1988),
MDV (Ross et al., 1989) and H C M V (Kouzarides et al., 1987) are also cleaved. Furthermore, a sequence, Arg-XArg-Arg/Lys--Ser/Ala, similar to the sequence at the VZV cleavage site, Arg-Thr-Arg-Arg--Ser, is present at virtually the same location in each of these gB glycoproteins. Conversely, this sequence is not found in the HSV-1 (Bzik et al., 1984) and EBV (Pellett et al., 1985) gB glycoproteins, which are not cleaved. The significance of this cleavage event is unknown. It does not appear, however, to be essential for replication in vitro, as strains of BHV-1 (Blewett & Misra, 1991) and H C M V (Spaete et al., 1990) that have been mutated at the cleavage site, and therefore encode an uncleavable gB glycoprotein, are still infectious. It is not known whether CHV gB is cleaved internally. However, the sequence Arg-Lys-ArgArg--Ser is present at approximately the same location in CHV as in VZV, FHV, EHV- 1, PRV, BHV- 1, MDV and H C M V (data not shown). Therefore, it is probable that CHV gB is proteolytically cleaved. Identification and sequencing o f the C H V g C gene
A radiolabelled probe containing the 3' end of the F H V gC (and gD) gene(s) did not hybridize to restriction enzyme fragments of CHV genomic D N A (see above). Therefore another method of analysis was used to identify the CHV gC (and gD) gene(s). CHV genomic fragments were randomly cloned into pBluescript SK. The nucleotide sequences of the termini of these fragments were determined and the predicted amino acid sequences of potential ORFs were analysed for homology against the SWISS-PROT (Release 20) amino acid database. Using this method, a 12 kb XbaI fragment encoding an O R F with homology to herpesvirus gC glycoproteins was identified. The nucleotide sequence of this O R F is presented in Fig. 2. The putative CHV gC
Sequence of CHV gB, gC and gD homologues
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CGAGc~CTAATTATTGGTTTGTATATGACTGTTGG~TTTGTTACATTTTTATTAAAA~T~TT~TTTTTTAAAcTATATTAC~GTTGTGT~TGTTTT~GTTTTAAATAAAG~ 120 AT~T~TCG~TT~A~ATTTATCA/~U~ACATTA~A~U~CC~CA~TTT~TAT~]~T~GGT~T~GTCAJU~T~GT1TTA~ATTTTTATCT~TATATGT~TTAT~240 M S F K N F Y L I Y Y ~ I 13 TTTTTATA~CTCGAT~T~CTTCGGCATCTACAT~C~cTT~CAC~TAC~T~TTCCM~TTCAGCAAAT~TCACCTGcTTCCATA~TAC~CTAT~CAAAACCTATAT I F I N S I I T S A S T S K P S T P T I I P T S A N E S P A S I D T T I T K P I
360 53
CTACAGAGGCAAAT~TTTAAAATCAGT~GTACCTC~TTAAACCACCTAAAAACTTAAAA~J~g~TTACTTAAAT~TAAATGTA~T~TGTTATTTATAGGCCATATTTTAGTC480 S T E A N M k K S V S T S I K P P K N k K K K L k K S K C R D N V I Y R P Y F S 93 ~TTAG~TT~CTGTACTAT~CTA/UU~GCA/d~TTT~GT~TCCTTT~TTGAGTTAT~GTTTAAAG~CTTTCTACATATMTAA/~CC~TGAAAATGTTGAAAGTTTAAJU~ 600 O L E I N C T I T K K O N L $ N p L ] E L W F K E L S T Y N K T N E N V E S L K 133 CAGATATATC~AAATATTTTATTATTTTCGACAAAAAAT~TAGT~T~CTTTTAT~T~TTTTTTATTAGGTATA~AAAJkTC~CCAGTAAATTATAAACTTTACGGTTCCC~T 720 T D I S K N I L L F S T K N N S D N F Y N D F L L G I Q N Q P V N Y K L Y G S Q 1~ TTTATGAT~TGGAAACATATTACTAAATATAAAGTCGGTT~CTTTA/~a~CCTCTG~TATATACTTGGAAACTATAT~TTCAAAT~TGAAAGTATTTTTGAAACTTTTAAAATTC ~0 F y D N G N I L L N I K S V D F K T S G I Y T W K L Y N S N N E S I F E T F K I 213 ~GTATATGCATATCATTCCCCAAATGTAAACTTAAAATCAAACCC~GTTTATAT~TGAAAACTACAGCGCTATTTGTAC~TAGC~TTACTTTCCATTG~TCTACGGAAATAT960 Q V y A y H S p N V N L K S N P S k Y N E N Y S A I C T I A N Y F P k E S T E I 253 TTTGGTTT~CGATGGA~CTATT~TAAAAAATATATAGATG~CTTATAGTGTATG~TT~CGGTCTTAT~CACG~A~TTC~TATTAT~CCTTCCCTTTT~GAAGCCATGG 1080 F W F N D G O p I D K K y I D E T y S V W I D G k I T R T S I L S L P F S E A M 293 ~GC~CCCCC~TTTGCGATGT~TGTT~TGGTATAAAAATTC~GG~ATCAAAAAAATTTTCAAATACCGTTATTCCAAAAGTTTACTATAAACCTTTTATATCTATAAAATTTG 1200 E S p p N k R C N V E W y K N S K A S K K F S N T V [ P K V Y Y K P F I S I K F 333
AT~TGGTTTAG~TATTTGTGATGCTAAATGTGTTTCCCGTGA~U~T~TAAATTAC~TGGT~GTTA~L4~TATACCTATAj~TGGTGATGATATTAT~GCGGCCCCTGTTT~CC 1320 0
N G
k
A
I
C D A
K
C V
S
R E
N N K
L
O W L
V
K
D
I
P
l
N G D D
I
I
S
G P
C
L
N
3~
AC~CTGGTTTGGTC~TATTCAAAATAAAATAGATATAT~G~TTAT~T~CCTGTTACCTATAAATGTTC~TTATTGGTTATCC~T~TTTTTCCC~CTTTTAT~TGAAAAGG 1440 H p G k V N I Q N K l D ] S D Y D E P V T Y K C S I ] G Y P I [ F P N F Y D E K 413 TGTTT~TG~ATCGGATGAAAATGTTAGT~T~TGTT~T~GTATTACCAC~T~TTGGTG~GCCATTTTTGTTATAGTATT~TTTTTAT~CAGC~TTATGTTTTTATTGTT 1560 V F D A S O E N V S K S M L [ S I T T l I G G A I F V I V L I F I T A L C F Y C 453 C~AAAAT~T~GATCT~TATC~TATTTACGTAAATG~TTATAT~TGTTATATTCGTGTTATTATGATTTAT~GTTCATCAAATTTAAAAATTTGTATAGTATT~TTTTTA 1~0 S K N N K I * 459
ATAGGGGTATCGTTT~TATGGCTCAGTTAGTTTT~cT~TATTC~TC~TGTGGAAAATAAAAATACTT~AT~C~c~GAAAC~CT~CTT~CCAGAAAAAATC~A M A Q L V L T D I p L E D V E N K N T S S D E E T T N L N g K K
T
1800 34
TGTC~TGTTTATGTGTTAcCCTTG~TTTTTTG~AGCTGG~TTTTATT~CCATAGcTGC~T~TTTTTACTTTTATTTTTACAGTACCATTAGAAATGCTTG~TCTATT~TTGT C Q C L C V T L G F F A A G I L L T I A A ] I F T F I F T V P L E M L G S I N C
1920 74
CCTCCATCTACATTTGGTATTGAT~TGTTTGTATC~C~TAAAAAAATCTATT~TTCTTATT~TTATCTAAAATATGTTATGATA~TTGTCAAATCCGAT~TCAGAGT p P S T F G I D N V C ] E P I K K S I N S Y S E l S K I C Y D R k S N P I N O S
2040 114
S
ACTATTAACTCCTTATT~CTGTTTT~TATGTTTGCA~TAAAAACTATGAAAATGTTTAT~TTGT~TAC~T~GTGAAAAAACATGT~TTCATC~TAGCTATTTGTCAAA~T 2160 T [ N S L L T V L N M F A D K N Y E N V Y N C N T H S E K T C N S S ] A I C Q T 154 AATCATCCACT~GTTCATTGGGAAATTTTGTTATTAAAATTAGAAAAATTTTTGGGTTTAAAT~T~TAAAAT~TAAACATTACTTTTTGTTTTTGTCTTTATTAAACAGTTGTA 2280 N H P L S S k G N F V I K I R K ] F G F K * 175
Fig. 2. Nucleotide sequence and predicted amino acid sequence of the CHV gC homologue and ORF 2. The putative transmembrane region, potential gIycosylation sites, TATA box, CAAT box and polyadenylation signal sequences are underlined. Nucleotides and predicted amino acid residues are numbered to the right of the sequence.
gene starts at position 201 and ends at position 1580. The predicted translation product is 459 amino acids long. Comparison of this amino acid sequence with that of gC glycoproteins from other herpesviruses revealed extensive homology, indicating that this ORF encodes the CHV gC homologue (Table 2).
Analysis of the CHV gC nucleotide sequence Numerous potential TATA box, CAAT box and polyadenylation signal sequences are found in the 5' and 3' non-coding regions of the CHV gC gene (Fig. 2). The nucleotide sequence surrounding the CHV gC initiation
codon (AAAATGA) is favourable with respect to Kozak's rules at position - 3 , but not at position +4. That the CHV gC gene does not follow Kozak's rules is not unusual, as the CHV gB gene (see above), as well as the gC genes of FHV (J.-C. Audonnet, unpublished results), EHV-I (Allen & Coogle, 1988) and VZV (Davison & Scott, 1986) also contain an unfavourable nucleotide at position + 4.
Analysis of the predicted CHV gC amino acid sequence The deduced amino acid sequence of the CHV gC homologue is presented in Fig. 2. Hydropathicity
2034
K. J. Limbach and others
T a b l e 2. Homology between the predicted amino acid sequences of nine herpesvirus gC glycoproteins*
CHV FHV EHV-1 EHV-4 PRV BHV-1 VZV MDV
FHV
EHV-I
EHV-4
PRV
BHV-I
VZV
MDV
HSV-1
44
32 32
34 33 81 -
27 29 31 32
27 31 32 31 37
29 28 30 31 27 29 -
27 25 27 25 25 25 22
25 23 27 27 29 27 22 23
.
-
.
.
.
.
* Values were obtained using the ALIGN Plus program and are expressed as percentage identity. The entire gC amino acid sequence was used. See Table 1 for alignment parameters. References: FHV (J.-C. Audonnet, unpublished results), EHV-1 (Allen & Coogle, 1988), EHV-4 (Nicolson & Onions, 1990), PRV (Robbins et al., 1986), BHV-1 (Fitzpatrick et al., 1989), VZV (Davison & Scott, 1986), MDV (Ihara et al., 1989) and HSV-1 (McGeoch et al., 1988).
