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Influenza A virus M2 ion channel protein: a structure-function analysis
L J Holsinger1, D Nichani, L H Pinto
1Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
Abstract:
A structure-function analysis of the influenza A virus M2 ion channel protein was performed. The M2 protein of human influenza virus A/Udorn/72 and mutants containing changes on one face of the putative alpha helix of the M2 transmembrane (TM) domain, several of which lead to amantadine resistance when found in virus, were expressed in oocytes of Xenopus laevis. The membrane currents of oocytes expressing mutant M2 ion channels were measured at both normal and low pH, and the amantadine-resistant mutant containing the change of alanine at residue 30 to threonine was found to have a significantly attenuated low pH activation response. The specific activity of the channel current of the amantadine-resistant mutants was investigated by measuring the membrane current of individual oocytes followed by quantification of the amount of M2 protein expressed in these single oocytes by immunoblotting analysis. The data indicate that changing residues on this face of the putative alpha helix of the M2 TM domain alters properties of the M2 ion channel. Some of the M2 proteins containing changes in the TM domain were found to be modified by addition of an N-linked carbohydrate chain at an asparagine residue that is membrane proximal and which is not modified in the wild-type M2 protein. These N-linked carbohydrate chains were further modified by addition of polylactosaminoglycan. A glycosylated M2 mutant protein (M2 + V, A30T) exhibited an ion channel activity with a voltage-activated, time-dependent kinetic component. Prevention of carbohydrate addition did not affect the altered channel activity. The ability of the M2 protein to tolerate deletions in the TM domain was examined by expressing three mutants (del29-31, del28-31, and del27-31) containing deletions of three, four, and five residues in the TM domain. No ion channel activity was detected from expression of M2 del29-31 and del27-31, whereas expression of M2 del28-31 resulted in an ion channel activity that was activated by hyperpolarization (and not low pH) and was resistant to amantadine block. Examination of the oligomeric form of M2 del28-31 indicated that the oligomer is different from wild-type M2, and the data were consistent with M2 del28-31 forming a pentamer.
Insights
Mutations in the influenza A virus M2 protein
Area of Science:
- Virology
- Ion Channel Biophysics
- Protein Structure-Function Analysis
Background:
- The M2 protein of influenza A virus forms an ion channel essential for viral replication.
- Amantadine resistance in influenza A virus is often linked to mutations in the M2 protein's transmembrane domain.
- Understanding M2 protein structure-function is crucial for developing antiviral strategies.
Purpose of the Study:
- To investigate the structure-function relationship of the influenza A virus M2 ion channel.
- To analyze the impact of mutations in the M2 transmembrane domain on channel activity and amantadine resistance.
- To explore the role of glycosylation and deletions in M2 protein function.
Main Methods:
- Expression of wild-type and mutant M2 proteins in Xenopus laevis oocytes.
- Measurement of membrane currents at varying pH levels.
- Immunoblotting analysis to quantify M2 protein expression.
- Examination of M2 protein oligomerization and glycosylation.
Main Results:
- An amantadine-resistant mutant (A30T) showed reduced low pH activation.
- Specific mutations in the M2 transmembrane domain altered ion channel properties.
- N-linked glycosylation of M2 protein did not affect altered channel activity.
- M2 deletion mutants exhibited varied channel activities, with M2 del28-31 showing hyperpolarization activation and amantadine resistance, forming a pentamer.
Conclusions:
- Residue changes in the M2 transmembrane domain significantly impact ion channel function and amantadine sensitivity.
- Glycosylation of the M2 protein does not rescue altered channel activity caused by mutations.
- Deletions within the M2 transmembrane domain can lead to novel channel activities and altered oligomeric states, offering insights into M2 channel assembly and function.