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Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
Published on: June 28, 2013
A dimer is the minimal proton-conducting unit of the influenza a virus M2 channel
Kenichi Kawano1, Yoshiaki Yano1, Katsumi Matsuzaki1
1Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachicho, Sakyo-ku, Kyoto 606-8501, Japan.
Abstract:
When influenza A virus infects host cells, its integral matrix protein M2 forms a proton-selective channel in the viral envelope. Although X-ray crystallography and NMR studies using fragment peptides have suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein in the living cells have not yet been assessed directly. In the present study, we utilized recently developed stoichiometric analytical methods based on fluorescence resonance energy transfer using coiled-coil labeling technique and spectral imaging, and we examined the relationship between the oligomeric states of full-length M2 and its channel activities in living cells. In contrast to previous models, M2 formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH. The antiviral drug amantadine hydrochloride inhibited both tetramerization and channel activity. The removal of cholesterol resulted in a significant decrease in the activity of the dimer. These results indicate that the minimum functional unit of the M2 protein is a dimer, which forms a complex with cholesterol for its function.
Insights
Influenza M2 protein forms proton channels. New research shows M2 functions as a dimer at neutral pH, converting to tetramers at acidic pH, and requires cholesterol for activity.
Area of Science:
- Virology
- Structural Biology
- Biophysics
Background:
- Influenza A virus M2 protein forms proton channels essential for viral replication.
- Previous studies suggested M2 forms stable tetramers, but this was based on fragment peptides and not assessed in living cells.
Purpose of the Study:
- To determine the oligomeric state and channel activity of full-length M2 protein in living cells.
- To investigate the influence of pH and cholesterol on M2 protein function.
Main Methods:
- Utilized stoichiometric analytical methods based on fluorescence resonance energy transfer (FRET).
- Employed coiled-coil labeling technique and spectral imaging in living cells.
- Assessed the impact of amantadine hydrochloride and cholesterol depletion on M2 channel activity.
Main Results:
- Full-length M2 protein forms proton-conducting dimers at neutral pH.
- M2 dimers convert to tetramers at acidic pH.
- Amantadine hydrochloride inhibits M2 tetramerization and channel activity.
- Cholesterol depletion significantly reduces M2 dimer activity.
Conclusions:
- The minimum functional unit of influenza A virus M2 protein is a dimer.
- M2 dimer function is dependent on cholesterol.
- pH-dependent oligomerization regulates M2 channel activity.
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