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Kinetics of proton transport into influenza virions by the viral M2 channel.

Ivanovic T, Rozendaal R, Floyd DL, Popovic M, van Oijen AM, Harrison SC - PLoS ONE (2012)

Bottom Line: Fusion-pore formation usually follows internal acidification but does not require it.The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second.The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m) of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

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Fluorescein-based single-virion assay for the study of internal virion acidification.A) Loading of virions with pH-sensitive (fluorescein, green) or pH-stable (SRB, red) fluorophores for detection of M2-mediated internal virion acidification (left) or HA-mediated viral membrane fusion (right; 15). Target synthetic bilayers incorporate sialic-acid receptors and a fluorescein pH indicator. B) Selected frames from a representative time-lapse movie monitoring fluorescein-loaded virions at pH 4.5 (t−7, 7 sec before the pH drop; t0, the time of the pH drop; t50, 50 seconds after the pH drop). A subsection (∼1/6) of the imaged ∼70×140 µm area is shown. The green circle marks a single virion analyzed further in C. C) A representative fluorescence intensity-versus-time single-virion trace (green line), the model used to fit the data (inset), best-fit line (black) and fit-derived Gaussian (blue). Fit-derived parameters: tc, Gaussian mean or the time at which the transition is half complete; w, Gaussian width, or 21/2 x the standard deviation of the underlying distribution. Onset time: O = to−(tc−w). Dissipation time: D = 2w, centered at tc.
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pone-0031566-g001: Fluorescein-based single-virion assay for the study of internal virion acidification.A) Loading of virions with pH-sensitive (fluorescein, green) or pH-stable (SRB, red) fluorophores for detection of M2-mediated internal virion acidification (left) or HA-mediated viral membrane fusion (right; 15). Target synthetic bilayers incorporate sialic-acid receptors and a fluorescein pH indicator. B) Selected frames from a representative time-lapse movie monitoring fluorescein-loaded virions at pH 4.5 (t−7, 7 sec before the pH drop; t0, the time of the pH drop; t50, 50 seconds after the pH drop). A subsection (∼1/6) of the imaged ∼70×140 µm area is shown. The green circle marks a single virion analyzed further in C. C) A representative fluorescence intensity-versus-time single-virion trace (green line), the model used to fit the data (inset), best-fit line (black) and fit-derived Gaussian (blue). Fit-derived parameters: tc, Gaussian mean or the time at which the transition is half complete; w, Gaussian width, or 21/2 x the standard deviation of the underlying distribution. Onset time: O = to−(tc−w). Dissipation time: D = 2w, centered at tc.

Mentions: We mounted target membranes incorporating influenza virus receptor and external fluorescein pH probe on dextran-functionalized glass cover slips over a TIRF objective [15]. We attached dye-loaded virus particles to these membranes and reduced the pH of the exterior to 4.5 immediately after the onset of imaging. Figure 1B shows selected frames from a time-lapse movie monitoring fluorescein-loaded virions. Before the external pH drop we see both the labeled virions (bright spots) and the external pH probe (diffuse background fluorescence) (Figure 1B, left). At the point we define as the reaction start time (to), more than 90% of the external-pH-probe fluorescence has dissipated (Figure 1B, middle), and dissipation of fluorescence from virions follows (Figure 1B, right).


Kinetics of proton transport into influenza virions by the viral M2 channel.

Ivanovic T, Rozendaal R, Floyd DL, Popovic M, van Oijen AM, Harrison SC - PLoS ONE (2012)

Fluorescein-based single-virion assay for the study of internal virion acidification.A) Loading of virions with pH-sensitive (fluorescein, green) or pH-stable (SRB, red) fluorophores for detection of M2-mediated internal virion acidification (left) or HA-mediated viral membrane fusion (right; 15). Target synthetic bilayers incorporate sialic-acid receptors and a fluorescein pH indicator. B) Selected frames from a representative time-lapse movie monitoring fluorescein-loaded virions at pH 4.5 (t−7, 7 sec before the pH drop; t0, the time of the pH drop; t50, 50 seconds after the pH drop). A subsection (∼1/6) of the imaged ∼70×140 µm area is shown. The green circle marks a single virion analyzed further in C. C) A representative fluorescence intensity-versus-time single-virion trace (green line), the model used to fit the data (inset), best-fit line (black) and fit-derived Gaussian (blue). Fit-derived parameters: tc, Gaussian mean or the time at which the transition is half complete; w, Gaussian width, or 21/2 x the standard deviation of the underlying distribution. Onset time: O = to−(tc−w). Dissipation time: D = 2w, centered at tc.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295812&req=5

pone-0031566-g001: Fluorescein-based single-virion assay for the study of internal virion acidification.A) Loading of virions with pH-sensitive (fluorescein, green) or pH-stable (SRB, red) fluorophores for detection of M2-mediated internal virion acidification (left) or HA-mediated viral membrane fusion (right; 15). Target synthetic bilayers incorporate sialic-acid receptors and a fluorescein pH indicator. B) Selected frames from a representative time-lapse movie monitoring fluorescein-loaded virions at pH 4.5 (t−7, 7 sec before the pH drop; t0, the time of the pH drop; t50, 50 seconds after the pH drop). A subsection (∼1/6) of the imaged ∼70×140 µm area is shown. The green circle marks a single virion analyzed further in C. C) A representative fluorescence intensity-versus-time single-virion trace (green line), the model used to fit the data (inset), best-fit line (black) and fit-derived Gaussian (blue). Fit-derived parameters: tc, Gaussian mean or the time at which the transition is half complete; w, Gaussian width, or 21/2 x the standard deviation of the underlying distribution. Onset time: O = to−(tc−w). Dissipation time: D = 2w, centered at tc.
Mentions: We mounted target membranes incorporating influenza virus receptor and external fluorescein pH probe on dextran-functionalized glass cover slips over a TIRF objective [15]. We attached dye-loaded virus particles to these membranes and reduced the pH of the exterior to 4.5 immediately after the onset of imaging. Figure 1B shows selected frames from a time-lapse movie monitoring fluorescein-loaded virions. Before the external pH drop we see both the labeled virions (bright spots) and the external pH probe (diffuse background fluorescence) (Figure 1B, left). At the point we define as the reaction start time (to), more than 90% of the external-pH-probe fluorescence has dissipated (Figure 1B, middle), and dissipation of fluorescence from virions follows (Figure 1B, right).

Bottom Line: Fusion-pore formation usually follows internal acidification but does not require it.The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second.The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m) of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

Show MeSH
Related in: MedlinePlus