Kinetics of pH-dependent fusion between influenza virus and liposomes

Stegmann, Toon; Hoekstra, Dick; Scherphof, Gerrit L.; Wilschut, Jan

doi:10.1021/bi00334a006

Cited by 168 publications

(128 citation statements)

References 39 publications

(82 reference statements)

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“…Stegmann et al (1986Stegmann et al ( , 1987) also found 100~ fusion activity of the X-47 strain towards erythrocyte ghosts and CL or PS liposomes, but only 20 to 45 ~ of the virions fused with PC/PE liposomes with or without disialoganglioside GDla (Stegmann et al, 1985(Stegmann et al, , 1989Nir et al, 1988). Sendal virus also showed complete fusion activity towards erythrocyte ghosts (Hoekstra & Klappe, 1986;Nir et al, 1986b) and cells , but had incomplete fusion activity towards several liposomes, including PS and phosphatidylglycerol (Amselem et al, 1986;Nir et al, 1986c).…”

Section: Discussionmentioning

confidence: 98%

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

Düzgüneş

Lima²,

Stamatatos³

et al. 1992

Journal of General Virology

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The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma ($49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4-9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5-4, different from that of the virus preincubated at low pH without target membranes.

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Section: Discussionmentioning

confidence: 98%

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

Düzgüneş

Lima²,

Stamatatos³

et al. 1992

Journal of General Virology

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“…5). Reported values for macroscopic fluorimetrically measured R18 dequenching half-times are also heterogeneous, with some time constants for influenza and other virus types being similar (1,3,17,18), while faster values in the range of 1-10 s have been observed and reported (2). From rates of respiratory syncytial virus budding and readsorption observed by using a differential interference contrast video-microscopy method (7), it was proposed that the fusion rate for an influenza-sized virus would be less than 1 s. In a preliminary description of video microscopy (19,20) monitoring fusion of R18-labeled virus to artificial bilayer membranes it was reported that rapid rates were observed and that influenza virus fusion was faster than Sendai virus fusion.…”

Section: Discussionmentioning

confidence: 99%

“…Considerable insight has been gained recently by studying the fusion of viruses with cell plasma membranes by spectrofluorometric methods (1)(2)(3)(4)(5)(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.…”

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confidence: 99%

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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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“…ployed the fluorescence resonance energy transfer (FRET)-based virus-liposome fusion assay (18) adapted to a 384-well plate reader format. In this assay, the FRET pair consisting of (N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine (NBD-PE; Molecular Probes) and rhodamine-phosphatidylethanolamine (Rh-PE; Avanti) is added to liposomes in quantities that allow transfer of the NBD emission energy to Rh-PE in the liposome.…”

mentioning

confidence: 99%

Cysteines Flanking the Internal Fusion Peptide Are Required for the Avian Sarcoma/Leukosis Virus Glycoprotein To Mediate the Lipid Mixing Stage of Fusion with High Efficiency

Delos¹,

Brecher

Chen³

et al. 2008

J Virol

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We previously showed that the cysteines flanking the internal fusion peptide of the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA) are important for infectivity and cell-cell fusion. Here we define the stage of fusion at which the cysteines are required. The flanking cysteines are dispensable for receptor-triggered membrane association but are required for the lipid mixing step of fusion, which, interestingly, displays a high pH onset and a biphasic profile. Second-site mutations that partially restore infection partially restore lipid mixing. These findings indicate that the cysteines flanking the internal fusion peptide of EnvA (and perhaps by analogy Ebola virus glycoprotein) are important for the foldback stage of the conformational changes that lead to membrane merger.The avian sarcoma/leukosis virus (ASLV) Env protein is unusual among class I fusion proteins in that it contains an internal fusion peptide, a characteristic it shares with filovirus (Ebola virus and Marburg virus) glycoproteins. These fusion peptides are flanked by two Cys residues, which, based on structural and mutagenesis evidence, likely form a disulfide bond (9, 21). The ASLV fusion peptide contains one proline, and the filovirus fusion peptides contain two prolines, at their centers. Previous work has shown that these Pro residues are important for fusion (4,8). The fusion peptide of EnvA is operationally defined as residues 22 to 37 of the transmembrane (TM) subunit. We previously provided evidence that the Pro in the middle of this peptide (P29) requires a ␤-turn structure (4) at some stage of fusion. We subsequently showed that the two Cys residues (C9 and C45) that define the likely disulfide-bonded loop encompassing the fusion peptide are required for infectivity and cell-cell fusion (5). In this study, we identified the specific stage of fusion that requires the Cys residues that flank the EnvA fusion peptide.To begin our studies, we asked if the defect was in the first step of fusion: target membrane binding. Accordingly, we determined the ability of murine leukemia virus pseudotyped virions (10) bearing either wild-type EnvA or an EnvA harboring double Cys-to-Ser mutations at both positions 9 and 45 of the TM subunit (referred to below as EnvAC9,45S) to bind to target membranes in a receptor-dependent manner (6, 7, 15). As seen in Fig. 1, wild-type murine leukemia virus pseudovirions bound liposomes and floated to the top of the gradient when either the quail (lane 1) or the chicken (lane 5) form of the soluble Tva receptor (sTva) was used (7). In the absence of sTva, all of the pseudovirions remained at the bottom of the gradient (Fig. 1, lane 12). Similar receptor-dependent membrane association was observed for EnvAC9,45S (Fig. 1). These data show that the Cys residues (C9 and C45) that define the loop encompassing the fusion peptide are not required for receptor-triggered binding of EnvA-bearing virus particles to target membranes.We then asked whether the cysteines are required for EnvA to reach the lipid mixing ...

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Kinetics of pH-dependent fusion between influenza virus and liposomes

Cited by 168 publications

References 39 publications

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

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

Cysteines Flanking the Internal Fusion Peptide Are Required for the Avian Sarcoma/Leukosis Virus Glycoprotein To Mediate the Lipid Mixing Stage of Fusion with High Efficiency

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