Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence

Clague, Michael J.; Schoch, Christian; Zech, Loren A.; Blumenthal, Robert

doi:10.1021/bi00457a028

Cited by 94 publications

(93 citation statements)

References 19 publications

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“…Therefore the results suggest that activation of fusion is rapid and occurs within this time frame for the majority of viruses at both pH 5.0 and 5.1. Such a time frame is compatible with the stopped-flow spectroscopic measurements of vesicular stomatitis (22) and influenza (23) …”

Section: Discussionsupporting

confidence: 61%

“…If either the actual virus fusion or the R18 diffusion rate, or both, were heterogeneous our current video techniques would fail to measure very rapid objects but would select for a slow population. Recent spectrophotometric stopped-flow measurements of vesicular stomatitis (22) and influenza (23) virus fusion to erythrocyte indicate a faster kinetic pattern. Therefore in those studies, presumably, the majority of viruses would consist of rapidly (<1 s) redistributing components.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

Lowy

Sarkar

Chen

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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We have used intensified video fluorescence microscopy and digital image processing to observe and quantitate influenza vrus (A/PR8/34/HlNl) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) were seen to undergo fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quantitative intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calculate the fraction of R18 dye molecules transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a tl, of 46 s, longer than expected for a free-diffusion model. The R18 loss was approximately twice as fast at pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was observed after the pH changes, suggesting that activation processes are faster than the time resolution, 1-5 s, of the current method.Considerable insight has been gained recently by studying the fusion of viruses with cell plasma membranes by spectrofluorometric methods (1-6). Virus particles are labeled with the lipid analogue octadecylrhodamine B (R18), which intercalates in the viral coat at concentrations that cause selfquenching ofthe fluorochrome. When viruses are attached to a target cell, lowering the pH below a critical level results in a rapid increase in the fluorescence intensity. This signal change is due to a decrease in dye surface concentration, resulting from transfer of the lipid probe from the virus coat to the erythrocyte surface during the fusion process (1-6).Advances in knowledge of the mechanism of virus-cell fusion have followed the development of methods which allowed increased resolution, in either time or space, of this process. Recently success has been reported in observing virus-cell interactions by using video-microscopy techniques (7,8). Quantitative fluorescence video microscopy techniques have the advantage of combining morphological and spectroscopic methods, which can result in a further enhancement of detecting and measuring the spatial and temporal characteristics of cell biological processes (9, 10). We report here the successful use of this method to both observe and quantitate fluorescence intensity changes associated with the fusion of single R18-labeled virus particles with cell membranes. Although the influenza virus particle size (0.1 ,am) is below the detection limit of the light microscope the presence of the R18 fluorescent probe molecules produces a signal which is within the range of intensified video camera systems. The ability to continuously monitor these changes on this microscopic scale has permitted a more detailed analysis of the kinetics of transfer of a virus coat lipid analogue during the fusion process than previously possible.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 1...

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Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

Lowy

Sarkar

Chen

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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“…These changes in the conformation of G protein are thought to associate with the fusion event (2,11,12), but the molecular mechanisms underlying VSV-induced cell fusion remain uncertain (11,(13)(14)(15).…”

Section: Discussionmentioning

confidence: 99%

VESICULAR STOMATITIS VIRUS-MEDIATED CELL FUSION SUBSEQUENT TO VIRUS ADSORPTION AT DIFFRENT pH VALUES

Nagata

Kurata

Ueno

et al. 1991

JJMSB

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SUMMARY:Vesicular stomatitis virus (VSV)-mediated cell fusion from without can be induced by transient exposure to low pH, subsequent to adsorption of VSV at neutral pH. To study the mechanism of VSV-induced cell fusion, we examined the effect of pH condition at virus adsorption on acid-inducible VSVmediated cell fusion. Although the binding of VSV to BHK-21 cells was most efficient under acidic condition (pH 5.7-6.3), extensive cell fusion was not observed under this condition. A temporary exposure to low pH after binding at neutral pH also decreased fusion activity. However, return to neutral pH for 2 min just after the acid binding restored the fusion activity. These results indicate the requirement of neutral pH condition for VSV-mediated cell fusion prior to the acid stimulation which induces conformational change of the virus glycoprotein into a fusogenic form.

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“…This is the main difference between rhabdoviruses and other viruses fusing at low pH, for which low pH-induced fusion inactivation is irreversible [29]. G can assume at least three different conformational states having different biochemical and biophysical characteristics [26,30]: the native, prefusion state detected at the viral surface above pH 7; the activated hydrophobic state, which interacts with the target membrane as a first step of the fusion [31]; and the post-fusion conformation, which is antigenically distinct from the native and activated states [32]. There is a pH-dependent equilibrium between the different states of G that is shifted toward the post-fusion state at low pH [32].…”

Section: Rhabdovirus Glycoprotein Gmentioning

confidence: 99%

“…The activation energy of the fusion process has been estimated to be in the range of 40 kcal/mol [26,70,71], most of which is required by enlargement of the initial fusion pore. For class I and class II fusion proteins, it has been proposed that the energy released during the irreversible fusogenic transition is used to achieve the energetically expensive membrane-fusion reaction [ 50,72,73], and experimental data suggest that in the case of HIV a single env glycoprotein trimer is sufficient for fusion [74].…”

Section: Cooperativity Between a Large Number Of Glycoproteinsmentioning

confidence: 99%

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

Roche

Albertini

Lepault

et al. 2008

Cell. Mol. Life Sci.

127

138

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Abstract. Glycoprotein G of the vesicular stomatitis virus (VSV) is involved in receptor recognition at the host cell surface and then, after endocytosis of the virion, triggers membrane fusion via a low pHinduced structural rearrangement. G is an atypical fusion protein, as there is a pH-dependent equilibrium between its pre-and post-fusion conformations. The atomic structures of these two conformations reveal that it is homologous to glycoprotein gB of herpesviruses and that it combines features of the previously characterized class I and class II fusion proteins.Comparison of the structures of G pre-and postfusion states shows a dramatic reorganization of the molecule that is reminiscent of that of paramyxovirus fusion protein F. It also allows identification of conserved key residues that constitute pH-sensitive molecular switches. Besides the similarities with other viral fusion machineries, the fusion properties and structures of G also reveal some striking particularities that invite us to reconsider a few dogmas concerning fusion proteins.

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Gating kinetics of pH-activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow measurements by dequenching of octadecylrhodamine fluorescence

Cited by 94 publications

References 19 publications

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

VESICULAR STOMATITIS VIRUS-MEDIATED CELL FUSION SUBSEQUENT TO VIRUS ADSORPTION AT DIFFRENT pH VALUES

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

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