N-terminal sequence analysis of equine herpesvirus 1 glycoproteins D and B and evidence for internal cleavage of the gene 71 product

Abstract

Signal cleavage sites of equine herpesvirus 1 (EHV-1) glycoproteins D and B (gD and gB) and an endoproteolytic cleavage site of EHV-1 gB were determined by N-terminal amino acid sequencing and compared with known cleavage sites of homologues in other herpesviruses. Signal cleavage of EHV-1 gD occurred between Arg 35 and Ala 36 in a region of basic amino acids resembling the endoproteolytic cleavage sites of viral glycoproteins, nine amino acids downstream of the predicted site, while EHV-1 gB was cleaved as predicted between Ala s5 and Val s~. Endoproteolytic cleavage of EHV-1 gB occurred between Arg 54s and Ala 5aa, 28 amino acids downstream of the cleavage site predicted from conserved sequences of other herpesvirus gB homologues. One interpretation of these data is that EHV-1 gB is cleaved internally at both sites, a possibility which was supported by the apparent molecular masses of the unglycosylated gB subunits produced in the presence of tunicamycin. This double cleavage would release a stretch of amino acids which is not present in sequenced gB molecules of other herpesviruses. Experiments with glycosylation inhibitors indicated that cleavage of EHV-1 gB can occur in the absence of glycosylation. N-terminal sequencing also determined that a 42 kDa EHV-1 glycoprotein was a product of internal cleavage of the protein encoded by gene 71. Staggered endoproteolytic cleavage after adjacent arginine residues 506 and 507 separates the 42 kDa Cterminal subunit containing all the cysteine residues from the serine and threonine rich N-terminal region.

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin

“…1). Our data are in agreement with a previously proposed model in which gB was predicted to be cleaved at the 544 RLHKR 548 motif and possibly also at the conserved furin cleavage site 518 RRRR 521 (Wellington et al, 1996a). One similarity with VZV is what appears to be partial cleavage of gB when the cognate furin cleavage recognition sites is mutated.…”

“…Although the site responsible for cleavage of EHV-1 gB into two subunits was believed to be a conserved furin cleavage motif ( 518 RRRR 521 ), an alternate endoproteolytic cleavage site ( 544 RLHK 547 ) was identified for EHV-1 gB. However, cleavage at 518 RRRR 521 was not excluded, suggesting that the 28 aa between both cleavage sites are removed (Wellington et al, 1996a). As there are no previous studies addressing the role of furin cleavage during EHV-1 replication, we addressed the question of whether abolishing individual or all possible furin cleavage sites of EHV-1 gB would have attenuating effects on virus infection.…”

Characterization of glycoprotein B of the gammaherpesvirus equine herpesvirus-2.

“…Another possible explanation may be that the 62 kDa protein is an alternatively cleaved form of the small subunit of gB. EHV-1 gB has been shown to have an additional proteolytic cleavage site 28 amino acids C-terminal of the expected cleavage site (Wellington et al, 1996). Following complete deglycosylation of gB, 50 and 47 kDa subunits were clearly visible.…”

“…The consensus sequence at the cleavage site of gB homologues that are endoproteolytically cleaved has been reported to be the basic tetrapeptide Arg-Xaa-(Arg\Lys)-ArgSer (Wellington et al, 1996) or Arg-(Thr\Ser\Arg)-(Lys\Arg)-Arg (Spaete et al, 1990). EHV-2.86\67 has a predicted endoproteolytic cleavage motif (Arg-Arg-Arg-Arg) at amino acids 431-435.…”

“…Further digestion with O-glycosidase did not increase the mobility of the neuraminidase-treated proteins and suggests that EHV-2 gB contains no O-linked oligosaccharides. As a control, EHV-1 gp300, a protein known to contain O-linked oligosaccharides, was reduced in molecular mass using this protocol, thus verifying the activity of the enzymes (data not shown) (Wellington et al, 1996). The addition of neuraminidase to digestions containing N-glycosidase F appeared to facilitate removal of N-linked oligosaccharides by N-glycosidase F. Treatment with neuraminidase, O-glycosidase and N-glycosidase F reduced the molecular masses of the 130, 89, 65 and 62 kDa forms to 97, 50, 49 and 47 kDa, respectively (Fig.…”