T a b l e 3. Homology between the predicted amino acid sequences of the ORFs
located adjacent to the gC gene in eight herpesviruses*
CHV FHV EHV-1 EHV-4 MDV HTV HSV- 1
FHV
EHV-1
EHV-4
MDV
HTV
197 (22) -
211 (22) 177 (24)
219 (21) 167 (18) 470 (50) . .
62 (4) 69 (4) 95 (8) 132 (8)
105 (13) 66 (4) 104 (9) 130 (11) 767 (75)
. -
.
. -
H S V - 1 VZV 53 (4) 40 (1) 79 (7) 60 (5) 83 (6) 91 (7) -
31 (0) 52 (1) 58 (3) 30 (0) 28 (0) 33 (0) 49 (2)
* Values were obtained using the FASTA and RDF2 programs (Pearson & Lipman, 1988)~ A ktup of 1 was used. Values in parentheses represent the number of standard deviations between the FASTA score and the mean of the scores obtained from 100 randomly permutated versions of the potentially related sequence. References: FHV (J.-C. Audonnet, unpublished results), EHV-1 (Telford et aL, 1992), EHV-4 (Nicolson & Onions, 1990), MDV (Ihara et al., 1989), HVT (Kato et aL, 1989), HSV-1 (McGeoch et al., 1988) and VZV (Davison & Scott, 1986).
analysis o f the p r e d i c t e d C H V g C a m i n o acid sequence revealed the presence o f two p r o m i n e n t h y d r o p h o b i c p e a k s ( d a t a n o t shown). T h e first p e a k , l o c a t e d at the N terminus, m a y r e p r e s e n t a p o t e n t i a l signal sequence. H o w e v e r , analysis w i t h P S I G N A L d i d n o t identify the N t e r m i n u s o f this p o l y p e p t i d e as a p o t e n t i a l signal sequence. Nevertheless, this r e g i o n does c o n t a i n elements o f a typical signal sequence, n a m e l y a basic N - t e r m i n a l region, a h y d r o p h o b i c core (residues 6 to 20) a n d a relatively p o l a r C - t e r m i n a l r e g i o n (Fig. 2). T h e second h y d r o p h o b i c p e a k , l o c a t e d at the C terminus, with a p r e d i c t e d m e m b r a n e - s p a n n i n g s e g m e n t b e t w e e n residues 426 a n d 451 (using the m e t h o d o f K l e i n et al., 1985), p r o b a b l y functions as a m e m b r a n e a n c h o r region. A l i g n m e n t o f the C H V g C a m i n o acid sequence with h o m o l o g o u s sequences f r o m o t h e r herpesviruses revealed a m o d e r a t e level o f similarity t h r o u g h o u t the entire sequence, with the e x c e p t i o n o f the N t e r m i n u s ( d a t a n o t shown). This a l i g n m e n t also revealed t h a t m o s t o f the cysteine residues are conserved. F o r example, C H V gC c o n t a i n s 10 cysteine residues, eight o f which are
c o n s e r v e d in all a l p h a h e r p e s v i r u s e s . T h e only cysteine residues in C H V gC t h a t are n o t c o n s e r v e d are l o c a t e d in the p u t a t i v e t r a n s m e m b r a n e o r i n t r a c e l l u l a r d o m a i n s . These results suggest t h a t the gC g l y c o p r o t e i n s m a y have similar t e r t i a r y structures. A l i g n m e n t with o t h e r g C sequences also revealed the relative c o n s e r v a t i o n o f p o t e n t i a l N - l i n k e d g l y c o s y l a t i o n sites. T h e significance o f this c o n s e r v a t i o n relative to the structure a n d f u n c t i o n o f the g C C H V g l y c o p r o t e i n is n o t k n o w n .
Identification and sequencing of O R F 2 N u c l e o t i d e sequence analysis o f the region d o w n s t r e a m f r o m the C H V gC gene revealed the presence o f a s e c o n d O R F (Fig. 2). This O R F ( O R F 2) starts at p o s i t i o n 1699 a n d ends at p o s i t i o n 2226. T h e p r e d i c t e d t r a n s l a t i o n p r o d u c t is 175 a m i n o acids long. C o m p a r i s o n o f this a m i n o acid sequence with the S W I S S - P R O T (Release 23) d a t a b a s e revealed h o m o l o g y with O R F s l o c a t e d d o w n s t r e a m f r o m o t h e r a l p h a h e r p e s v i r u s g C genes ( T a b l e 3). These h o m o l o g y scores suggest t h a t the O R F s l o c a t e d
Sequence of CHV gB, gC and gD homologues
2035
~ATATTT~T~AACTATTATGAAA~TTCTTAT~CTTATTTGTTTTTATTAAA~GGGTTGGGTTG~TTTTAAAATTACATAC~TGTATTMGAATT~CAT~TAAAG~CA~ACCCAT
120
GAAAAACA~TTAAATTCTAT~TG~CG~TT~TTTTCTCATTTT~GTTGCTAC~CTT~T~TAGTA3~aUL.~GATTAAA~TT~TATTTATCTTATTTTAT~TT~C~
240
M I
K L L
F I
L
F Y
F N P
13
T~CTGGATATAAATGGGTAGACCCTCCTCGTAGGTAT~TTACACCGTTTT~GAAT~TTCCA~TATT~CAAATCC~TG~TCCTTCTAAAAACGCTGAAGTT~GGTATGT~CTT 360 I
T
G
y
K
~
V
D
p
p
R
R
y
N
y
T
V
L
R
M
I
P
D
I
P
N
P
M
D
P
$
K
N
A
E
V
R
Y
V
T
53
CTACTGACCCATGTGATATGGTTGCTTT~TTTCT~TC~AAATATAGAATcTAC~TTAAAAc~TTCAATTTGTGCAAAAGAAAAAATTTTAC~TGCATCTCTTAGTTGGTTTAAAG 480 S
T
0
p
C
0
M
V
A
L
I
S
N
P
N
I
E
S
T
I
K
T
I
Q
F
V
Q
K
K
K
F
Y
N
A
S
L
S
W
F
K
93
TTGGAGAT~TTGTACATATCC~TATATTT~TTC~TATTTTGATTGT~TCCT~AAA~TTTGG~TATGTTTAAAAA~T~TCCAGATTTTTGGAAAC~ATCGTTAGTTGGTT600 V
G
D
O
C
T
y
p
I
y
L
I
Q
Y
F
D
C
D
P
Q
R
E
F
G
]
C
L
K
R
S
P
D
F
W K
P
S
L
V
G
133
ACACATTTTT~CTGAT~TGAATTGGGATTAGTTTTAGCTGCCCCCGCTC~TTT~TC~GGTC~TATA~CGGGTTATTCAAATTGAAAATG~GTTTTTTATACT~TTTTATGG 720 y
T
F
L
T
D
D
E
L
G
L
V
L
A
A
P
A
P
F
N
Q
G
Q
Y
R
R
V
I
Q
I
E
N
E
V
F
Y
T
D
F
M
1~
TTC~TTACCACGAGAAACTTGTTATTTTT~TAAAGAA~TAAATTTG~CC~CTTTTATGGAAT~TGT~GGAATCTA~TCTGTAGG~TCAAAAGTTGAC~TG~CTTTTTT 840 V
Q
L
P
R
E
T
C
Y
F
S
K
E
D
K
F
E
P
T
F
M
E
W
C
K
E
S
R
S
V
G
A
S
K
V
D
D
E
L
F
213
AT~TAAATAGAGCTGGTC~CCAAACCCTGCTTAAATATTATGTTATTAAAGATTTTTATAGACTT~CGGTA~GAACCTCCAATAAAATTTAAAG~GCT~TTA~TACGATATA~CAT ~0 y
L
N
R
A
G
p
Q
T
L
k
K
y
y
V
I
K
D
F
Y
R
L
N
G
R
E
P
P
I
K
F
K
E
A
k
R
Y
D
I
P
253
AT~AGTG~TGAT~ATTTGATGATGAATTA~CAT~GGCCACATATTAGT~TA~TATT~TA~JU~CTATTAAAGAAATTGTAAATCTTG~GATTATTTTAAAAATACAAATGTTA 1080 y
K
V
N
O
K
F
D
D
E
k
P
S
R
p
H
I
S
N
T
I
N
K
T
I
K
E
I
V
N
L
E
D
Y
F
K
N
T
N
V
293
TAGATACTA~TACCCC~CAC~TAAAT~TACCC~AAAAAATAT~CCGTGGG~TTGTTAT~TTATATT~T~TACTATTTAT~TTGGATTTTTTGTTTATAAAAGACAAAAAA 1200 I
D
T
T
T
p
T
p
I
N
N
T
P
K
N
I
T
V
G
I
V
I
I
]
L
I
I
L
F
I
I
G
F
F
V
Y
K
R Q
K
333
TATAT~T~T~ATAAAAAATT~c~cAAATGTTTAGCCTTTA~AAATT~TTTACAG~TAAAC~CTGGGCGGT~TTTTGTTT~TAAAAATTCATGTAcCTAC~TTTTATTcAC 1320 I Y N N Y K K L T T N V * 345
Fig. 3. Nucleotide sequence and predicted amino acid sequence of the C H V gD homologue. The putative signal sequence, transmembrane region, potential glycosylation sites and polyadenylation signal sequences are underlined. Nucleotides and predicted amino acid residues are numbered to the right of the sequence.
downstream from the gC gene in CHV, FHV, EHV-1, EHV-4, MDV, herpesvirus of turkey (HVT) and possibly HSV-1 are evolutionarily related. Conversely, the ORF (gene 13) located next to the VZV gC gene does not exhibit significant homology with any of the other comparably positioned ORFs. Furthermore, VZV gene 13 is oriented on the genome in the opposite direction relative to all the other ORF 2-like genes (Davison & Scott, 1986). These results are consistent with the proposed functions of the proteins encoded by these two groups of genes; VZV gene 13 encodes a thymidylate synthetase (Davison & Scott, 1986), whereas the HSV-1 ORF 2-like gene (UL45) encodes a putative virion protein (Visalli & Brandt, 1993). Therefore, it appears that the O R F located next to the gC gene in CHV, FHV, EHV-1, EHV-4, MDV, HVT and possibly HSV-1 encodes a protein that is structurally and functionally unrelated to the protein encoded downstream from the VZV gC homologue.
Analysis of the CHV ORF 2 nucleotide sequence Numerous potential TATA box, CAAT box and polyadenylation signal sequences are found in the 5' and 3' non-coding regions of ORF 2 (Fig. 2). The nucleotide sequence surrounding the ORF 2 initiation codon (AATATGG) is favourable with respect to Kozak's rules at positions - 3 and +4.
