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Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel.

ABSTRACT: The A/M2 protein of influenza A virus forms a tetrameric proton-selective pH-gated ion channel. The H(37)xxxW(41) motif located in the channel pore is responsible for its gating and proton selectivity. Channel activation most likely involves protonation of the H37 residues, while the conductive state of the channel is characterized by two or three charged His residues in a tetrad. A/M2 channel activity is inhibited by the antiviral drug amantadine. Although a large number of functional amantadine-resistant mutants of A/M2 have been observed in vitro, only a few are observed in highly transmissible viruses in the presence or absence of amantadine. We therefore examined 49 point mutants of the pore-lining residues, representing both natural and nonnatural variants. Their ion selectivity, amantadine sensitivity, specific activity, and pH-dependent conductance were measured in Xenopus oocytes. These measurements showed how variations in the sequence lead to variations in the proton conduction. The results are consistent with a multistep mechanism that allows the protein to fine-tune its pH-rate profile over a wide range of proton concentrations, hypothesized to arise from different protonation states of the H37 tetrad. Mutations that give native-like conductance at low pH as well as minimal leakage current at pH 7.0 were surprisingly rare. Moreover, the results are consistent with a location of the amantadine-binding site inside the channel pore. These findings have helped to define the set of functionally fit mutants that should be targeted when considering the design of novel drugs that inhibit amantadine-resistant strains of influenza A virus.

SUBMITTER: Balannik V

PROVIDER: S-EPMC4924815 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel.

Balannik Victoria V Carnevale Vincenzo V Fiorin Giacomo G Levine Benjamin G BG Lamb Robert A RA Klein Michael L ML Degrado William F WF Pinto Lawrence H LH

Biochemistry 20100201 4

The A/M2 protein of influenza A virus forms a tetrameric proton-selective pH-gated ion channel. The H(37)xxxW(41) motif located in the channel pore is responsible for its gating and proton selectivity. Channel activation most likely involves protonation of the H37 residues, while the conductive state of the channel is characterized by two or three charged His residues in a tetrad. A/M2 channel activity is inhibited by the antiviral drug amantadine. Although a large number of functional amantadin ...[more]

PMID: 20028125

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Project description:BACKGROUND:A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a "mixing vessel" for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. RESULTS:To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2-cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2-cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2-cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (Vmid) values in Boltzmann modeling for oocytes injected with DM2-cMyc RNA or a buffer were -152 and -200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by Vmid for CM2 and DM2 were -141 and -146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were -21 and -22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower Vmid values while showing no change in reversal potential. CONCLUSION:The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule.

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Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel.

Publications

Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel.

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