Synthesis and processing of equine herpesvirus type 1 glycoprotein 14

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.

Section: Effect Of Glycosylation On Cleavage Of Ehv-1 Gbmentioning

confidence: 99%

Section: Effect Of Glycosylation On Cleavage Of Ehv-1 Gbmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Wellington

Gooley

Love

et al. 1996

“…Information about EHV-1 gB is limited, but it appears to be a 138 kDa protein that, when fully glycosylated, is proteolytically cleaved into two subunits (77 and 55 kDa). These subunits are linked by a disulfide bond(s) to form a 145 kDa complex (Sullivan et al, 1989). 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.…”

Section: Introductionmentioning

confidence: 99%

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

Spiesschaert

Stephanowitz

Krause

et al. 2016

Glycoprotein B (gB) of equine herpesvirus type 1 (EHV-1) is predicted to be cleaved by furin in a fashion similar to that of related herpesviruses. To investigate the contribution of furin-mediated gB cleavage to EHV-1 growth, canonical furin cleavage sites were mutated. Western blot analysis of mutated EHV-1 gB showed that it was cleaved at two positions, 518 RRRR 521 and 544 RLHK 547 , and that the 28 aa between the two sites were removed after cleavage. Treating infected cells with either convertase or furin inhibitors reduced gB cleavage efficiency. Further, removal of the first furin recognition motif did not affect in vitro growth of EHV-1, while mutation of the second motif greatly affected virus growth. In addition, a second possible signal peptide cleavage site was identified for EHV-1 gB between residues 98 and 99, which was 13 aa downstream of that previously identified.

“…It has been shown to be the homologue of herpes simplex virus (HSV) glycoprotein B (gB) and is regulated as a fl-y class protein (Whalley et al, 1989;Sullivan et al, 1989). A model of gpl4 processing proposes that a 980 amino acid polypeptide of M r l18K is co-translationally Nglycosylated to generate a 138K precursor molecule.…”

mentioning

confidence: 99%

Expression of equine herpesvirus type 1 glycoprotein gp14 in Escherichia coli and in insect cells: a comparative study on protein processing and humoral immune responses

Osterrieder

Wagner

Pfeffer

et al. 1994

The extracellular portion (amino acids 1 to 844) of the equine herpesvirus type 1 (EHV-1) glycoprotein gpl4, the homologue of gB of herpes simplex virus, was expressed in Escherichia coli and in insect cells using a recombinant baculovirus. Immunoblot analysis revealed that the recombinant E. coli expressed a fusion protein ofM r 135K which was composed of the truncated gpl4 and the maltose-binding protein (MBP) provided by the vector and a 90K protein lacking the MBP moiety. Both proteins were sequestered within the cells in form of inclusion bodies. Infection of insect cells with the recombinant baculovirus resulted in the production of a ll5K to ll8K glycoprotein which was cleaved intracellularly into two subunits ofM r 55K and 63K to 65K. The cleaved subunits were secreted into the cell culture supernatant and formed disulphide-linked dimers of M r 120K to 122K. The recombinant proteins produced in E. coli and in insect cells elicited EHV-l-specific antibodies in goats as demonstrated by Western blot analysis. The gpl4 expressed in insect cells induced antibodies with virus-neutralizing activity. In contrast, the truncated gpl4 expressed by E. coli failed to elicit neutralizing antibodies. The results suggest that posttranslational modification of the EHV-1 gpl4 may be important for the expression of epitopes necessary for the induction of neutralizing antibodies.