Identification and sequencing of the CHV gD gene Employing the same method used to map the CHV gC homologue, a 7 kb PstI fragment encoding an ORF with homology to herpesvirus gD glycoproteins was identified. The nuc|eotide sequence of this ORF is presented in Fig. 3. The putative CHV gD gene starts at position 201 and ends at position 1238. The predicted translation product is 345 amino acids long. Comparison of this amino acid sequence with the sequence of other gD glycoproteins revealed extensive homology, indicating that this ORF encodes the CHV gD homologue (Table 4).
Table 4. Homology between the predicted amino acid sequences of six herpesvirus gD glycoproteins*
CHV FHV EHV-1 PRV BHV- 1
FHV
EHV-1
PRV
BHV-1
HSV-1
45 -
35 31
-
-
27 30 26 -
34 34 27 37
21 24 21 27 24
* Values were obtained using the A L I G N Plus program and are expressed as percentage identity. The entire gD amino acid sequence was used. See Table 1 for alignment parameters. References : F H V (J.C. Audonnet, unpublished results), EHV-I (Flowers et al., 1991), PRV (Petrovskis et al., 1986), BHV-I (Tikoo et al., 1990) and HSV-I (Lasky & Dowbenko, 1984).
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K. J. Limbach and others
Analysis o f the C H V gD nucleotide sequence
Potential polyadenylation signal sequences are found downstream from the CHV gD gene. However, TATA box and CAAT box sequences are not found upstream from the CHV gD gene, although numerous TATA boxlike sequences are found (Fig. 3). The nucleotide sequence surrounding the CHV gD initiation codon (AAAATGA) is favourable with respect to Kozak's rules at position - 3 , but not at position + 4 (Fig. 3). This is not unusual, as the CHV gB and gC genes (see above), as well as gD genes of FHV (J.-C. Audonnet, unpublished results), EHV-1 (Audonnet et al., 1990; Flowers et al., 1991), PRV (Petrovskis et al., 1986) and BHV-1 (Tikoo et al., 1990) also contain an unfavourable nucleotide at position + 4.
to that of other alphaherpesviruses. For example, the ORF located immediately upstream from the CHV gB gene has homology with gene 30 of VZV (Davison & Scott, 1986) and with UL28 of HSV-1 (McGeoch et al., 1988), both of which are located immediately upstream from the gB homologue in those viruses (data not shown). ORF 2, located immediately downstream from the CHV gC gene, has homology with the ORFs located immediately downstream from the gC homologue in FHV (J.-C. Audonnet, unpublished results), EHV-1 (Telford et al., 1992), EHV-4 (Nicolson & Onions, 1990), HVT (Kato et al., 1989) and perhaps HSV-1 (McGeoch et al., 1988) (see above). Additionally, the ORF located immediately downstream from the CHV gD gene has homology with the gI gene of EHV-1 (Audonnet et al., 1990) and the gp63 gene of PRV (Petrovskis et al., 1986), both of which are located immediately downstream from the gD homologue in those viruses (data not shown).
Analysis of the predicted C H V gD amino acid sequence
The deduced amino acid sequence of the CHV gD homologue is presented in Fig. 3. Hydropathicity analysis of the predicted CHV gD amino acid sequence revealed the presence of two prominent hydrophobic peaks (data not shown). The first peak, located at the N terminus, may represent a potential signal sequence. In fact, PSIGNAL identifies a potential cleavage site between residues 16 and 17. The second hydrophobic peak, located near the C terminus, with a predicted membrane-spanning segment between residues 309 and 328 (using the method of Klein et al., 1985), probably functions as a membrane anchor region. Alignment of the CHV gD amino acid sequence with homologous sequences from other herpesviruses revealed a moderate level of similarity throughout the entire sequence, with the exception of the N terminus (data not shown). This alignment also revealed that the vast majority of cysteine residues are perfectly conserved. For example, CHV gD contains six cysteine residues, all of which are perfectly conserved in all alphaherpesviruses. These results suggest that the gD glycoproteins may have similar tertiary structures. This alignment also revealed that the potential N-linked glycosylation sites are well conserved. It is not known whether the CHV gD glycosylation sites are utilized. However, it is known that all the potential HSV-1 gD glycosylation sites are used (Sodora et al., 1991).
Genomic organization
The gB, gC and gD genes were not mapped to specific locations on the CHV genome. However, nucleotide sequence analyses of the regions flanking these genes indicate that the genomic organization of CHV is similar
Discussion In this report we present the nucleotide sequence of the CHV gB, gC and gD genes. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequence of these putative glycoproteins with the gB, gC and gD amino acid sequences of other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. This analysis also revealed that the homology among gB homologues is greater than that among gC or gD homologues, suggesting that the structural and functional constraints on gB may be greater than those on gC or gD. Alignment of homologous gB, gC and gD polypeptides revealed that the vast majority of cysteine residues are perfectly conserved. These results suggest that these cysteine residues, due to their ability to form disulphide bonds, are important in maintaining the structural and functional integrity of the gB, gC and gD glycoproteins. In HSV-1 gD, it has been shown that cysteine 1 forms a disulphide bond with cysteine 5, that cysteine 2 forms a disulphide bond with cysteine 6 and that cysteine 3 forms a disulphide bond with cysteine 4 (Long et al., 1992). Furthermore, it has been shown that a mutation of any of these residues has a profound effect on the conformation, processing and function of the resulting glycoprotein (Wilcox et al., 1988; Long et al., 1990). Therefore, the conservation of cysteine residues in these glycoproteins may have structural significance. The high degree of homology among the gB, gC and gD homologues also suggests that these glycoproteins have common functions. It has been shown that the
Sequence of C H V gB, gC and gD homologues BHV-1 gB homologue can rescue a gB- PRV strain, indicating that these two glycoproteins are functionally equivalent (Kopp & Mettenleiter, 1992). Alignment of the gB, gC and gD amino acid sequences also revealed that potential N-linked glycosylation sites are somewhat conserved. N-linked glycosylation is thought to play a role in a variety of functions, such as maintenance of protein conformation and protection against proteolytic degradation. The biological significance of N-linked carbohydrates on herpesvirus glycoproteins is not completely understood. For example, tunicamycin treatment of HSV-l-infected cells has been shown to inhibit the production of infectious virions (Pizer et al., 1980). In addition, endoglycosidase treatment of HSV-1 virions has been shown to decrease infectivity (Kuhn et al., 1988). On the other hand, N-linked glycosylation of HSV-1 gD does not appear to be absolutely essential, since mutagenesis of the glycosylation sites on this glycoprotein does not affect infectivity (Sodora et al., 1991). Therefore, although the glycosylation sites on the gB, gC and gD glycoproteins are relatively well conserved, proper glycosylation of each of these polypeptides may not be absolutely essential. The G + C content of herpesviruses varies from 33 to 75 % (Roizman, 1982). It has been suggested that this extensive variability is due to a non-selective mutational bias based on the presence (or absence) of virus-encoded or -induced enzymes involved in nucleotide metabolism (Honess, 1984). For example, VZV and herpesvirus saimiri both have a relatively low G + C content (46 %) and both encode an enzyme, thymidylate synthetase, which is involved in TTP synthesis (Davison & Scott, 1986; Honess et al., 1986). HSV-1, HCMV and EBV, on the other hand, have relatively high G + C content (68 %, 57% and 60%, respectively) and do not appear to encode a thymidylate synthetase (Honess et al., 1986). CHV has been determined by DNA density analysis to have the lowest G + C content of any herpesvirus, 33 % (Plummer et al., 1969; Roizman, 1982), a value which is consistent with the relatively low G + C content of the nucleotide sequence presented in this report (29 %). It is not known whether CHV encodes an enzyme involved in nucleotide metabolism. However, it is known that the ORF located immediately downstream from the CHV gC gene is not homologous to VZV thymidylate synthetase. Therefore, if CHV contains a thymidylate synthetase gene, it is not found at the same genomic location as that of VZV. The parameters of a protective immune response against a CHV infection have not been completely elucidated. It is known that newborn pups exposed to CHV usually die without forming CHV-specific neutralizing antibodies. It is also known that maternal anti-
2037
bodies or treatment with immune serum from seropositive dogs can protect puppies from a fatal CHV infection (Carmichael, 1970). Therefore, it appears that serum neutralizing antibodies can protect puppies against a fatal CHV infection. Three CHV glycoproteins, gp145/112, gp80 and gp47, are known to elicit CHV neutralizing antibodies (Xuan et al., 1991). The genes encoding these glycoproteins have not been identified. It is possible that these antigens are encoded by the gB, gC and gD genes identified in this paper. Since several reports have indicated that an immune response against gB, gC and/or gD can provide protection of target species animals against a herpesvirus challenge (Babiuk et al., 1987; Nazarian et al., 1992; Riviere et al., 1992; Brockmeier et al., 1993), it is hoped that the isolation of the CHV gB, gC and gD genes will lead to the development of an efficacious CHV vaccine. We thank Gerard Johnson for his invaluable assistance with the computer-based analyses and manipulations presented in this report. We also thank James Tartaglia and Gerard Johnson for their critical review of this manuscript.
References ALLEN, G. P. & COOGLE,L. D. (1988). Characterization of an equine herpesvirus type l gene encoding a glycoprotein (gp13) with homology to herpes simplex virus glycoprotein C. Journal of Virology 62, 285~2858. API'EL, M. (1987). Canine herpesvirus. In Virus Infections of Vertebrates, vol. 1, Virus Infections of Carnivores, pp. 5 15. Edited by M. Appel. Amsterdam/Oxford/New York/Tokyo: Elsevier Science Publishers. AUDONNET, J.-C., WINSLOW, J., ALLEN, G. & PAOLETTI,E. (1990). Equine herpesvirus type 1 unique short fragment encodes glycoproteins with homology to herpes simplex virus type 1 gD, gI and gE. Journal of General Virology 71, 2969-2978.
BABIUK,L. A., L'ITALIEN,J., VANDRUNENLITTEL-VANDEN HURK, S., ZAMB, T., LAWMAN,M.J., HUGHES, G. & GIFFORD,G. A. (1987). Protection of cattle from bovine herpesvirus type 1 (BHV1) infection by immunization with individual viral glycoproteins. Virology 159, 57 66. BAUSE, E. (1983). Structural requirements of N-glycosylation of proteins. Biochemical Journal 209, 331-336. BLEWEa'T, E. L. & M~SRA,V. (1991). Cleavage of the bovine herpesvirus glycoprotein B is not essential for its function. Journal of General Virology 72, 2083-2090. BLOBEL, G. (1980). Intracellular protein topogenesis. Proceedings of the National Academy of Sciences, U.S.A. 77, 149(~1500.
BROCKMEIER,S., LAGER,K., TARTAGLIA,J., RIVIERE,M., PAOLETTI,E. & MENGELING,W. (1993). Vaccination of pigs against pseudorabies with highly attenuated vaccinia (NYVAC) recombinant viruses. Veterinary Microbiology 38, 41 58. BZlK, D. J., Fox, B. A., DELUCA, N. A. & PERSON, S. (1984). Nucleotide sequence specifying the glycoprotein gene, gB, of herpes simplex virus type 1. Virology 133, 301-314. CARMICHAEL,L. (1970). Herpesvirus canis: aspects of pathogenesis and immune response. Journal of the American Veterinary Medical Association 156, 1714-1721. CARMICHAEL,L., STRANDBERG,J. & BARNES,F. (1965). Identification of a cytopathogenic agent infectious for puppies as a canine herpesvirus.
