Hydrophobic Residues That Form Putative Fusion Loops of Epstein-Barr Virus Glycoprotein B Are Critical for Fusion Activity

Abstract

To test the importance of the hydrophobic residues within the putative Epstein-Barr virus (EBV) glycoprotein B (gB) fusion loops in membrane fusion, WY 112-113 and WLIW [193][194][195][196] were mutated into alanine, glutamic acid, or the analogous residues from herpes simplex virus type 1 (HSV-1) gB (HR and RVEA). All gB variants exhibited cell surface expression, demonstrating that the substitutions did not perturb gB trafficking. None of six gB variants was, however, capable of mediating fusion with either epithelial or B cells. These data demonstrate that the bulky and hydrophobic EBV loop residues, which differ from the more hydrophilic HSV-1 residues and appear more compatible with membrane insertion, are essential for EBV gB-dependent fusion.Envelope glycoprotein B (gB) and glycoproteins H and L (gH/gL) form the core fusion machinery of all herpesviruses (32). The mechanism by which the three glycoproteins function to orchestrate membrane fusion is not fully understood. In varicella-zoster virus, cytomegalovirus, and human herpesvirus 8 (HHV-8), the action of gB or the gH/gL complex alone can result in fusion, although at a lower level than when all three glycoproteins are present (6,17,25). A truncated variant of Epstein-Barr virus (EBV) gB mediates fusion with epithelial cells at levels up to 60% of what is observed when gB, gH, and gL are transfected together (23,25). In the case of herpes simplex virus type 1 (HSV-1), the gH/gL complex seems to be responsible for the formation of a hemifusion intermediate, whereas gB is required to resolve the intermediate and complete fusion (33). The involvement of multiple proteins distinguishes herpesviruses from most other viruses where membrane merger is typically mediated by one fusion protein (16).Glycoprotein B is highly conserved throughout the herpesvirus family. HSV-1 gB exhibits 86% sequence identity with HSV-2 gB and 29% with EBV gB, while EBV and HHV-8 gB share 40% sequence identity. Although HSV-1 gB does not share any similarity with the fusion protein (G) of vesicular stomatitis virus (VSV) at the protein sequence level, the structural homology between the two proteins is notable (Fig. 1A) (11, 29). The only available structure of HSV-1 gB (11) was proposed to represent a postfusion conformation based on the similarity with the postfusion form of G.Fusion peptides of class I and II fusion proteins are rich in hydrophobic and aromatic residues and directly insert into the membrane after the conformational change is triggered. The residues critical for the ability of VSV G protein to cause fusion fall within two internal regions and give rise to a bipartite fusion peptide made of WY [72][73] and YA 116-117 (7, 35, 37). The conformation of the two fusion loops resembles the typical hairpin fold adopted by fusion peptides of class II fusion proteins (16). Regions structurally homologous to the fusion peptide of G were proposed to form putative fusion loops in HSV-1 gB (11). Most of the corresponding residues in HSV-1 gB, however, are not hydrophobic an...

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

“…Indeed, these loops are much less hydrophobic than the amino-terminal fusion peptides of class I proteins (even when the three fusion domains of G are grouped together in the post-fusion conformation). That these loops are indeed an essential part of the membrane interacting motif is consistent with previous mutagenesis work performed on rhabdoviruses [59,60] (Table 1) and has since been confirmed for both VSV G [61] (Table 1) and Herpesviruses gB [62,63].…”

Molecular Architecture of the Bipartite Fusion Loops of Vesicular Stomatitis Virus Glycoprotein G, a Class III Viral Fusion Protein

“…In contrast, the fusion loops of class II fusion proteins and herpesvirus gB are buried at an oligomeric interface. Interestingly, mutation of the hydrophobic residues of the gB fusion loop did not cause any generalized problem in protein trafficking to the cell surface (48,49). In contrast, we found that even conservative changes in the hydrophobic residues of the VSV G fusion loops caused significant problems in protein transport to the cell surface.…”

“…Both VSV (Rhadboviridae) and HSV-1 (Herpesviridae) belong to an extended family of viruses with equivalent fusion domains. In the case of the herpesviruses, the Epstein-Barr virus and human herpesvirus 6, 7, and 8 gB homologs all appear to have more exposed tryptophan residues (49), with in one case (Epstein-Barr virus) three tryptophan residues present (50). Animal rhabdoviruses in general have a single tryptophan residue in their fusion loops.…”

Cleavage and Secretion of Epstein-Barr Virus Glycoprotein 42 Promote Membrane Fusion with B Lymphocytes

“…After 6 h, the medium was changed, and at 24 h posttransfection, cells were detached with Versene and transferred to Corning 96-well plates, using 3 wells per sample. After incubation for 24 h at 37°C, cells were washed with PBS-ABC (PBS with 0.89 g of CaCl 2 and 0. Immunoprecipitation.…”