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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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Kinetics of internal virion acidification.A) Proton-concentration dependence of inverse onset (left) and dissipation times (right). Error bars are propagated from the standard error of the mean (SEM) of measured onset and dissipation times as SEM(mean time)/(mean time)2. B) Number of M2 channels per virion does not scale with virion size. Upper and lower left – representative negative-stain electron micrographs of long (L) and short (S) virion fractions. All kinetic measurements in Figures 1 to 4 used the long-virion fraction. Upper right – long-virion length histogram distribution as estimated from multiple negative stain electron micrographs (see Materials and Methods). Lower right – SDS-PAGE of two-fold serial dilutions of long (4L, 2L, 1L) and short (2S, 1S) virion fractions and immunoblotting using anti-HA1 antibody (upper panel, HA0 and HA1) or anti-M2 antibody (lower panel, M2).
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pone-0031566-g004: Kinetics of internal virion acidification.A) Proton-concentration dependence of inverse onset (left) and dissipation times (right). Error bars are propagated from the standard error of the mean (SEM) of measured onset and dissipation times as SEM(mean time)/(mean time)2. B) Number of M2 channels per virion does not scale with virion size. Upper and lower left – representative negative-stain electron micrographs of long (L) and short (S) virion fractions. All kinetic measurements in Figures 1 to 4 used the long-virion fraction. Upper right – long-virion length histogram distribution as estimated from multiple negative stain electron micrographs (see Materials and Methods). Lower right – SDS-PAGE of two-fold serial dilutions of long (4L, 2L, 1L) and short (2S, 1S) virion fractions and immunoblotting using anti-HA1 antibody (upper panel, HA0 and HA1) or anti-M2 antibody (lower panel, M2).

Mentions: To probe the mechanism of proton conductance by M2 we determined the onset time and duration of fluorescein signal dissipation over a range of proton concentrations from 6 to 100 µM (pH range 5.2 to 4). As expected for pH-activated processes, both onset and dissipation times decrease with increased proton concentration. Overall rates for both onset and dissipation (inverse mean onset and dissipation times) obey Michaelis-Menten-like kinetics; fitting the rate data toyields pKm values of 4.44+/−0.06 (R2 = 0.994) and 4.72+/−0.03 (R2 = 0.998), respectively (Figure 4A). These values correspond to the measured pKa of the fourth protonation event in the channel histidine tetrad [9], and the observed pH dependence closely mimics that measured for the purified M2 protein reconstituted in liposomes and triggered to conduct protons at pHs ranging from 5 to 3.4 [13]. By distinguishing between onset times (related to the probability of channel activation) and dissipation times (related to both the probability of channel activation and proton flux through an activated channel) and by showing essentially the same dependence of rates on proton concentration, we conclude that channel activation and proton conductance are related events, limited by the concentration of the fully protonated channel histidines.


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)

Kinetics of internal virion acidification.A) Proton-concentration dependence of inverse onset (left) and dissipation times (right). Error bars are propagated from the standard error of the mean (SEM) of measured onset and dissipation times as SEM(mean time)/(mean time)2. B) Number of M2 channels per virion does not scale with virion size. Upper and lower left – representative negative-stain electron micrographs of long (L) and short (S) virion fractions. All kinetic measurements in Figures 1 to 4 used the long-virion fraction. Upper right – long-virion length histogram distribution as estimated from multiple negative stain electron micrographs (see Materials and Methods). Lower right – SDS-PAGE of two-fold serial dilutions of long (4L, 2L, 1L) and short (2S, 1S) virion fractions and immunoblotting using anti-HA1 antibody (upper panel, HA0 and HA1) or anti-M2 antibody (lower panel, M2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0031566-g004: Kinetics of internal virion acidification.A) Proton-concentration dependence of inverse onset (left) and dissipation times (right). Error bars are propagated from the standard error of the mean (SEM) of measured onset and dissipation times as SEM(mean time)/(mean time)2. B) Number of M2 channels per virion does not scale with virion size. Upper and lower left – representative negative-stain electron micrographs of long (L) and short (S) virion fractions. All kinetic measurements in Figures 1 to 4 used the long-virion fraction. Upper right – long-virion length histogram distribution as estimated from multiple negative stain electron micrographs (see Materials and Methods). Lower right – SDS-PAGE of two-fold serial dilutions of long (4L, 2L, 1L) and short (2S, 1S) virion fractions and immunoblotting using anti-HA1 antibody (upper panel, HA0 and HA1) or anti-M2 antibody (lower panel, M2).
Mentions: To probe the mechanism of proton conductance by M2 we determined the onset time and duration of fluorescein signal dissipation over a range of proton concentrations from 6 to 100 µM (pH range 5.2 to 4). As expected for pH-activated processes, both onset and dissipation times decrease with increased proton concentration. Overall rates for both onset and dissipation (inverse mean onset and dissipation times) obey Michaelis-Menten-like kinetics; fitting the rate data toyields pKm values of 4.44+/−0.06 (R2 = 0.994) and 4.72+/−0.03 (R2 = 0.998), respectively (Figure 4A). These values correspond to the measured pKa of the fourth protonation event in the channel histidine tetrad [9], and the observed pH dependence closely mimics that measured for the purified M2 protein reconstituted in liposomes and triggered to conduct protons at pHs ranging from 5 to 3.4 [13]. By distinguishing between onset times (related to the probability of channel activation) and dissipation times (related to both the probability of channel activation and proton flux through an activated channel) and by showing essentially the same dependence of rates on proton concentration, we conclude that channel activation and proton conductance are related events, limited by the concentration of the fully protonated channel histidines.

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