Proceedings of the Society for Experimental Biology and Medicine 120, 644-650.
CORDEN, J., WASYLYK, B., BUCHWALDER,A., SASSONE-CORSI,P.,
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KEDINGER, C. & CHAMBON, P. (1980). Promoter sequences of eukaryotic protein-coding genes. Science 209, 1406-1414. DAVISON,A. J. & SCOTT,J. E. (1986). The complete DNA sequence of varicella-zoster virus. Journal of General Virology 67, 1759 1816. FITZPATRICK, D. R., BABIUK,L. A. & ZAMB, T. J. (1989). Nucleotide sequence of bovine herpesvirus type 1 glycoprotein gill, a structural model for gill as a new member of the immunoglobulin superfamily, and implications for the homologous glycoproteins of other herpesviruses. Virology 173, 46-57. FLOWERS, C., EASTMAN,E. & O'CALLAGHAN,D. (1991). Sequence analysis of a glycoprotein D gene homolog within the unique short segment of the EHV-1 genome. Virology 180, 175 184. HONESS, R.W. (1984). Herpes simplex and 'the herpes complex': diverse observations and a unifying hypothesis. Journal of General Virology 65, 2077-2107. HONESS, R.W., BODEMER,W., CAMERON,K.R., NILLER, H.-H. & FLECKENSTEIN, B. (1986). The A + T rich genome of herpesvirus saimiri contains a highly conserved gene for thymidylate synthase. Proceedings of the National Academy of Sciences, U.S.A. 83, 3604-3608. IHARA, T., KATO, A., UEDA, S., ISHIHAMA,A. & HIRAI, K. (1989). Comparison of the sequence of the secretory glycoprotein A (gA) gene in Md5 and BC-1 strains of Marek's disease virus type 1. Virus Genes 3, 127 140. KATO, A., SATO, I., IHARA,T., UEDA, S., ISHIHAMA,A. & HIRAI, K. (1989). Homologies between herpesvirus of turkey and Marek's disease virus type-1 DNAs within two co-linearly arranged open reading frames, one encoding glycoprotein A. Gene 84, 399-405. KELLER, P. M., DAVISON,A. J., LOWE, R. S., BENNETT,C. D. & ELLIS, R. W. (1986). Identification and structure of the gene encoding gplI, a major glycoprotein ofvaricella-zoster virus. Virology 152, 181-191. KLEIN, P., KANEHISA,M. & DELISI, C. (1985). The detection and classification of membrane-spanning proteins. Biochimica et biophysica acta 815, 468~476. KoPP, A. & METTENLEITER,T. (1992). Stable rescue of a glycoprotein glI deletion mutant of pseudorabies virus by glycoprotein gl bovine herpesvirus 1. Journal of Virology 66, 2754-2762. KOUZARIDES, T., BANKIER, A.T., SATCHWELL, S.C., WESTON, K., TOMLINSON,P. & BARRELL,B. G. (1987). Large-scale rearrangement of homologous regions in the genomes of HCMV and EBV. Virology 157, 397-413. KOZAK, M. (1986). Point mutations define a sequence flanking the AUG inhibitor codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292. KidHN, J. M., LING, B. R., BROSSMER,R., MUNK, K. & BRAUN,R. W. (1988). Removal of N-linked carbohydrates decreases the infectivity of herpes simplex virus type 1. Journal of General Virology 69, 284~2858. LASKY, L. A. & DOWBENKO,D. J. (1984). DNA sequence analysis of the type-common glycoprotein D genes of herpes sinlplex virus types 1 and 2. DNA 3, 23 29. LONG, D., COHEN, G., MUGGERIDGE,M. & EISENBERG, R. (1990). Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function. Journal of Virology 64, 5542-5552. LONG, D., WILCOX, W., ABRAMS, W., COHEN, G. & EISENBERG, R. (1992). Disulfide bond structure of glycoprotein D of herpes simplex virus types 1 and 2. Journal of Virology 66, 6668-6685. McGEOCH, D.J., DALRYMPLE, M.A., DAVISON, A.J., DOLAN, A., FRAME, M. C., MCNAB, D., PERRY, L. J., SCOTT,J. E. & TAYLOR,P. (1988). The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. Journal of"General Virology 69, 1531 1574. MAEDA,K., HORIMOTO,T., NORIMINE,J., KAWAGUCHI,Y., TOMONAGA, K., NIIKURA, M., KAI, C., TAKAHASHI,E. & MIKAMI, T. (1992). Identification and nucleotide sequence of a gene in feline herpesvirus type 1 homologous to the herpes simplex virus gene encoding the glycoprotein B. Archives of Virology 127, 38~397. MANIATIS, T., FRITSCH, E.F. & SAMBROOK, J. (1982). Molecular Cloning." A Laboratory Manual. New York: Cold Spring Harbor Laboratory. NAZARIAN,K., LEE, L., YANAGIDA,N. & OGAWA,R. (1992). Protection
against Marek's disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek's disease virus. Journal of Virology 66, 1409 1413. NICOLSON, L. & ONIONS,D. E. (1990). The nucleotide sequence of the equine herpesvirus 4 gC gene homologue. Virology 179, 378-387. PEARSON,W. R. & LIPMAN, D. J. (1988). Improved tools for biological sequence comparison. Proceedings of the National Academy of Sciences, U.S.A. 85, 2444-2448. PELLETT, P. E., BIGGIN, M.D., BARRELL,B. & ROIZMAN, B. (1985). Epstein-Barr virus genome may encode a protein showing significant amino acid and predicted secondary structure homology with glycoprotein B of herpes simplex virus 1. Journal of Virology 56, 807 813. PETROVSKIS, E., TIMMINS, J., ARMENTROUT, M., MARCHIOLI, C., YANCEY, R. & POST, L. (1986). DNA sequence of the gene for pseudorabies virus gpS0, a glycoprotein without N-linked glycosylation. Journal of Virology 59, 216-223. PIZER, L., COHEN,G. & EISENBERG,R. (1980). Effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production. Journal of Virology 34, 142-153. PLUMMER,G., GOODHEART,C., HENSON,D. & BOWLING,C. (1969). A comparative study of the DNA density and behavior in tissue cultures of fourteen different herpesviruses. Virology 39, 134-137. PROUDFOOT, N.J. & BROWNLEE,G.G. (1976). 3'-Noncoding region sequences in eukaryotic messenger RNA. Nature, London 263, 211-214. RIVIERE, M., TARTAGLIA, J., PERKUS, M.E., NORTON, E.K., BONGERMINO, C.M., LACOSTE, F., DURET, C., DESMETTRE,P. & PAOLETTI,E. (1992). Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants. Journal of Virology 66, 3424-3434. ROBBINS, A., WATSON, R., W•EALY, M., HAYS, W. & ENQUIST, L. (1986). Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C. Journal of Virology 58, 339 347. ROBBINS, A.K., DORNEY, D.J., WATHEN, M.W., WHEALY, M.E., GOLD, C., WATSON,R. J., HOLLAND,L. E., WEED, S. D., LEVINE,M., GLORIOSO,J. C. & ENQUIST,L. W. (1987). The pseudorabies virus glI gene is closely related to the gB.glycoprotein gene of herpes simplex virus. Journal of Virology 61, 2691 2701. ROIZMAN, B. (1982). The family Herpesviridae: general description, taxonomy, and classification. In The Herpesviruses, vol. 1, pp. 1 23. Edited by B. Roizman. New York & London: Plenum Press. Ross, L. J. N., SANDERSON,M., SCOTT, S. D., BINNS, M. M., DOLL, T. & MILNE, B. (1989). Nucleotide sequence and characterization of the Marek's disease virus homologue of glycoprotein B of herpes simplex virus. Journal of General Virology 70, 1789-1804. SODORA, D., COHEN, G., MUGGERIDGE,M. & EISENBERG,R. (1991). Absence of asparagine-linked oligosaccharides from glycoprotein D of herpes simplex virus type 1 results in a structurally altered but biologically active protein. Journal of Virology 65, 442~4431. SPAETE,R., SAXENA,A., SCOTT,P., LONG, G., PROBERT,W., BRITT,W., GIBSON, W., RASMUSSEN, L. & PACHL, C. (1990). Sequence requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne. Journal of Virology 64, 2922-2931. TARTAGLIA,J., WINSLOW,J., GOEBEL,S., JOHNSON,G. P., TAYLOR,J. & PAOLETTI, E. (1990). Nucleotide sequence analysis of a 10.5 kbp HindllI fragment of fowlpox virus : relatedness to the central portion of the vaccinia virus HindlII D region. Journal of General Virology 71, 1517 1524. TELFORD, E.A., WATSON, M.S., McBRiDE, K. & DAVISON, A.J. (1992). The DNA sequence of equine herpesvirus-1. Virology 189, 304-316. TIKOO, S. K., FITZPATRICK,D. R., BABIUK,L. A. & ZAMB,T. J. (1990). Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein glV in transfected bovine cells. Journal of Virology 64, 5132-5142. VISALLI, R. J. & BRANDT,C. R. (1993). The HSV-1 UL45 18 kDa gene product is a true late protein and a component of the virion. Virus Research 29, 167-178. YON HEDNE, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 14, 4683-4690.
Sequence of C H V gB, gC and gD homologues 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. WHITBECK,J. C., BELLO,L. J. 8z LAWRENCE,W. C. (1988). Comparison of the bovine herpesvirus 1 gI gene and the herpes simplex virus type 1 gB gene. Journal of Virology 62, 331%3327. WILCOX, W. C., LONG, D., SODORA,D. L., EISENBERG,R. J. & COHEN,
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G. H. (1988). The contribution of cysteine residues to antigenicity and extent of processing of herpes simplex virus type 1 glycoprotein D. Journal of Virology 62, 1941-1947. XUAN, X., HORIMOTO,T., LIMCUMPAO,J. A., TAKUMI,A., TOHYA,Y., TA~AHASHI,E. & MIKAMI, T. (1991). Neutralizing determinants of canine herpesvirus as defined by monoclonal antibodies. Archives of Virology 116, 185-195.
(Received 17 August 1993; Accepted 9 February 1994)
2029
Nucleotide sequence of the genes encoding the canine herpesvirus gB, gC and gD homologues Keith J. L i m b a c h , M . Paullin L i m b a c h , Darryl C o n t e and E n z o P a o l e t t i * Virogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy, N e w York 12180-8349, U.S.A.
