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, ...

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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. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164].