Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope

Abstract

Studies with molecular and immunological techniques identified and mapped the transcript encoding glycoprotein D (gD) of equine herpesvirus 1 KyA, as well as two continuous gD antigenic determinants. Three mRNA species of 5.5, 3.8, and 1.7 kb overlap the gD open reading frame and are transcribed from the DNA strand encoding gD. Northern (RNA) blot hybridization with both DNA clones and riboprobes, as well as S1 nuclease analyses, showed the 3.8-kb mRNA to encode gD and to be synthesized as a late (beta-gamma) transcript. The 3.8-kb gD mRNA initiates within the US segment 91 and 34 nucleotides downstream of the CCAAT and TATA elements, respectively, and encodes a potential polypeptide of 392 amino acids. The termination site of this transcript maps within the terminal repeat at a site also used by the 5.5-kb mRNA and the IR6-encoded 1.2-kb mRNA, such that these three transcripts form a 3'-coterminal nested set. The extended size (2,250 nucleotides) of the 3' untranslated region of the gD transcript and its termination within the terminal repeat may result from the deletion of 3,859 bp, which eliminates two consensus polyadenylation signals downstream of the gD open reading frame of EHV-1 KyA. Use of antisera to synthetic peptides of 19 amino acids (residues 4 to 22) and 20 amino acids (residues 267 to 285) in Western immunoblot analyses revealed that gD is present in EHV-1 virions as a 55-kDa polypeptide. In addition, these antisera detected the 55-kDa protein as well as 58- and 47-kDa polypeptides in infected-cell extracts at late times of infection. Residues 4 to 22 make up a continuous neutralizing epitope of gD, since incubation of equine herpesvirus 1 with the anti-19-mer serum prior to infection results in reduced numbers of plaques and reduced levels of virus-encoded thymidine kinase. Complement is not required for neutralization mediated by the anti-19-mer serum.