The nucleotide sequence of the genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues was determined. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequences of CHV gB, gC and gD with the homologous sequences from other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. Alignment of the homologous gB,
gC and gD amino acid sequences indicates that most of the cysteine residues are conserved, suggesting that these glycoproteins possess similar tertiary structures. The nucleotide sequence of the open reading frame downstream from the CHV gC gene was also determined. The predicted amino acid sequence of this putative polypeptide appears to be homologous to a family of proteins encoded downstream from the gC gene in most, although not all, alphaherpesviruses.
Introduction
virulent PRV challenge (Brockmeier et al., 1993). Furthermore, vaccinia virus recombinants expressing PRV gII and gp50, or gII, gIII (gC) and gp50 have been shown to elicit a higher level of protection than recombinants expressing gII or gp50 alone, suggesting a potential synergistic effect with these glycoproteins (Riviere et al., 1992). The results of these studies indicate that an immune response against gB, gC a n d / o r gD can protect target species animals against a herpesvirus challenge. In this communication, we report the nucleotide sequence of the genes encoding the CHV gB, gC and gD homologues. This information will form a database upon which potential CHV vaccine candidates can be developed.
Canine herpesvirus (CHV) causes a fatal haemorrhagic disease in neonatal puppies and a self-limiting, usually subclinical, upper respiratory tract infection in adult dogs (Appel, 1987). Little is known about the genomic structure of CHV. Restriction enzyme maps of the CHV genome and nucleotide sequence of CHV genes have not been published. In particular, genes encoding immunologically pertinent proteins have not been characterized. During a herpesvirus infection, the majority of the immune response is directed against the viral envelope glycoproteins. These antigens have been shown to elicit both humoral and cellular immune responses. The contribution of the individual glycoproteins in eliciting a protective immune response, however, is not completely understood. Several reports have indicated that immunization with the herpesvirus gB, gC a n d / o r gD glycoproteins can induce a protective immune response. For example, immunization with purified bovine herpesvirus type 1 (BHV-1) gI (gB), gIII (gC) a n d / o r gIV (gD) has been shown to protect cattle against a BHV1/ Pasteurella haemolytica challenge (Babiuk et al., 1987). In addition, immunization with a fowlpox virus recombinant expressing Marek's disease virus (MDV) gB has been shown to protect chickens against a virulent MDV challenge (Nazarian et al., 1992). Immunization with NYVAC (highly attenuated vaccinia virus) recombinants expressing pseudorabies virus (PRV) gII (gB) or gp50 (gD) have also been shown to protect swine against a 0001-2025 © 1994 SGM
Methods Preparation of genomic CHV DNA. CHV (obtained from L. Carmichael, Cornell University, Ithaca, N.Y., U.S.A.) was propagated on Madin-Darbycaninekidney(MDCK)cells(ATCC CCL34). Viral DNA was isolated using standard methods (Tartaglia et al., 1990). DNA hybridization. CHV genomic DNA was digested with restriction enzymes,run on agarose gels and transferredto Gene-Screen membranes(New EnglandNuclear)under conditionsrecommendedby the manufacturers.Hybridizationswere performedat 44 °C, 53 °C or 59°C in 1M-NaC1, 1% SDS and 10% dextran sulphate. The hybridization probe included a 1800bp BamHI-XbaI fragment, containingan internal segmentof the felineherpesvirus(FHV) gB gene (Maeda et al., 1992), a 950 bp BamHI-EcoRI fragmentcontainingthe 3' end of the FHV gC gene (J.-C. Audonnet,unpublishedresults), and
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K. J. Limbach and others
a 970 bp BamHI-HindIIl fragmentcontainingthe 3' end of the FHV gD gene (J:C. Audonnet, unpublished results). Cloning and DNA sequencing. CHV genomic fragments were subcloned into pBluescript SK (Stratagene). Plasmid DNA was prepared and manipulatedusing standard techniques(Maniatis et al., 1982). Nucleotide sequencing was performed on both strands with double-stranded plasmid templates, using the modified T7 DNA polymerase, Sequenase Version 2.0 (U.S. BiochemicalCorporation), and standard protocols recommended by the manufacturer. M13 forward and reverse primers were used to obtain initial sequence; and custom primers, prepared with a Biosearch 8700 or an Applied Biosystems380B oligonucleotidesynthesizer,were used for subsequent reactions. DNA and amino acid sequence analyses. DNA and amino acid sequence analyses were performed with PC/GENE (IntelliGenetics), ALIGN Plus (Scientificand Educational Software) and IBI-Pustell (International Biotechnologies)softwarepackages.Homologysearches were conducted on the SWlSS-PROT (Release 20 and 23) (lntelliGenetics) databases, using the FASTA program (Pearson & Lipman, 1988).
Results Identification and sequencing of the C H V gB gene
Hybridization of restriction enzyme-digested CHV genomic DNA with a radiolabelled probe containing the FHV gB, gC and gD genes (Maeda et at., 1992; J.C. Audonnet, unpublished results) identified one complementary sequence (data not shown). A 6 kb XbaI fragment containing this sequence was cloned into pBluescript SK and the nucleotide sequence of the hybridizing region was determined (Fig. 1). An open reading frame (ORF) starting at position 201 and ending at position 2840 was identified. The predicted translation product of this ORF is 879 amino acids long. Comparison of this amino acid sequence with the SWISS-PROT (Release 20) database revealed significant homology with the gB glycoprotein of numerous herpesviruses. Additional analyses revealed that the predicted CHV gene product was more similar to the gB glycoprotein of alphaherpesviruses, such as herpes simplex virus type 1 (HSV-1), than those of beta- or gammaherpesviruses, such as human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV) (Table 1). These results indicate that CHV should be classified as an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties (Carmichael et at., 1965; Roizman, 1982).
Analysis o f the C H V gB nucleotide sequence
The 5' and 3' non-coding regions of the CHV gB gene contain numerous R N A polymerase II regulatory sequence motifs, such as TATA box, CAAT box and
polyadenylation signal sequences (Corden et al., 1980; Proudfoot & Brownlee, 1976) (Fig. 1). The nucleotide sequence surrounding the initiation codon has been shown to affect the efficiency of translation initiation (Kozak, 1986). In particular, the sequence ( A / G ) N N A T G G has been found to be most efficient. Therefore, according to Kozak's rules, the nucleotide sequence surrounding the CHV gB initiation codon (AGTATGT) is favourable at position - 3 , but not at position + 4 (Fig. 1). The fact that the CHV gB gene does not follow Kozak's rules is not unusual. The FHV (Maeda et al., 1992), PRV (Robbins et al., 1987), varicella-zoster virus (VZV) (Keller et al., 1986), MDV (Ross et al., 1989) and HSV-1 (Bzik et al., 1984) gB genes also contain a pyrimidine at position + 4. Analysis of the predicted C H V gB amino acid sequence
The deduced amino acid sequence of the CHV gB homologue is presented in Fig. 1. Hydropathicity analysis of this amino acid sequence revealed the presence of two prominent hydrophobic peaks (data not shown). The first peak, located at the N terminus, may represent a potential signal sequence. N-terminal signal sequences initiate transport across the endoplasmic reticulum membrane and are critical for the proper posttranslational modification and targeting of glycoproteins (Blobel, 1980). Signal sequences vary in length from about 15 to 30 residues and usually consist of a basic Nterminal region, a central hydrophobic region and a short, relatively polar C-terminal region. In addition, the cleavage site usually conforms to the - 3, - 1 rule, where the residue at position - 1 is small (Ala, Ser, Gly, Cys, Thr or Gln) and the residue at position - 3 is not aromatic (Phe, His, Tyr or Trp), charged (Asp, Glu, Lys or Arg) or large and polar (Asn or Gln), and residues - 3 to + 1 are not Pro residues (von Heijne, 1986). Although analysis with PSIGNAL, a P C / G E N E program designed to detect eukaryotic signal sequences, did not identify the N terminus of CHV gB as a potential signal sequence, this region does have elements consistent with typical signal sequences, namely a hydrophobic core (residues 2 to 17) and a relatively polar C-terminal region (Fig. 1). That PSIGNAL does not detect a signal sequence in the N-terminal region of CHV gB is not unique. This algorithm also fails to detect a signal sequence in the Nterminal region of the VZV gB homologue. The second, very broad, hydrophobic peak is located near the C terminus, with predicted membrane-spanning segments between amino acid residues 725 and 741, and 747 and 771 (using the method of Klein et al., 1985), and probably functions as a membrane anchor region. It has been hypothesized that the transmembrane domain of HSV-1 gB, as well as other gB homologues, traverses the
Sequence of CH V gB, gC and gD homologues
2031
TTTTCTGGATTTCAGCTATGTCCTTCGGGAGTTTATAT~CTTAT~GAAAACTGTCCTTTGGTA~CAGTTTTACAAAGCGGTGTAAATTGCGAAATTGGACC~CTA~CTGTM~
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GTAGAATT~AA~TG~AT~TGT~T~TTCAAAAT~GAGA~CCCTTTG~T~GTTATG~TT~TCC~CTAGTGTGGCTTCGGTTGCTATGGAT~pd~p~GAG~TTCAGCAC 1800 S
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TAT~ATTTAAAGCACTAG~TC~T~CTATATT~G~CTTGGGGAAAAT~T~TATTAGTAG~C~GCT~TT~CcATGTACAGCT~CCAT~GATATT ] S F K A L E N S T N Y ] E G 0 L G E N N E l l V E R K L I E P C T
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TTACAAAc~TATCTCTT~CATCGTGGTc~AAAA~ACAAA~GTTTGAAAAA~GT~d~TGAAAGT~GTTT~TAJU~TTT~TATTA~GTAAAATTTTCTGACTCTGC~ [ T N l S l K H R G P K Y K R l K N V N E N E $ K V *
2~0 879
~ACTTT~T~TAT~TAT~TTTTAGAJ~ATTTTA~T~ATTTTATTATCTTTTAT~CCTC~CTATTTAT~G~T~T~TG~ATTT~TG~GGTGCCTGTATAT~CTACT~
3000
Fig. l. Nucleotide sequence and predicted amino acid sequence of the CHV gB homologue. The putative cleavage site, transmembrane regions, potential glycosylation sites, TATA box, CAAT box and polyadenylation signal sequences are underlined. Nucleotides and predicted amino acid residues are numbered to the right of the sequence.
2032
K. J. Limbach and others
Table 1. Homology between the predicted amino acid sequences o f 10 herpesvirus gB glycoproteins* FHV CHV FHV EHV-1 PRV BHV-1 VZV MDV HSV-1 HCMV
EHV-1 PRV BHV-1 VZV MDV
78
. . .
61 57 . .
61 59 52 . .
.
59 58 52 63 -
.
. .
.
52 48 45 48 46 49
. .
.
55 51 47 52 52
HSV-1 HCMV EBV
.
.
.
