Bovine herpesvirus 1 U-s open reading frame 4 encodes a glycoproteoglycan

Sequence analysis of the short unique (U-s) segment of the bovine herpesvirus I (BHV-1) genome predicted that the U-s open reading frame (ORF) 4 encodes a protein with homology to glycoprotein G (gG) of other alpha-herpesviruses (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, 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 61 and 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, 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 high molecular-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 herpesvirus encoded protein.