Analysis of the Pore Structure of the Influenza A Virus M 2 Ion Channel by the Substituted-Cysteine Accessibility Method

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Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.Influenza A and B viruses are enveloped viruses with a genome consisting of eight segments of negative-strand RNA. RNA segment 7 of both influenza A and B viruses encodes the M2 integral membrane protein (A/M2 and BM2, respectively) that functions as a pH-sensitive proton channel and is essential for virus replication (1-5). A/M2 and BM2 proteins both consist of a small N-terminal ectodomain (24 and 7 residues, respectively), a single transmembrane domain, 19 amino acids long, and a cytoplasmic tail (54 and 83 residues, respectively) (5). Although A/M2 and BM2 proteins have similar structural and functional properties, the only homology between their amino acid sequences is found in the HXXXW motif of the membrane-spanning region. This motif plays a critical role in the ion channel activity (4, 6 -8). The A/M2 channel activity is inhibited by the antiviral drug amantadine (9, 10) but unfortunately, amantadine does not inhibit the BM2 channel activity (5, 11).The oligomeric state of the A/M2 channel had been investigated by several approaches. First, A/M2 was shown to form homo-oligomers, most likely homotetramers, by cross-linking and sedimentation experiments (12, 13). Site-specific mutagenesis studies demonstrated that the oligomeric structure of A/M2 channels is stabilized by disulfide bonds (14). Finally, by functional analysis of mixed oligomers of known composition, the active oligomeric form of A/M2 protein was shown to be a tetramer (15). Most recently the atomic stru...

Section: Discussionsupporting

confidence: 70%

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Balannik

Lamb

Pinto

2008

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“…Recent crystallographic (13) and NMR (14) studies of the structure of the A/M2 protein have suggested similar mechanisms for proton conduction and activation but quite different mechanisms for inhibition by amantadine. An essential foundation for both of these studies was prior biochemical information about the A/M2 channel, particularly its pore-lining residues (15)(16)(17) and its active oligomeric state (18). Although the active oligomeric state of the BM2 channel is known to be a tetramer (19), the pore-lining residues of the channel have not yet been identified.…”

Section: Sermentioning

confidence: 99%

“…12 , Ser 16 , or Trp 23 . Based on these experimental data, a BM2 transmembrane domain model is proposed.…”

Section: Sermentioning

confidence: 99%

Identification of the Pore-lining Residues of the BM2 Ion Channel Protein of Influenza B Virus

Soto

Ohigashi

et al. 2008

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The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleoprotein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. . Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.

“…Therefore, we performed cysteinescanning mutagenesis for the residues that form the cytoplasmic-proximal ␣-helical turns of the TM domain (42,62). We used the presence of outward currents into alkaline solutions as an indication that the gate was open.…”

Section: Fig 2 Outward Current Of M 2 -W41f Mutant Protein Is Amantmentioning

confidence: 99%

The Gate of the Influenza Virus M2 Proton Channel Is Formed by a Single Tryptophan Residue

Tang¹,

Zaitseva²,

Lamb

et al. 2002

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230

319

The influenza virus M 2 proton-selective ion channel is known to be essential for acidifying the interior of virions during virus uncoating in the lumen of endosomes. The M 2 protein is a homotetramer that contains four 19-residue transmembrane (TM) domains. These TM domains are multifunctional, because they contain the channel pore and also anchor the protein in membranes. The M 2 protein is gated by pH, and thus we have measured pH-gated currents, the accessibility of the pore to Cu 2؉ , and the effect of a protein-modifying reagent for a series of TM domain mutant M 2 proteins. The results indicate that gating of the M 2 ion channel is governed by a single side chain at residue 41 of the TM domain and that this property is mediated by an indole moiety. Unlike many ion channels where the gate is formed by a whole segment of a protein, our data suggest a model of striking simplicity for the M 2 ion channel protein, with the side chain of Trp 41 blocking the pore of the M 2 channel when pH out is high and with this side chain leaving the pore when pH out is low. Thus, the Trp 41 side chain acts as the gate that opens and closes the pore.The prediction that the influenza A virus M 2 protein has a proton-selective ion channel activity (Refs. 1 and 2 and reviewed in Ref.3) arose from a coupling of various observations on the life cycle of influenza virus. The M 2 protein is an integral membrane protein that is expressed at the plasma membrane of influenza virus-infected cells and is incorporated in small amounts into budding virions (4, 5). Studies on the mechanism of action of the anti-viral drug, amantadine (1-aminoadamantine hydrochloride), indicated that viral escape mutants resistant to the drug mapped to the transmembrane (TM) 1 domain of the M 2 protein (6) and that amantadine affected two steps in the life cycle, virus uncoating and virus maturation. The effect of amantadine on inhibition of uncoating is general to all strains of influenza A virus (7, 8) (reviewed in Refs. 3, 9 -11). When a virion has entered the cell by receptor-mediated endocytosis and the virus particle is in the acidic environment of the endosomal lumen, the M 2 ion channel is activated and conducts protons across the viral membrane. The lowered internal virion pH is thought to weaken protein-protein interactions between the viral matrix protein (M1) and the ribonucleoprotein (RNP) core (7,(12)(13)(14)(15)). In the presence of amantadine, influenza virus uncoating is incomplete, because the M1 protein is not released from the RNPs and the RNPs fail to enter the nucleus. Normally, influenza virus RNPs are transcribed and replicated in the nucleus (reviewed in Ref. 10). For some influenza virus subtypes, amantadine inhibits a "late" step in virus replication. The M 2 ion channel activity is activated during transport of the M 2 protein through the exocytic pathway; this ion channel activity raises the lumenal pH of the trans Golgi network (TGN), equilibrating pH with that of the cytoplasm (1, 17-22). Thus, the intralumenal pH of the TGN i...