50 48 44 50 48 48 48
29 27 27 29 28 30 30 28
27 27 26 29 28 28 29 29 32
* Values were obtained usingthe ALIGN Plus program and are expressedas percentageidentity. The entire gB amino acid sequence was used. The alignment parameters were: mismatch penalty = 2, open gap penalty = 4, extended gap penalty = 1. References: FHV (Maeda et al., 1992), EHV-1 (Whalleyet al., 1989),PRV (Robbins et al., 1987), BHV-I (Whitbecket al., 1988), VZV (Kelleret al., 1986),MDV (Ross et al, 1989),HSV-1 (Bziket al., 1984),HCMV (Kouzarides et al., 1987)and EBV (Pellett et al., 1985).
membrane three times (Pellett et al., 1985). As hydropathicity analysis of the C-terminal region of CHV gB reveals the presence of at least two membrane-spanning regions, it is possible that CHV gB and HSV-1 gB have similar transmembrane structures. Alignment of the CHV gB amino acid sequence with similar sequences from other herpesviruses revealed extensive homology throughout the entire sequence, with the exception of the N terminus, a region surrounding the putative cleavage site (see below) and a region near the C terminus (data not shown). This alignment also revealed that most of the cysteine residues are conserved. For example, CHV gB contains 11 cysteine residues, 10 of which are conserved in all alpha-, beta- and gammaherpesviruses. In fact, the only cysteine residue in CHV gB that is not conserved is found near the N terminus and may be located in the putative signal sequence. These results suggest that the gB glycoproteins may have similar tertiary structures. N-linked oligosaccharides can be added to Asn residues which have the sequence Asn-X-Ser/Thr, where X is not a Pro residue (Bause, 1983). Alignment of the CHV gB amino acid sequence with other gB sequences revealed that potential N-linked glycosylation sites are relatively well conserved (data not shown). It is not known whether glycosylation at these sites is structurally or functionally significant. The gB glycoprotein o f most herpesviruses is cleaved internally during maturation, with the subsequent peptides being held together by disulphide bonds. The VZV gB homologue (gpII), for example, is cleaved between Arg and Ser residues, resulting in two glycoproteins of approximately 60K (Keller et al., 1986). The gB glycoproteins o f FHV (Maeda et al., 1992), equine herpesvirus type 1 (EHV-1) (Whalley et al., 1989), PRV (Robbins et al., 1987), BHV-1 (Whitbeck et al., 1988),
MDV (Ross et al., 1989) and H C M V (Kouzarides et al., 1987) are also cleaved. Furthermore, a sequence, Arg-XArg-Arg/Lys--Ser/Ala, similar to the sequence at the VZV cleavage site, Arg-Thr-Arg-Arg--Ser, is present at virtually the same location in each of these gB glycoproteins. Conversely, this sequence is not found in the HSV-1 (Bzik et al., 1984) and EBV (Pellett et al., 1985) gB glycoproteins, which are not cleaved. The significance of this cleavage event is unknown. It does not appear, however, to be essential for replication in vitro, as strains of BHV-1 (Blewett & Misra, 1991) and H C M V (Spaete et al., 1990) that have been mutated at the cleavage site, and therefore encode an uncleavable gB glycoprotein, are still infectious. It is not known whether CHV gB is cleaved internally. However, the sequence Arg-Lys-ArgArg--Ser is present at approximately the same location in CHV as in VZV, FHV, EHV- 1, PRV, BHV- 1, MDV and H C M V (data not shown). Therefore, it is probable that CHV gB is proteolytically cleaved. Identification and sequencing o f the C H V g C gene
A radiolabelled probe containing the 3' end of the F H V gC (and gD) gene(s) did not hybridize to restriction enzyme fragments of CHV genomic D N A (see above). Therefore another method of analysis was used to identify the CHV gC (and gD) gene(s). CHV genomic fragments were randomly cloned into pBluescript SK. The nucleotide sequences of the termini of these fragments were determined and the predicted amino acid sequences of potential ORFs were analysed for homology against the SWISS-PROT (Release 20) amino acid database. Using this method, a 12 kb XbaI fragment encoding an O R F with homology to herpesvirus gC glycoproteins was identified. The nucleotide sequence of this O R F is presented in Fig. 2. The putative CHV gC
Sequence of CHV gB, gC and gD homologues
2033
CGAGc~CTAATTATTGGTTTGTATATGACTGTTGG~TTTGTTACATTTTTATTAAAA~T~TT~TTTTTTAAAcTATATTAC~GTTGTGT~TGTTTT~GTTTTAAATAAAG~ 120 AT~T~TCG~TT~A~ATTTATCA/~U~ACATTA~A~U~CC~CA~TTT~TAT~]~T~GGT~T~GTCAJU~T~GT1TTA~ATTTTTATCT~TATATGT~TTAT~240 M S F K N F Y L I Y Y ~ I 13 TTTTTATA~CTCGAT~T~CTTCGGCATCTACAT~C~cTT~CAC~TAC~T~TTCCM~TTCAGCAAAT~TCACCTGcTTCCATA~TAC~CTAT~CAAAACCTATAT I F I N S I I T S A S T S K P S T P T I I P T S A N E S P A S I D T T I T K P I
360 53
CTACAGAGGCAAAT~TTTAAAATCAGT~GTACCTC~TTAAACCACCTAAAAACTTAAAA~J~g~TTACTTAAAT~TAAATGTA~T~TGTTATTTATAGGCCATATTTTAGTC480 S T E A N M k K S V S T S I K P P K N k K K K L k K S K C R D N V I Y R P Y F S 93 ~TTAG~TT~CTGTACTAT~CTA/UU~GCA/d~TTT~GT~TCCTTT~TTGAGTTAT~GTTTAAAG~CTTTCTACATATMTAA/~CC~TGAAAATGTTGAAAGTTTAAJU~ 600 O L E I N C T I T K K O N L $ N p L ] E L W F K E L S T Y N K T N E N V E S L K 133 CAGATATATC~AAATATTTTATTATTTTCGACAAAAAAT~TAGT~T~CTTTTAT~T~TTTTTTATTAGGTATA~AAAJkTC~CCAGTAAATTATAAACTTTACGGTTCCC~T 720 T D I S K N I L L F S T K N N S D N F Y N D F L L G I Q N Q P V N Y K L Y G S Q 1~ TTTATGAT~TGGAAACATATTACTAAATATAAAGTCGGTT~CTTTA/~a~CCTCTG~TATATACTTGGAAACTATAT~TTCAAAT~TGAAAGTATTTTTGAAACTTTTAAAATTC ~0 F y D N G N I L L N I K S V D F K T S G I Y T W K L Y N S N N E S I F E T F K I 213 ~GTATATGCATATCATTCCCCAAATGTAAACTTAAAATCAAACCC~GTTTATAT~TGAAAACTACAGCGCTATTTGTAC~TAGC~TTACTTTCCATTG~TCTACGGAAATAT960 Q V y A y H S p N V N L K S N P S k Y N E N Y S A I C T I A N Y F P k E S T E I 253 TTTGGTTT~CGATGGA~CTATT~TAAAAAATATATAGATG~CTTATAGTGTATG~TT~CGGTCTTAT~CACG~A~TTC~TATTAT~CCTTCCCTTTT~GAAGCCATGG 1080 F W F N D G O p I D K K y I D E T y S V W I D G k I T R T S I L S L P F S E A M 293 ~GC~CCCCC~TTTGCGATGT~TGTT~TGGTATAAAAATTC~GG~ATCAAAAAAATTTTCAAATACCGTTATTCCAAAAGTTTACTATAAACCTTTTATATCTATAAAATTTG 1200 E S p p N k R C N V E W y K N S K A S K K F S N T V [ P K V Y Y K P F I S I K F 333
AT~TGGTTTAG~TATTTGTGATGCTAAATGTGTTTCCCGTGA~U~T~TAAATTAC~TGGT~GTTA~L4~TATACCTATAj~TGGTGATGATATTAT~GCGGCCCCTGTTT~CC 1320 0
N G
k
A
I
C D A
K
C V
S
R E
N N K
L
O W L
V
K
D
I
P
l
N G D D
I
I
S
G P
C
L
N
3~
AC~CTGGTTTGGTC~TATTCAAAATAAAATAGATATAT~G~TTAT~T~CCTGTTACCTATAAATGTTC~TTATTGGTTATCC~T~TTTTTCCC~CTTTTAT~TGAAAAGG 1440 H p G k V N I Q N K l D ] S D Y D E P V T Y K C S I ] G Y P I [ F P N F Y D E K 413 TGTTT~TG~ATCGGATGAAAATGTTAGT~T~TGTT~T~GTATTACCAC~T~TTGGTG~GCCATTTTTGTTATAGTATT~TTTTTAT~CAGC~TTATGTTTTTATTGTT 1560 V F D A S O E N V S K S M L [ S I T T l I G G A I F V I V L I F I T A L C F Y C 453 C~AAAAT~T~GATCT~TATC~TATTTACGTAAATG~TTATAT~TGTTATATTCGTGTTATTATGATTTAT~GTTCATCAAATTTAAAAATTTGTATAGTATT~TTTTTA 1~0 S K N N K I * 459
ATAGGGGTATCGTTT~TATGGCTCAGTTAGTTTT~cT~TATTC~TC~TGTGGAAAATAAAAATACTT~AT~C~c~GAAAC~CT~CTT~CCAGAAAAAATC~A M A Q L V L T D I p L E D V E N K N T S S D E E T T N L N g K K
T
1800 34
TGTC~TGTTTATGTGTTAcCCTTG~TTTTTTG~AGCTGG~TTTTATT~CCATAGcTGC~T~TTTTTACTTTTATTTTTACAGTACCATTAGAAATGCTTG~TCTATT~TTGT C Q C L C V T L G F F A A G I L L T I A A ] I F T F I F T V P L E M L G S I N C
1920 74
CCTCCATCTACATTTGGTATTGAT~TGTTTGTATC~C~TAAAAAAATCTATT~TTCTTATT~TTATCTAAAATATGTTATGATA~TTGTCAAATCCGAT~TCAGAGT p P S T F G I D N V C ] E P I K K S I N S Y S E l S K I C Y D R k S N P I N O S
2040 114
S
ACTATTAACTCCTTATT~CTGTTTT~TATGTTTGCA~TAAAAACTATGAAAATGTTTAT~TTGT~TAC~T~GTGAAAAAACATGT~TTCATC~TAGCTATTTGTCAAA~T 2160 T [ N S L L T V L N M F A D K N Y E N V Y N C N T H S E K T C N S S ] A I C Q T 154 AATCATCCACT~GTTCATTGGGAAATTTTGTTATTAAAATTAGAAAAATTTTTGGGTTTAAAT~T~TAAAAT~TAAACATTACTTTTTGTTTTTGTCTTTATTAAACAGTTGTA 2280 N H P L S S k G N F V I K I R K ] F G F K * 175
Fig. 2. Nucleotide sequence and predicted amino acid sequence of the CHV gC homologue and ORF 2. The putative transmembrane region, potential gIycosylation sites, TATA box, CAAT box and polyadenylation signal sequences are underlined. Nucleotides and predicted amino acid residues are numbered to the right of the sequence.
gene starts at position 201 and ends at position 1580. The predicted translation product is 459 amino acids long. Comparison of this amino acid sequence with that of gC glycoproteins from other herpesviruses revealed extensive homology, indicating that this ORF encodes the CHV gC homologue (Table 2).
