The BHV-5 strain N569 (BHV-5/N569) homolog to the BHV-1 US4 gene was sequenced and characterized. RNA analyses showed that a 1.8-kb mRNA which contains the BHV-5/N569 US4 open reading frame initiates 55 nucleotides upstream from the predicted translational start codon and terminates 17 nucleotides downstream from the consensus sequence for polyadenylation. Comparison of the deduced amino acid sequences of the predicted US4 encoded proteins of BHV-5/N569 and BHV-1 strain Schönböken (BHV-1/Schö) revealed 75% identity. An antiserum, raised in rabbits after infection with a BHV-5/N569 US4 ORF expressing recombinant vaccinia virus, specifically precipitated a 65-kDa protein and a diffusely migrating protein species with an apparent molecular mass between 90 and > 240 kDa from the supernatant of BHV-5/ N569 infected cells. Treatment of immmunprecipitated proteins with chondroitinase AC demonstrated that the latter contains glycosaminoglycans. The mobility of the BHV-5/N569 US4 gene products was identical to the BHV-1 US4 ORF encoded glycoprotein G (gG) and glycoproteoglycan G (gpgG; G. M. Keil, T. Engelhardt, A. Karger, and M. Enz. J. Virol. 70, 3032-3038, 1996) and were therefore named BHV-5 gG and BHV-5 gpgG. Immunoprecipitations with sera from BHV-1 infected cattle indicated a type-specific immune response to gG, since these sera failed to react with vaccinia virus-expressed gG-5 but recognized vaccinia virus-expressed gG-1.
Sequence analysis of the short unique (U S ) segment of the bovine herpesvirus 1 (BHV-1) genome predicted that the U S open reading frame (ORF) 4 encodes a protein with homology to glycoprotein G (gG) of other alphaherpesviruses (P. Leung-Tack, J.-C. Audonnet, and M. Riviere, Virology 199:409-421, 1994). RNA analysis showed that the U S ORF4 is contained within two transcripts of 3.5 and 1.8 kb. The 3.5 kb RNA represents a structurally bicistronic RNA which encompasses the U S ORF3 and U S ORF4, whereas the 1.8-kb RNA constitutes the monocistronic U S ORF4 mRNA. To identify the predicted BHV-1 gG, recombinant vaccinia virus expressing the U S ORF4 was used to raise specific antibodies in rabbits. The antiserum recognized a 65-kDa polypeptide and a very diffusely migrating species of proteins with an apparent molecular mass of between 90 and greater than 240 kDa in supernatants of BHV-1-infected cells which was also precipitated together with 61and 70-kDa polypeptides from cell-associated proteins. The specificity of the reaction was demonstrated by the absence of these proteins from the supernatant of cells infected with the U S ORF4 deletion mutant BHV-1/ gp1-8. Treatment of the immunoprecipitated proteins with glycosidases and chondroitinase AC showed that the 65-kDa protein constitutes gG, which contains both N-and O-linked carbohydrates, and that the highmolecular-mass proteins contain glycosaminoglycans linked to a 65-kDa glycoprotein that is antigenically related to gG. These molecules were therefore named glycoproteoglycan G (gpgG). Pulse chase experiments indicated that gG and gpgG were processed from a common precursor molecule with an apparent molecular mass of 61 kDa via a 70-kDa intermediate. Both gG and gpgG could not be found associated with purified virions. In summary, our results identify the BHV-1 gG protein and demonstrate the presence of a form of posttranslational modification, glycosamino-glycosylation, that has not yet been described for a herpesvirusencoded protein.
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