Membrane Fusion Induced by Vesicular Stomatitis Virus Depends on Histidine Protonation

Abstract

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. VSV-induced membrane fusion occurs at a very narrow pH range, between 6.2 and 5.8, suggesting that His protonation is required for this process. To investigate the role of His in VSV fusion, we chemically modified these residues using diethylpyrocarbonate (DEPC). We found that DEPC treatment inhibited membrane fusion mediated by VSV in a concentration-dependent manner and that the complete inhibition of fusion was fully reversed by incubation of modified virus with hydroxylamine. Fluorescence measurements showed that VSV modification with DEPC abolished pHinduced conformational changes in G protein, suggesting that His protonation drives G protein interaction with the target membrane at acidic pH. Mass spectrometry analysis of tryptic fragments of modified G protein allowed the identification of the putative active His residues. Using synthetic peptides, we showed that the modification of His-148 and His-149 by DEPC, as well as the substitution of these residues by Ala, completely inhibited peptide-induced fusion, suggesting the direct participation of these His in VSV fusion.Membrane fusion is an essential step in the entry of enveloped viruses into their host cells (1-3). Virus-induced fusion is always mediated by viral surface glycoprotein and may occur through two different general mechanisms: (i) surface fusion between viral envelope and host cell plasma membrane after virus interaction with its cellular receptor, and (ii) fusion of endosomal membrane with viral envelope after virus particle internalization by receptor-mediated endocytosis. In the latter case, fusion is triggered by conformational changes in viral glycoproteins induced by the decrease in the pH of the endosomal medium.Vesicular stomatitis virus (VSV) 1 is a member of Rhabdoviridae family, genus Vesiculovirus. Rhabdoviruses contain helically wound ribonucleocapisid surrounded by a lipid bilayer through which spikes project. These spikes are formed by trimers of a single type of glycoprotein, named G protein. VSV enters into the cell by endocytosis followed by low pH-induced membrane fusion in the endosome (4, 5), which is catalyzed by VSV G protein (6). A common feature of viral fusion proteins is that they bear a highly conserved hydrophobic fusion domain, which is most often located at the N terminus of the polypeptide chain (7). However, VSV G protein does not contain an apolar amino acid sequence similar to the fusion peptides found in other viruses, suggesting alternative mechanisms involved in VSV-induced membrane fusion.We have shown recently (8) that VSV-induced fusion depends on a dramatic structure reorganization of G protein, which occurs within a very narrow pH range, close to 6.0. In addition, ...

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

“…Low pH-induced protonation of these residues leads to a cluster of positive charges that might trigger the movement of the fusion domain toward the target membrane. This is consistent with the observed inhibition of VSV fusion by diethylpyrocarbonate, a reagent known to modify specifically histidine residues [67]. Conversely, in the post-fusion state [54] a large number of acidic amino acids are brought close together in the central six-helix bundle.…”

Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo

“…VSV-G induced membrane fusion occurs, however, at considerably higher pH, 5.8-6.2. 24 Indeed, large syncytia formation was observed in infected insect cells with VSV-G baculovirus. The pH of the insect cell medium remained Truncated VSV-G protein improves baculovirus transduction efficiency MU Kaikkonen et al unaltered and was 6.2 after three days of infection.…”

Shuttle of lentiviral vectors via transplanted cells in vivo

“…VSV-induced membrane fusion occurs at a very narrow pH range and can be avoided by different pH or other factors. [18][19][20] It is conceivable that the culture medium used during the transwell experiments allows release of already cellbound but not fused lentiviral particles. Whether this is a unique property of VSV-G pseudotyped viral particles or can also be observed when using other viral protein envelopes remains to be determined.…”