Analysis of the CHV gC nucleotide sequence Numerous potential TATA box, CAAT box and polyadenylation signal sequences are found in the 5' and 3' non-coding regions of the CHV gC gene (Fig. 2). The nucleotide sequence surrounding the CHV gC initiation
codon (AAAATGA) is favourable with respect to Kozak's rules at position - 3 , but not at position +4. That the CHV gC gene does not follow Kozak's rules is not unusual, as the CHV gB gene (see above), as well as the gC genes of FHV (J.-C. Audonnet, unpublished results), EHV-I (Allen & Coogle, 1988) and VZV (Davison & Scott, 1986) also contain an unfavourable nucleotide at position + 4.
Analysis of the predicted CHV gC amino acid sequence The deduced amino acid sequence of the CHV gC homologue is presented in Fig. 2. Hydropathicity
2034
K. J. Limbach and others
T a b l e 2. Homology between the predicted amino acid sequences of nine herpesvirus gC glycoproteins*
CHV FHV EHV-1 EHV-4 PRV BHV-1 VZV MDV
FHV
EHV-I
EHV-4
PRV
BHV-I
VZV
MDV
HSV-1
44
32 32
34 33 81 -
27 29 31 32
27 31 32 31 37
29 28 30 31 27 29 -
27 25 27 25 25 25 22
25 23 27 27 29 27 22 23
.
-
.
.
.
.
* Values were obtained using the ALIGN Plus program and are expressed as percentage identity. The entire gC amino acid sequence was used. See Table 1 for alignment parameters. References: FHV (J.-C. Audonnet, unpublished results), EHV-1 (Allen & Coogle, 1988), EHV-4 (Nicolson & Onions, 1990), PRV (Robbins et al., 1986), BHV-1 (Fitzpatrick et al., 1989), VZV (Davison & Scott, 1986), MDV (Ihara et al., 1989) and HSV-1 (McGeoch et al., 1988).
T a b l e 3. Homology between the predicted amino acid sequences of the ORFs
located adjacent to the gC gene in eight herpesviruses*
CHV FHV EHV-1 EHV-4 MDV HTV HSV- 1
FHV
EHV-1
EHV-4
MDV
HTV
197 (22) -
211 (22) 177 (24)
219 (21) 167 (18) 470 (50) . .
62 (4) 69 (4) 95 (8) 132 (8)
105 (13) 66 (4) 104 (9) 130 (11) 767 (75)
. -
.
. -
H S V - 1 VZV 53 (4) 40 (1) 79 (7) 60 (5) 83 (6) 91 (7) -
31 (0) 52 (1) 58 (3) 30 (0) 28 (0) 33 (0) 49 (2)
* Values were obtained using the FASTA and RDF2 programs (Pearson & Lipman, 1988)~ A ktup of 1 was used. Values in parentheses represent the number of standard deviations between the FASTA score and the mean of the scores obtained from 100 randomly permutated versions of the potentially related sequence. References: FHV (J.-C. Audonnet, unpublished results), EHV-1 (Telford et aL, 1992), EHV-4 (Nicolson & Onions, 1990), MDV (Ihara et al., 1989), HVT (Kato et aL, 1989), HSV-1 (McGeoch et al., 1988) and VZV (Davison & Scott, 1986).
analysis o f the p r e d i c t e d C H V g C a m i n o acid sequence revealed the presence o f two p r o m i n e n t h y d r o p h o b i c p e a k s ( d a t a n o t shown). T h e first p e a k , l o c a t e d at the N terminus, m a y r e p r e s e n t a p o t e n t i a l signal sequence. H o w e v e r , analysis w i t h P S I G N A L d i d n o t identify the N t e r m i n u s o f this p o l y p e p t i d e as a p o t e n t i a l signal sequence. Nevertheless, this r e g i o n does c o n t a i n elements o f a typical signal sequence, n a m e l y a basic N - t e r m i n a l region, a h y d r o p h o b i c core (residues 6 to 20) a n d a relatively p o l a r C - t e r m i n a l r e g i o n (Fig. 2). T h e second h y d r o p h o b i c p e a k , l o c a t e d at the C terminus, with a p r e d i c t e d m e m b r a n e - s p a n n i n g s e g m e n t b e t w e e n residues 426 a n d 451 (using the m e t h o d o f K l e i n et al., 1985), p r o b a b l y functions as a m e m b r a n e a n c h o r region. A l i g n m e n t o f the C H V g C a m i n o acid sequence with h o m o l o g o u s sequences f r o m o t h e r herpesviruses revealed a m o d e r a t e level o f similarity t h r o u g h o u t the entire sequence, with the e x c e p t i o n o f the N t e r m i n u s ( d a t a n o t shown). This a l i g n m e n t also revealed t h a t m o s t o f the cysteine residues are conserved. F o r example, C H V gC c o n t a i n s 10 cysteine residues, eight o f which are
c o n s e r v e d in all a l p h a h e r p e s v i r u s e s . T h e only cysteine residues in C H V gC t h a t are n o t c o n s e r v e d are l o c a t e d in the p u t a t i v e t r a n s m e m b r a n e o r i n t r a c e l l u l a r d o m a i n s . These results suggest t h a t the gC g l y c o p r o t e i n s m a y have similar t e r t i a r y structures. A l i g n m e n t with o t h e r g C sequences also revealed the relative c o n s e r v a t i o n o f p o t e n t i a l N - l i n k e d g l y c o s y l a t i o n sites. T h e significance o f this c o n s e r v a t i o n relative to the structure a n d f u n c t i o n o f the g C C H V g l y c o p r o t e i n is n o t k n o w n .
Identification and sequencing of O R F 2 N u c l e o t i d e sequence analysis o f the region d o w n s t r e a m f r o m the C H V gC gene revealed the presence o f a s e c o n d O R F (Fig. 2). This O R F ( O R F 2) starts at p o s i t i o n 1699 a n d ends at p o s i t i o n 2226. T h e p r e d i c t e d t r a n s l a t i o n p r o d u c t is 175 a m i n o acids long. C o m p a r i s o n o f this a m i n o acid sequence with the S W I S S - P R O T (Release 23) d a t a b a s e revealed h o m o l o g y with O R F s l o c a t e d d o w n s t r e a m f r o m o t h e r a l p h a h e r p e s v i r u s g C genes ( T a b l e 3). These h o m o l o g y scores suggest t h a t the O R F s l o c a t e d
Sequence of CHV gB, gC and gD homologues
2035
~ATATTT~T~AACTATTATGAAA~TTCTTAT~CTTATTTGTTTTTATTAAA~GGGTTGGGTTG~TTTTAAAATTACATAC~TGTATTMGAATT~CAT~TAAAG~CA~ACCCAT
120
GAAAAACA~TTAAATTCTAT~TG~CG~TT~TTTTCTCATTTT~GTTGCTAC~CTT~T~TAGTA3~aUL.~GATTAAA~TT~TATTTATCTTATTTTAT~TT~C~
240
M I
K L L
F I
L
F Y
F N P
13
T~CTGGATATAAATGGGTAGACCCTCCTCGTAGGTAT~TTACACCGTTTT~GAAT~TTCCA~TATT~CAAATCC~TG~TCCTTCTAAAAACGCTGAAGTT~GGTATGT~CTT 360 I
T
G
y
K
~
V
D
p
p
R
R
y
N
y
T
V
L
R
M
I
P
D
I
P
N
P
M
D
P
$
K
N
A
E
V
R
Y
V
T
53
CTACTGACCCATGTGATATGGTTGCTTT~TTTCT~TC~AAATATAGAATcTAC~TTAAAAc~TTCAATTTGTGCAAAAGAAAAAATTTTAC~TGCATCTCTTAGTTGGTTTAAAG 480 S
T
0
p
C
0
M
V
A
L
I
S
N
P
N
I
E
S
T
I
K
T
I
Q
F
V
Q
K
K
K
F
Y
N
A
S
L
S
W
F
K
93
TTGGAGAT~TTGTACATATCC~TATATTT~TTC~TATTTTGATTGT~TCCT~AAA~TTTGG~TATGTTTAAAAA~T~TCCAGATTTTTGGAAAC~ATCGTTAGTTGGTT600 V
G
D
O
C
T
y
p
I
y
L
I
Q
Y
F
D
C
D
P
Q
R
E
F
G
]
C
L
K
R
S
P
D
F
W K
P
S
L
V
G
133
ACACATTTTT~CTGAT~TGAATTGGGATTAGTTTTAGCTGCCCCCGCTC~TTT~TC~GGTC~TATA~CGGGTTATTCAAATTGAAAATG~GTTTTTTATACT~TTTTATGG 720 y
T
F
L
T
D
D
E
L
G
L
V
L
A
A
P
A
P
F
N
Q
G
Q
Y
R
R
V
I
Q
I
E
N
E
V
F
Y
T
D
F
M
1~
TTC~TTACCACGAGAAACTTGTTATTTTT~TAAAGAA~TAAATTTG~CC~CTTTTATGGAAT~TGT~GGAATCTA~TCTGTAGG~TCAAAAGTTGAC~TG~CTTTTTT 840 V
Q
L
P
R
E
T
C
Y
F
S
K
E
D
K
F
E
P
T
F
M
E
W
C
K
E
S
R
S
V
G
A
S
K
V
D
D
E
L
F
213
AT~TAAATAGAGCTGGTC~CCAAACCCTGCTTAAATATTATGTTATTAAAGATTTTTATAGACTT~CGGTA~GAACCTCCAATAAAATTTAAAG~GCT~TTA~TACGATATA~CAT ~0 y
L
N
R
A
G
p
Q
T
L
k
K
y
y
V
I
K
D
F
Y
R
L
N
G
R
E
P
P
I
K
F
K
E
A
k
R
Y
D
I
P
253
AT~AGTG~TGAT~ATTTGATGATGAATTA~CAT~GGCCACATATTAGT~TA~TATT~TA~JU~CTATTAAAGAAATTGTAAATCTTG~GATTATTTTAAAAATACAAATGTTA 1080 y
K
V
N
O
K
F
D
D
E
k
P
S
R
p
H
I
S
N
T
I
N
K
T
I
K
E
I
V
N
L
E
D
Y
F
K
N
T
N
V
293
TAGATACTA~TACCCC~CAC~TAAAT~TACCC~AAAAAATAT~CCGTGGG~TTGTTAT~TTATATT~T~TACTATTTAT~TTGGATTTTTTGTTTATAAAAGACAAAAAA 1200 I
D
T
T
T
p
T
p
I
N
N
T
P
K
N
I
T
V
G
I
V
I
I
]
L
I
I
L
F
I
I
G
F
F
V
Y
K
R Q
K
333
TATAT~T~T~ATAAAAAATT~c~cAAATGTTTAGCCTTTA~AAATT~TTTACAG~TAAAC~CTGGGCGGT~TTTTGTTT~TAAAAATTCATGTAcCTAC~TTTTATTcAC 1320 I Y N N Y K K L T T N V * 345
Fig. 3. Nucleotide sequence and predicted amino acid sequence of the C H V gD homologue. The putative signal sequence, transmembrane region, potential glycosylation sites and polyadenylation signal sequences are underlined. Nucleotides and predicted amino acid residues are numbered to the right of the sequence.
downstream from the gC gene in CHV, FHV, EHV-1, EHV-4, MDV, herpesvirus of turkey (HVT) and possibly HSV-1 are evolutionarily related. Conversely, the ORF (gene 13) located next to the VZV gC gene does not exhibit significant homology with any of the other comparably positioned ORFs. Furthermore, VZV gene 13 is oriented on the genome in the opposite direction relative to all the other ORF 2-like genes (Davison & Scott, 1986). These results are consistent with the proposed functions of the proteins encoded by these two groups of genes; VZV gene 13 encodes a thymidylate synthetase (Davison & Scott, 1986), whereas the HSV-1 ORF 2-like gene (UL45) encodes a putative virion protein (Visalli & Brandt, 1993). Therefore, it appears that the O R F located next to the gC gene in CHV, FHV, EHV-1, EHV-4, MDV, HVT and possibly HSV-1 encodes a protein that is structurally and functionally unrelated to the protein encoded downstream from the VZV gC homologue.
