Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication, and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo

Abstract

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-ͦ-X, where ͦ indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Varicella-zoster virus (VZV), an alphaherpesvirus, causes chicken pox (varicella) as a primary infection and shingles (zoster) upon reactivation from infected ganglia in humans (reviewed in reference 16).Although not yet investigated in VZV, herpesvirus entry requires fusion of the virus envelope with cell membranes governed by viral glycoprotein B (gB) and gH/gL, which are conserved across the family Herpesviridae (12,27,57). gB is the most conserved glycoprotein, with its function as a fusion protein well documented for several of the herpesviruses (10,19,38,48,51,52).Open reading frame 31 (ORF31) codes for the 931 amino acids of VZV gB (18, 37). The successive N-and O-linked glycosylation plus the sialation and sulfation of VZV gB yields a mature protein with a molecular mass of approximately 140 kDa (45). Upon maturation, gB is cleaved, presumably by cellular proteases, into two polypeptides of 66 and 68 kDa. Intracellular trafficking of gB was shown to be dependent upon amino acid motifs in the cytoplasmic domain (24-26). In transfection studies, gB was transported to the cellular surface where it was endocytosed and localized to the trans-Golgi, where envelopment of viral particles is thought to occur.The structures of gB in the two human alphaherpesviruses, VZV and herpes simplex virus type 1 (HSV-1), are likely to be very similar as they have 49% amino acid identity (reviewed in reference 16). The ectodomain of HSV-1 gB was shown to form a spike that consisted of trimers with the structural homology to gG of vesicular stomatitis virus (28). Heldwein et al. (28) proposed that HSV-1 gB is a class II fusion protein based on homology to VSV G. The herpesvirus gB monomer was divided into five domains, I to V. Domain I consisted of a continuous amino acid sequence that folded into a pleckstrin homology-like domain, while domain II was comprised of two discontinuous segments, which also had a pleckstrin homologylike domain. A loop region exposed to the exterior of gB connected domain II with domain III. Domain III was comprised of three discontinuous segments and connected to the external loop by a long ␣ helix that ended in a central coiled coil. Domain IV crowned gB and was connected to domain V, which stretched from the top to the bottom of the gB monomer, forming ...

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

“…For example, smaller plaque sizes were reported for BHV-1 suggesting a role for proteolytic cleavage of gB in cell-to-cell spread (Kopp et al, 1994). Furthermore, reduced syncytium formation and attenuated replication in human skin xenografts were seen for PRV and VZV, respectively (Okazaki, 2007;Oliver et al, 2009). Reduced cell fusion was also observed for EBV in epithelial and B-cells but not in fibroblasts, indicating a conserved function of furin-mediated cleavage among gB homologues from different herpesvirus subfamilies (Sorem & Longnecker, 2009;Strive et al, 2002).…”

“…Previous studies done on furin cleavage of gB homologues of other herpesviruses have made evident differences in mutant viruses when evaluated for their growth properties in vitro (Glauser et al, 2013;Kopp et al, 1994;Okazaki, 2007;Oliver et al, 2009;Sorem & Longnecker, 2009;Strive et al, 2002). Cleavage of gB has been shown to be important for syncytium formation in the case of PRV (Okazaki, 2007) and for cell-to-cell spread in the case of BHV-1 (Kopp et al, 1994).…”

“…One similarity with VZV is what appears to be partial cleavage of gB when the cognate furin cleavage recognition sites is mutated. Previously, two alternative cleavage sites in proximity to the furin recognition site were proposed to be responsible for partial cleavage of VZV gB, but the functionality of these motifs was not tested (Oliver et al, 2009). For other members of the genus Varicellovirus, such as PRV and BHV-1, no conclusion with regard to additional cleavage sites can be drawn, since they were evaluated by deleting large portions of gB (BHV-1) or only shown using chemical inhibitors (PRV) (Kopp et al, 1994;Okazaki, 2007).…”

“…Across most of the members of the herpesvirus subfamilies, gB seems to possess a conserved cleavage site Arg-X-Lys/ Arg-Arg targeted by the cellular protease furin (Backovic et al, 2007;Glauser et al, 2013;Okazaki, 2007;Oliver et al, 2009;Sorem & Longnecker, 2009). EHV-1 gB was predicted ( Fig.…”

“…The function of the furin cleavage motif of glycoprotein B (gB) was evaluated for several herpesviruses. Abolishing furin cleavage is dispensable for in vitro replication of human cytomegalovirus (HCMV), bovine herpesvirus 1 (BHV-1), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), murid herpesvirus 4 (MuHV-4) and pseudorabies virus (PRV) (Glauser et al, 2013;Kopp et al, 1994;Okazaki, 2007;Oliver et al, 2009;Sorem & Longnecker, 2009;Strive et al, 2002). However, changes in phenotype were seen for several of these viruses.…”