Identification and nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4

M P Riggio, A A Cullinane, D E Onions
1989 Journal of Virology  
The nucleotide sequence of the glycoprotein gB gene of equine herpesvirus 4 (EHV-4) was determined. The gene was located within a BamHI genomic library by a combination of Southern and dot-blot hybridization with probes derived from the herpes simplex virus type 1 (HSV-1) gB DNA sequence. The predominant portion of the coding sequences was mapped to a 2.95-kilobase BamHI-EcoRI subfragment at the left-hand end of BamHI-C. Potential TATA box, CAT box, and mRNA start site sequences and the
more » ... ional initiation codon were located in the BamHI M fragment of the virus, which is located immediately to the left of BamHI-C. A polyadenylation signal, AATAAA, occurs nine nucleotides past the chain termination codon. Translation of these sequences would give a 110-kilodalton protein possessing a 5' hydrophobic signal sequence, a hydrophiic surface domain containing 11 potential N-linked glycosylation sites, a hydrophobic transmembrane domain, and a 3' highly charged cytoplasmic domain. A potential internal proteolytic cleavage site, Arg-Arg/Ser, was identified at residues 459 to 461. Analysis of this protein revealed amino acid sequence homologies of 47% with HSV-1 gB, 54% with pseudorabies virus gpII, 51% with varicella-zoster virus gpII, 29% with human cytomegalovirus gB, and 30% with Epstein-Barr virus gB. Alignment of EHV-4 gB with HSV-1 (KOS) gB further revealed that four potential N-linked glycosylation sites and all 10 cysteine residues on the external surface of the molecules are perfectly conserved, suggesting that the proteins possess similar secondary and tertiary structures. Thus, we showed that EHV-4 gB is highly conserved with the gB and gpII glycoproteins of other herpesviruses, suggesting that this glycoprotein has a similar overall function in each virus. * Corresponding author. were determined with DNA probes derived from the HSV-1 gB gene. In this report, we present the nucleotide sequence of the EHV-4 gB gene and report that this protein shares amino acid homology with the gB glycoproteins of HSV-1 (8, 31), Epstein-Barr virus (29), and human cytomegalovirus (11) and the gpII glycoproteins of pseudorabies virus (PRV) (34) and varicella-zoster virus (VZV) (21). MATERIALS AND METHODS Recombinant DNA methods. The EcoRI F restriction fragment of HSV-1 (19) cloned in plasmid pACYC184 (26) was kindly provided by J. B. Clements (MRC Institute of Virology, Glasgow), and this recombinant plasmid was designated pACYC-EcoRI(F). The entire HSV-1 gB gene was excised from plasmid pACYC-EcoRI(F) as a 3.3-kilobase (kb) XhoI-KpnI fragment, which was subsequently directionally cloned into the XhoI and KpnI sites of pIC20R to generate the plasmid pICgB. Digestion of pICgB with NarI yielded a 650-base-pair fragment (corresponding to amino acids 121 to 336 of HSV-1 gB), and digestion of pICgB with PstI yielded an 810base-pair fragment (corresponding to amino acids 530 to 799 of HSV-1 gB), which were used as 5' and 3' HSV-1 gB DNA hybridization probes, respectively, in dot-blot and Southern blot analyses (see Fig. 2 ). The genomic library of EHV-4 strain 1942 (12) contained the 13.6-kb BamHI C fragment in the BamHI site of pUC9. The 2.95-kb BamHI-EcoRI fragment at the left end of BamHI-C was excised from pUC9 as a 2.95-kb EcoRI fragment and cloned into the EcoRI site of Bluescript M13+ vector. This recombinant plasmid was designated pBSgB. Bacterial strains. Escherichia coli JM83 and JM101 were used for recombinant DNA experiments and were grown in L broth medium (L broth contains 10 g of tryptone, 5 g of 1123
doi:10.1128/jvi.63.3.1123-1133.1989 fatcat:zkkpxk4zgza37pdaabd6aljjli