Analysis of the CHV ORF 2 nucleotide sequence Numerous potential TATA box, CAAT box and polyadenylation signal sequences are found in the 5' and 3' non-coding regions of ORF 2 (Fig. 2). The nucleotide sequence surrounding the ORF 2 initiation codon (AATATGG) is favourable with respect to Kozak's rules at positions - 3 and +4.
Identification and sequencing of the CHV gD gene Employing the same method used to map the CHV gC homologue, a 7 kb PstI fragment encoding an ORF with homology to herpesvirus gD glycoproteins was identified. The nuc|eotide sequence of this ORF is presented in Fig. 3. The putative CHV gD gene starts at position 201 and ends at position 1238. The predicted translation product is 345 amino acids long. Comparison of this amino acid sequence with the sequence of other gD glycoproteins revealed extensive homology, indicating that this ORF encodes the CHV gD homologue (Table 4).
Table 4. Homology between the predicted amino acid sequences of six herpesvirus gD glycoproteins*
CHV FHV EHV-1 PRV BHV- 1
FHV
EHV-1
PRV
BHV-1
HSV-1
45 -
35 31
-
-
27 30 26 -
34 34 27 37
21 24 21 27 24
* Values were obtained using the A L I G N Plus program and are expressed as percentage identity. The entire gD amino acid sequence was used. See Table 1 for alignment parameters. References : F H V (J.C. Audonnet, unpublished results), EHV-I (Flowers et al., 1991), PRV (Petrovskis et al., 1986), BHV-I (Tikoo et al., 1990) and HSV-I (Lasky & Dowbenko, 1984).
2036
K. J. Limbach and others
Analysis o f the C H V gD nucleotide sequence
Potential polyadenylation signal sequences are found downstream from the CHV gD gene. However, TATA box and CAAT box sequences are not found upstream from the CHV gD gene, although numerous TATA boxlike sequences are found (Fig. 3). The nucleotide sequence surrounding the CHV gD initiation codon (AAAATGA) is favourable with respect to Kozak's rules at position - 3 , but not at position + 4 (Fig. 3). This is not unusual, as the CHV gB and gC genes (see above), as well as gD genes of FHV (J.-C. Audonnet, unpublished results), EHV-1 (Audonnet et al., 1990; Flowers et al., 1991), PRV (Petrovskis et al., 1986) and BHV-1 (Tikoo et al., 1990) also contain an unfavourable nucleotide at position + 4.
to that of other alphaherpesviruses. For example, the ORF located immediately upstream from the CHV gB gene has homology with gene 30 of VZV (Davison & Scott, 1986) and with UL28 of HSV-1 (McGeoch et al., 1988), both of which are located immediately upstream from the gB homologue in those viruses (data not shown). ORF 2, located immediately downstream from the CHV gC gene, has homology with the ORFs located immediately downstream from the gC homologue in FHV (J.-C. Audonnet, unpublished results), EHV-1 (Telford et al., 1992), EHV-4 (Nicolson & Onions, 1990), HVT (Kato et al., 1989) and perhaps HSV-1 (McGeoch et al., 1988) (see above). Additionally, the ORF located immediately downstream from the CHV gD gene has homology with the gI gene of EHV-1 (Audonnet et al., 1990) and the gp63 gene of PRV (Petrovskis et al., 1986), both of which are located immediately downstream from the gD homologue in those viruses (data not shown).
Analysis of the predicted C H V gD amino acid sequence
The deduced amino acid sequence of the CHV gD homologue is presented in Fig. 3. Hydropathicity analysis of the predicted CHV gD amino acid sequence revealed the presence of two prominent hydrophobic peaks (data not shown). The first peak, located at the N terminus, may represent a potential signal sequence. In fact, PSIGNAL identifies a potential cleavage site between residues 16 and 17. The second hydrophobic peak, located near the C terminus, with a predicted membrane-spanning segment between residues 309 and 328 (using the method of Klein et al., 1985), probably functions as a membrane anchor region. Alignment of the CHV gD amino acid sequence with homologous sequences from other herpesviruses revealed a moderate level of similarity throughout the entire sequence, with the exception of the N terminus (data not shown). This alignment also revealed that the vast majority of cysteine residues are perfectly conserved. For example, CHV gD contains six cysteine residues, all of which are perfectly conserved in all alphaherpesviruses. These results suggest that the gD glycoproteins may have similar tertiary structures. This alignment also revealed that the potential N-linked glycosylation sites are well conserved. It is not known whether the CHV gD glycosylation sites are utilized. However, it is known that all the potential HSV-1 gD glycosylation sites are used (Sodora et al., 1991).
Genomic organization
The gB, gC and gD genes were not mapped to specific locations on the CHV genome. However, nucleotide sequence analyses of the regions flanking these genes indicate that the genomic organization of CHV is similar
Discussion In this report we present the nucleotide sequence of the CHV gB, gC and gD genes. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequence of these putative glycoproteins with the gB, gC and gD amino acid sequences of other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. This analysis also revealed that the homology among gB homologues is greater than that among gC or gD homologues, suggesting that the structural and functional constraints on gB may be greater than those on gC or gD. Alignment of homologous gB, gC and gD polypeptides revealed that the vast majority of cysteine residues are perfectly conserved. These results suggest that these cysteine residues, due to their ability to form disulphide bonds, are important in maintaining the structural and functional integrity of the gB, gC and gD glycoproteins. In HSV-1 gD, it has been shown that cysteine 1 forms a disulphide bond with cysteine 5, that cysteine 2 forms a disulphide bond with cysteine 6 and that cysteine 3 forms a disulphide bond with cysteine 4 (Long et al., 1992). Furthermore, it has been shown that a mutation of any of these residues has a profound effect on the conformation, processing and function of the resulting glycoprotein (Wilcox et al., 1988; Long et al., 1990). Therefore, the conservation of cysteine residues in these glycoproteins may have structural significance. The high degree of homology among the gB, gC and gD homologues also suggests that these glycoproteins have common functions. It has been shown that the
Sequence of C H V gB, gC and gD homologues BHV-1 gB homologue can rescue a gB- PRV strain, indicating that these two glycoproteins are functionally equivalent (Kopp & Mettenleiter, 1992). Alignment of the gB, gC and gD amino acid sequences also revealed that potential N-linked glycosylation sites are somewhat conserved. N-linked glycosylation is thought to play a role in a variety of functions, such as maintenance of protein conformation and protection against proteolytic degradation. The biological significance of N-linked carbohydrates on herpesvirus glycoproteins is not completely understood. For example, tunicamycin treatment of HSV-l-infected cells has been shown to inhibit the production of infectious virions (Pizer et al., 1980). In addition, endoglycosidase treatment of HSV-1 virions has been shown to decrease infectivity (Kuhn et al., 1988). On the other hand, N-linked glycosylation of HSV-1 gD does not appear to be absolutely essential, since mutagenesis of the glycosylation sites on this glycoprotein does not affect infectivity (Sodora et al., 1991). Therefore, although the glycosylation sites on the gB, gC and gD glycoproteins are relatively well conserved, proper glycosylation of each of these polypeptides may not be absolutely essential. The G + C content of herpesviruses varies from 33 to 75 % (Roizman, 1982). It has been suggested that this extensive variability is due to a non-selective mutational bias based on the presence (or absence) of virus-encoded or -induced enzymes involved in nucleotide metabolism (Honess, 1984). For example, VZV and herpesvirus saimiri both have a relatively low G + C content (46 %) and both encode an enzyme, thymidylate synthetase, which is involved in TTP synthesis (Davison & Scott, 1986; Honess et al., 1986). HSV-1, HCMV and EBV, on the other hand, have relatively high G + C content (68 %, 57% and 60%, respectively) and do not appear to encode a thymidylate synthetase (Honess et al., 1986). CHV has been determined by DNA density analysis to have the lowest G + C content of any herpesvirus, 33 % (Plummer et al., 1969; Roizman, 1982), a value which is consistent with the relatively low G + C content of the nucleotide sequence presented in this report (29 %). It is not known whether CHV encodes an enzyme involved in nucleotide metabolism. However, it is known that the ORF located immediately downstream from the CHV gC gene is not homologous to VZV thymidylate synthetase. Therefore, if CHV contains a thymidylate synthetase gene, it is not found at the same genomic location as that of VZV. The parameters of a protective immune response against a CHV infection have not been completely elucidated. It is known that newborn pups exposed to CHV usually die without forming CHV-specific neutralizing antibodies. It is also known that maternal anti-
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bodies or treatment with immune serum from seropositive dogs can protect puppies from a fatal CHV infection (Carmichael, 1970). Therefore, it appears that serum neutralizing antibodies can protect puppies against a fatal CHV infection. Three CHV glycoproteins, gp145/112, gp80 and gp47, are known to elicit CHV neutralizing antibodies (Xuan et al., 1991). The genes encoding these glycoproteins have not been identified. It is possible that these antigens are encoded by the gB, gC and gD genes identified in this paper. Since several reports have indicated that an immune response against gB, gC and/or gD can provide protection of target species animals against a herpesvirus challenge (Babiuk et al., 1987; Nazarian et al., 1992; Riviere et al., 1992; Brockmeier et al., 1993), it is hoped that the isolation of the CHV gB, gC and gD genes will lead to the development of an efficacious CHV vaccine. We thank Gerard Johnson for his invaluable assistance with the computer-based analyses and manipulations presented in this report. We also thank James Tartaglia and Gerard Johnson for their critical review of this manuscript.
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(Received 17 August 1993; Accepted 9 February 1994)
