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

Abstract

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.

Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins

“…Full‐length and truncated versions of gG encoded by both BHV‐1 and BHV‐5 were secreted and bound both CCL19 and CXCL8, confirming that gG from these viruses encodes the vCKBP. BHV‐1 gG consists of a glycosylated form (gG; 65 kDa) and a complex glycoproteoglycan of higher molecular mass (gpgG; 90 to >240 kDa) which is modified by the addition of chondroitin sulfate (Keil et al ., 1996), and this was consistent with chemokines cross‐linking to a diffusely migrating species of proteins between 60 and >175 kDa (Figure 1A). These high molecular size forms were also observed after chemokine cross‐linking with BHV‐5, RanHV‐1 and CerHV‐1 samples, but not with CapHV‐1.…”

“…A unique feature not found previously in vCKBPs is that gG is a membrane protein, anchored by a C‐terminal transmembrane domain, which may be secreted after proteolytic cleavage. Secretion of gG has been demonstrated during infections with EHV‐1, EHV‐4, BHV‐1, BHV‐5 and HSV‐2 (Marsden et al ., 1984; Su et al ., 1987; Crabb et al ., 1992; Engelhardt and Keil, 1996; Keil et al ., 1996; Drummer et al ., 1998). The finding of chemokine binding activity secreted from cells infected with EHV‐3, RanHV‐1, CapHV‐1 and CerHV‐1 suggests that gG from these viruses is also proteolytically cleaved and secreted.…”

“…Alphaherpesvirus gG was first characterized in HSV‐2 as a secreted 92 kDa glycoprotein (Marsden et al ., 1984; Su et al ., 1987). Homologues of gG were found in HSV‐1 (Richman et al ., 1986), EHV‐1 (Drummer et al ., 1998), EHV‐3 (Hartley et al ., 1999), EHV‐4 (Crabb et al ., 1992; Drummer et al ., 1998), BHV‐1 (Keil et al ., 1996) and BHV‐5 (Engelhardt and Keil, 1996), but the gG gene is absent in VZV (Gomi et al ., 2002).…”

The Class II Membrane Glycoprotein G of Bovine Respiratory Syncytial Virus, Expressed from a Synthetic Open Reading Frame, Is Incorporated into Virions of Recombinant Bovine Herpesvirus 1

“…The schematic representation of the prototype orientation of the BHV-1 genome is shown above the HindIII restriction fragment map of BHV-1 strain Schönböken (7,27). The wild-type HindIII L fragment is enlarged, and the location and direction of transcription of genes encoding the putative protein kinase (PK) and glycoproteins G (gG), D (gD), I (gI), and E (gE) are indicated by arrows (15,25,40,45). Relevant restriction enzyme cleavage sites are marked.…”

“…As with other large DNA viruses, interest exists in the use of recombinant BHV-1 as an improved live vaccine against BHV-1 infection (1,21,42) or as a vector for bi-or multivalent vaccines against BHV-1 and additional bovine pathogens (17,18). To date, incorporation of heterologous genes into the genome of BHV-1 has concentrated mainly on the expression of the procaryotic lacZ gene to identify essential and nonessential genes or as a reporter gene for analytical studies (3,8,12,15,20,29,37,38,45). Recently, BHV-1 has been used to express biologically active bovine interleukins (21,32) and glycoproteins of pseudorabiesvirus (19,31).…”