Vesicular stomatitis virus (VSV) infection depends on the fusion of viral and cellular membranes, which is mediated by virus spike glycoprotein G at the acidic environment of the endosomal compartment. VSV G protein does not contain a hydrophobic amino acid sequence similar to the fusion peptides found among other viral glycoproteins, suggesting that membrane recognition occurs through an alternative mechanism. Here we studied the interaction between VSV G protein and liposomes of different phospholipid composition by force spectroscopy, isothermal titration calorimetry (ITC), and fluorescence spectroscopy. Force spectroscopy experiments revealed the requirement for negatively charged phospholipids for VSV binding to membranes, suggesting that this interaction is electrostatic in nature. In addition, ITC experiments showed that VSV binding to liposomes is an enthalpically driven process. Fluorescence data also showed the lack of VSV interaction with the vesicles as well as inhibition of VSV-induced membrane fusion at high ionic strength. Intrinsic fluorescence measurements showed that the extent of G protein conformational changes depends on the presence of phosphatidylserine (PS) on the target membrane. Although the increase in PS content did not change the binding profile, the rate of the fusion reaction was remarkably increased when the PS content was increased from 25 to 75%. On the basis of these data, we suggest that G protein binding to the target membrane essentially depends on electrostatic interactions, probably between positive charges on the protein surface and negatively charged phospholipids in the cellular membrane. In addition, the fusion is exothermic, indicating no entropic constraints to this process.

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, ...

We describe a stable and sensitive HIV evaluation system, which discriminates HIV-specific membrane fusion and early transcription events and is suitable for high-throughput inhibitor screening. A human lymphocytic line, constitutively producing infectious HIV-1, serves as Env-positive donor. A second indicator cell line carries a silent HIV-1 LTR lacZ reporter plasmid. A bicellular cocultivation setup allows titration and standardization of "fusion-induced gene stimulation (FIGS)" events. With few manipulations aspects of fusion and/or LTR induction can be distinguished and simultaneously assayed. Anti-Env-V3 antibodies prevent fusion and subsequent lacZ induction, and a Tat-specific inhibitor blocks only lacZ induction in a dose dependent manner without affecting membrane fusion. The LTR reporter is readily activated by Tat from HIV-1, HIV-2, or SIV and it responds to exogenous Tat protein. The reporter system is sensitive enough to detect single infection events on pre-seeded layers of indicator cells, which renders it potentially useful for direct virus quantification in patients' material. Moreover, our system allows to control and normalize DNA transfection efficiencies of HIV-derived plasmids. This aspect is particularly valuable for studies of RT- and protease-inhibitors and resistances, where p24 or supernatant reverse transcriptase, otherwise standard virus readouts, can be directly affected by inhibitors or mutations.

Vaccine discovery stands out as one of the public health interventions that has achieved the greatest impact in world's health. Vaccination is the most effective means of disease prevention, especially for viral infections. Starting with the use of smallpox vaccine by Jenner in the late 1700s, the technology for vaccine development has seen numerous advances. Currently, vaccines available for human viral illness are based on live attenuated (e.g. measles, mumps, and rubella), inactivated (e.g. hepatitis A) and recombinant (e.g. hepatitis B) viruses. Among these, inactivated vaccines are known for their easy production and safety. The present article reviews the literature and patents related to the mechanisms used for viral inactivation, mainly chemical and physical procedures, including the novel strategies that are currently being explored and that have been recently patent protected.

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins. Correspondence