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http://www.biophysj.org/cgi/content/full/74/1/54
ABSTRACT
The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30°C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30°C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30°C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion
RESULTS
The inactivation kinetics for the virus were obtained by incubating virions at pH 4.9 and 30°C for the time periods indicated in minutes next to each curve and then adding them to liposomes at pH 7.4. The measurements are referenced to the time the liposome-virus mixture was acidified to pH 4.9. For a 0-min incubation, lipid mixing was complete in ~6 min. Incubation for 2 min was also complete in ~6 min. There was a 30-40% decrease in the rate of lipid mixing after a 10-min incubation, and the virus was completely inactive after a 60-min incubation at low pH.
ABSTRACT
The kinetics of low-pH induced fusion of influenza virus with liposomes have been compared to changes in the morphology of influenza hemagglutinin (HA). At pH 4.9 and 30°C, the fusion of influenza A/PR/8/34 virus with ganglioside-bearing liposomes was complete within 6 min. Virus preincubated at pH 4.9 and 30°C in the absence of liposomes for 2 or 10 min retained most of its fusion activity. However, fusion activity was dramatically reduced after 30 min, and virtually abolished after a 60-min preincubation. Cryo-electron microscopy showed that the hemagglutinin spikes of virions exposed to pH 4.9 at 30°C for 10 min underwent no major morphological changes. After 30 min, however, the spike morphology changed dramatically, and further changes occurred for up to 60 min after exposure to low pH. Because the morphological changes occur at a rate corresponding to the loss of fusion activity, and because these changes are much slower than the rate at which fusion occurs, we conclude that the morphologically altered HA is inactive with respect to fusion-promoting activity. Molecular modeling studies indicate that the formation of an extended coiled coil within the HA trimer, as proposed for HA at low pH, requires a major conformational change in HA, and that the morphological changes we observe are consistent with the formation of an extended coiled coil. These results imply that the crystallographically determined low-pH form of HA does occur in the intact virus, but that this form is not a precursor of viral fusion. It is speculated that the motion to the low-pH form may be responsible for the membrane destabilization leading to fusion
RESULTS
The inactivation kinetics for the virus were obtained by incubating virions at pH 4.9 and 30°C for the time periods indicated in minutes next to each curve and then adding them to liposomes at pH 7.4. The measurements are referenced to the time the liposome-virus mixture was acidified to pH 4.9. For a 0-min incubation, lipid mixing was complete in ~6 min. Incubation for 2 min was also complete in ~6 min. There was a 30-40% decrease in the rate of lipid mixing after a 10-min incubation, and the virus was completely inactive after a 60-min incubation at low pH.
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