Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains

Abstract

The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, Dl and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants,1 H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.

Entry of Rhabdoviruses Into Animal Cells

“…61 Analysis of VSV G demonstrated that mutation of another highly conserved region, between residues 118-139, abolished fusion activity or modified the pH of fusion activation. 59,62,63 Other studies have also demonstrated that altering region 395 to 418 for VSV, 64 and 392 to 396 in RABV, 65 have an influence on glycoprotein G-mediated fusion. In addition, double mutants within region 118-139 and region 395-418 of VSV show an additive inhibition of fusion activity.…”

The Many Mechanisms of Viral Membrane Fusion Proteins

“…The idea of loop structures for internal fusion peptides is further supported by the known looped structure of the TBE E and SFV EI fusion peptides (Rey et al 1995;Allison et al 2001;Lescar et al 2001). In some cases, two or more noncontiguous sequence loops may function as a collective fusion peptide (Gaudin et al 1999a;Li et al 1993).…”

Internalization and Fusion Mechanism of Vesicular Stomatitis Virus and Related Rhabdoviruses

“…Other mutations in this region, such as F125(109)Y and D137(121)N, shifted the optimal pH of G protein mediated cell-cell fusion [48]. However, mutations at other conserved regions, for example, amino acids 181-212(165-196) and 395-418(379-402) located near the carboxyl terminus, could also affect protein fusion [49,50]. Although the aforementioned results shed light on the architecture of G protein fusion machinery, mutagenesis studies in the absence of a high-resolution protein structure may not identify the true viral fusion peptide; however, they may instead recognize amino acids or regions involved in protein conformational changes during the fusion event or those which are involved in stabilizing trimer organization.…”

“…It is hypothesized that the membrane-proximal region might interact with the fusion peptide proximal segment to stabilize the membrane-interactive end of the trimer hairpins [77]. For VSV G protein, the deletion of the last 13 amino acids in the ectodomain; that is, N449(433)-W461(445) dramatically reduced cell-cell fusion activity and viral infectivity [78] and the insertion of a three amino acid linker at positions 410(394) and 415(399) abolished membrane fusion [49]. However, the double mutation of G131(115)A and G404(388) A, which are adjacent to the fusion loop II and the membraneproximal region, was more fusogenic than the two individual mutations alone, suggesting that these two regions might act synergistically during viral fusion [79].…”