Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block.

ABSTRACT Ion channel proteins are common constituents of cells and have even been identified in some viruses. For example, the M2 protein of influenza A virus has proton ion channel activity that is thought to play an important role in viral replication. Because direct support for this function is lacking, we attempted to generate viruses with defective M2 ion channel activity. Unexpectedly, mutants with apparent loss of M2 ion channel activity by an in vitro assay replicated as efficiently as the wild-type virus in cell culture. We also generated a chimeric mutant containing an M2 protein whose transmembrane domain was replaced with that from the hemagglutinin glycoprotein. This virus replicated reasonably well in cell culture but showed no growth in mice. Finally, a mutant lacking both the transmembrane and cytoplasmic domains of M2 protein grew poorly in cell culture and showed no growth in mice. Thus, influenza A virus can undergo multiple cycles of replication without the M2 transmembrane domain responsible for ion channel activity, although this activity promotes efficient viral replication.

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Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus

Molecules ◽

10.3390/molecules25122903 ◽

2020 ◽

Vol 25 (12) ◽

pp. 2903

Author(s):

Maggie C. Duncan ◽

Pascal Amoa Onguéné ◽

Ibuki Kihara ◽

Derrick N. Nebangwa ◽

Maya E. Naidu ◽

...

Keyword(s):

Ion Channel ◽

Influenza A Virus ◽

Influenza A ◽

Influenza Viruses ◽

Drug Resistant ◽

M2 Protein ◽

Virtual Screen ◽

Chebulagic Acid ◽

Virus Strains

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.

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Generation of recombinant influenza A virus without M2 ion-channel protein by introduction of a point mutation at the 5′ end of the viral intron

Journal of General Virology ◽

10.1099/vir.0.80727-0 ◽

2005 ◽

Vol 86 (5) ◽

pp. 1447-1454 ◽

Cited By ~ 22

Author(s):

T. K. W. Cheung ◽

Y. Guan ◽

S. S. F. Ng ◽

H. Chen ◽

C. H. K. Wong ◽

...

Keyword(s):

Cell Culture ◽

Ion Channel ◽

Influenza A Virus ◽

Influenza A ◽

Gene Mutations ◽

M2 Protein ◽

M Gene ◽

Recombinant Viruses ◽

Channel Protein ◽

Splicing Site

The aim of this study was to inhibit influenza virus M2 protein expression by mutating the splicing signal of the M gene. Mutations were introduced into the GU dinucleotide sequence at the 5′-proximal splicing site of the M gene (corresponding to nt 52–53 of M cRNA). Transfected cells expressing mutated M viral ribonucleoproteins failed to generate M2 mRNA. Interestingly, recombinant viruses with mutations at the dinucleotide sequence were viable, albeit attenuated, in cell culture. These recombinants failed to express M2 mRNA and M2 protein. These observations demonstrated that the GU invariant dinucleotide sequence at the 5′-proximal splicing site of M gene is essential for M2 mRNA synthesis. These results also indicated that the M2 ion-channel protein is critical, but not essential, for virus replication in cell culture. This approach may provide a new way of producing attenuated influenza A virus.

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Influenza A Virus with Defective M2 Ion Channel Activity as a Live Vaccine

Virology ◽

10.1006/viro.2002.1508 ◽

2002 ◽

Vol 299 (2) ◽

pp. 266-270 ◽

Cited By ~ 21

Author(s):

Tokiko Watanabe ◽

Shinji Watanabe ◽

Hiroshi Kida ◽

Yoshihiro Kawaoka

Keyword(s):

Ion Channel ◽

Influenza A Virus ◽

Influenza A ◽

Live Vaccine ◽

Channel Activity

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Design and Pharmacological Characterization of Inhibitors of Amantadine-Resistant Mutants of the M2 Ion Channel of Influenza A Virus

Biochemistry ◽

10.1021/bi9014488 ◽

2009 ◽

Vol 48 (50) ◽

pp. 11872-11882 ◽

Cited By ~ 75

Author(s):

Victoria Balannik ◽

Jun Wang ◽

Yuki Ohigashi ◽

Xianghong Jing ◽

Emma Magavern ◽

...

Keyword(s):

Ion Channel ◽

Influenza A Virus ◽

Influenza A ◽

Pharmacological Characterization ◽

Resistant Mutants

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Influenza A Virus M2 Protein: Proton Selectivity of the Ion Channel, Cytotoxicity, and a Hypothesis on Peripheral Raft Association and Virus Budding

Protein Reviews - Viral Membrane Proteins: Structure, Function, and Drug Design ◽

10.1007/0-387-28146-0_9 ◽

2007 ◽

pp. 113-130 ◽

Cited By ~ 2

Author(s):

Cornelia Schroeder ◽

Tse-I Lin

Keyword(s):

Ion Channel ◽

Influenza A Virus ◽

Influenza A ◽

M2 Protein ◽

Virus Budding

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Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

Journal of Virology ◽

10.1128/jvi.03253-14 ◽

2014 ◽

Vol 89 (4) ◽

pp. 1975-1985 ◽

Cited By ~ 24

Author(s):

Esmeralda Alvarado-Facundo ◽

Yamei Gao ◽

Rosa María Ribas-Aparicio ◽

Alicia Jiménez-Alberto ◽

Carol D. Weiss ◽

...

Keyword(s):

Influenza Virus ◽

Cell Surface ◽

Avian Influenza ◽

Influenza A Virus ◽

Conformational Changes ◽

Influenza A ◽

Low Ph ◽

Proton Channel ◽

Channel Activity ◽

M2 Protein

ABSTRACTThe influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability.IMPORTANCEInfluenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable.

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Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

Journal of Virology ◽

10.1128/jvi.76.3.1391-1399.2002 ◽

2002 ◽

Vol 76 (3) ◽

pp. 1391-1399 ◽

Cited By ~ 187

Author(s):

Makoto Takeda ◽

Andrew Pekosz ◽

Kevin Shuck ◽

Lawrence H. Pinto ◽

Robert A. Lamb

Keyword(s):

Influenza Virus ◽

Ion Channel ◽

Influenza A Virus ◽

Influenza A ◽

Antiviral Drug ◽

Influenza Viruses ◽

Channel Activity ◽

Efficient Replication ◽

Tm Domain ◽

Golgi Network

ABSTRACT The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656–5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del29–31) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del29–31 virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del29–31 virus. We found that the M2-del29–31 virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del29–31 virus in 4 days after cocultivation of a 100:1 ratio of M2-del29–31 virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle.

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