The active oligomeric state of the minimalistic influenza virus M
            <sub>2</sub>
            ion channel is a tetramer

Sakaguchi, Takemasa; Tu, Qiang; Pinto, Lawrence H.; Lamb, Robert A.

doi:10.1073/pnas.94.10.5000

Cited by 155 publications

(136 citation statements)

References 26 publications

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“…Conductance levels of the 6,000-Da protein encoded by ␣-viruses cover a range from Ϸ50 pS to Ϸ800 pS (30). Other viral ion channels such as the tetrameric proton channel M2 from influenza A virus (31,32) exhibit conductance levels in the femtosiemen range (33,34). Channel-forming peptides show conductance levels as high as the ones we observed for HCV p7 when five or more transmembrane helices assemble to form a pore (26,27).…”

Section: N-7-oxanonyl-6-deoxy-dgjmentioning

confidence: 57%

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

Pavlović

Neville²,

Argaud³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

335

302

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We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.H epatitis C virus (HCV) is the major cause of chronic hepatitis with a significant risk of end-stage liver cirrhosis and hepatocellular carcinoma (1). HCV belongs to the family Flaviviridae, which consists of three genera: flaviviruses, pestiviruses, and hepaciviruses. In the absence of both a suitable small animal model and a reliable in vitro infectivity assay for HCV, potential antiviral drugs initially have been tested by using a related pestivirus, bovine viral diarrhea virus (BVDV) (2). BVDV in vitro infectivity assays were used to demonstrate that long-alkyl-chain iminosugar derivatives containing either the glucose analogue deoxynojirimycin (DNJ) or the galactose analogue deoxygalactonojirimycin (DGJ) are potent antiviral inhibitors (3).DNJ derivatives inhibit endoplasmic reticulum (ER) ␣-glucosidases I and II (4, 5), and this inhibition leads to the misfolding of many host-and virus-encoded glycoproteins, including the envelope glycoproteins of BVDV (6) and HCV (7). Previous experiments have shown that the antiviral effect of the longalkyl-chain derivative N-nonyl-DNJ (NN-DNJ) is more pronounced than that of the short-alkyl-chain derivative N-butyl-DNJ (NB-DNJ), although the latter achieves a more effective ER ␣-glucosidase inhibition in cellulo. In addition, long-alkylchain DGJ derivatives that are not recognized by and do not inhibit ER ␣-glucosidases also show potent antiviral activity (3). Therefore, ER ␣-glucosidase inhibition does not correlate directly with the observed antiviral effect and is ruled out as the sole antiviral mechanism.The additional mechanism of action apparently is associated with the length of the alkyl side chain, because the short-chain N-butyl-DGJ (NB-DGJ) shows no antiviral activity, whereas the long-alkyl-chain derivative NN-DGJ is a potent inhibitor (3).The predominant antiviral mechanism is proposed to be mediated directly or indirectly by an effect of the long-alkyl side chains on the membrane and͞or membrane proteins, because treatment with long-alkyl-chain iminosugars affects the dimerization of viral membrane glycoproteins and alters the membrane glycoprotein composition of secreted BVDV virions but does not influence either viral RNA replication or protein synthesis (3).We decided to investigate the small membrane-spanning protein p7 as a potential target of long-alkyl-chain iminosugar derivatives, because flaviviruses such as dengue virus and Japanese encephalitis virus (8), which do not contain p7, are not inhibited by long-alkyl-chain DGJ derivatives, whereas pestiviruses are (3). Pesti-and hepaciviruses both contain the p7 protein.Most functional data about p7 are derived from the pestivirus p7, a 70-aa protein very similar to HCV p7. Functional data hav...

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Section: N-7-oxanonyl-6-deoxy-dgjmentioning

confidence: 57%

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

Pavlović

Neville²,

Argaud³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

335

302

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“…This conductance is in agreement with the values found for the TM domain of the peptide in our study. M2 is formed by a pair of covalently linked dimers (57,58). M2 expressed in Spodoptera frugiperda (Sf9) cells and reconstituted in lipid membranes exhibits conductances >25 pS (59).…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Peptide NB of Influenza B: A Simulation, Structure, and Conductance Study

et al. 2000

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The putative transmembrane segment of the ion channel forming peptide NB from influenza B was synthesized by standard solid-phase peptide synthesis. Insertion into the planar lipid bilayer revealed ion channel activity with conductance levels of 20, 61, 107, and 142 pS in a 0.5 M KCl buffer solution. In addition, levels at -100 mV show conductances of 251 and 413 pS. A linear current-voltage relation reveals a voltage-independent channel formation. In methanol and in vesicles the peptide appears to adopt an R-helical-like structure. Computational models of R-helix bundles using N ) 4, 5, and 6 NB peptides per bundle revealed water-filled pores after 1 ns of MD simulation in a solvated lipid bilayer. Calculated conductance values [using HOLE (Smart et al. (1997) Biophys. J. 72, 1109-1126] of ca. 20, 60, and 90 pS, respectively, suggested that the multiple conductance levels seen experimentally must correspond to different degrees of oligomerization of the peptide to form channels.In the genome of influenza B, RNA segment 6 encodes two integral membrane proteins, NB and NA. NB is a protein of ca. 100 amino acids. It has ca. 18 extramembraneous residues on its N-terminal end, a 22-residue membrane spanning segment, and a ca. 60 residue long C-terminal end (1, 2). NB is expressed at the surface of infected cells (3)(4)(5). In the virion the N-terminal site is located at the external surface. Experiments with gold immunolabeled freezefractured virions indicate that the C-terminus is located at the inside of the virion (4). By analogy with the M2 protein of influenza A, it was suggested (reviewed in ref 6) that NB might be a channel-forming protein.Conductance measurements with purified NB expressed in Escherichia coli and reconstituted in lipid bilayers reveal a conductance of 45 pS. However, when NB is incorporated in liposomes and then added to planar lipid bilayers, the conductance ranged from ca. 10 pS at very low NB concentration in the liposomes to several hundred picosiemens for higher concentrations (7). At neutral pH the channels are more permeable for sodium ions than chloride ions (P Na /P Cl ≈ 9 at pH 6.5). In asymmetrical NaCl solutions at pH 2.5 the permeability is higher for chloride ions (P Cl / P Na ≈ 4). A recent study on NB expressed in the plasmalemma of MEL 1 cells showed that the channel peptides induce a Na + -dependent proton current and a pH-activated Cl -current (8).One way in which single integral membrane proteins can form ion channels is via oligomerization to form a bundle of transmembrane helices. For the analogous channel peptide in influenza A, M2, it is believed that a tetrameric R-helix bundle is the minimal form of the proton-permeable ion channel (9-11). For NB the number of subunits in the bundle is not known. Purification of NB on gels using nonreducing conditions revealed the formation of disulfide-linked dimers and higher oligomers (2). Molecular modeling studies on bundles of the transmembrane parts of the peptide have been performed in vacuo with four, five, and si...

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“…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

Journal of Biological Chemistry

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233

319

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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...

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The active oligomeric state of the minimalistic influenza virus M ₂ ion channel is a tetramer

Cited by 155 publications

References 26 publications

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

Transmembrane Peptide NB of Influenza B: A Simulation, Structure, and Conductance Study

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

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The active oligomeric state of the minimalistic influenza virus M 2 ion channel is a tetramer

Cited by 155 publications

References 26 publications

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives

Transmembrane Peptide NB of Influenza B: A Simulation, Structure, and Conductance Study

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

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The active oligomeric state of the minimalistic influenza virus M ₂ ion channel is a tetramer