Lawrence H. Pinto

Influenza virus M2 protein has ion channel activity

The influenza virus M2 protein was expressed in Xenopus laevis oocytes and shown to have an associated ion channel activity selective for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of oocyte membranes. Mutations in the M2 membrane-spanning domain that confer viral resistance to amantadine produced currents that were resistant to the drug. Analysis of the currents of these altered M2 proteins suggests that the channel pore is formed by the transmembrane domain of the M2 protein. The wild-type M2 channel was found to be regulated by pH. The wild-type M2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection.

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

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.

Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus

The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2+ and 3+ with a pKa near 6. A 1.65 Å resolution X-ray structure of the transmembrane protein (residues 25–46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

Permeation and Activation of the M2 Ion Channel of Influenza A Virus

The M2 ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, Vrev. We have confirmed the high proton selectivity of the channel (1.5–2.0 × 106) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of Vrev with proton equilibrium potential; 2) measurement of pHin and Vrev while Na+ out was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M2-expressing cells with cells exposed to a protonophore. Increased currents at low pHout are due to true activation and not merely increased [H+]out because increased pHout stops the outward current of acidified cells. Although the proton conductance is the biologically relevant conductance in an influenza virus-infected cell, experiments employing methods 1–3 show that the channel is also capable of conducting NH4 +, probably by a different mechanism from H+.

Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel

Influenza A and B viruses contain proton-selective ion channels, A/M2 and BM2, respectively, and the A/M2 channel activity is inhibited by the drugs amantadine and its methyl derivative rimantadine. The structure of the pore-transmembrane domain has been determined by both x-ray crystallography [Stouffer et al. (2008) Nature 451:596–599] and by NMR methods [Schnell and Chou (2008) Nature 451:591–595]. Whereas the crystal structure indicates a single amantadine molecule in the pore of the channel, the NMR data show four rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40–45 with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24–36 into BM2, yielded a pH-activated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.

Influenza Virus M2 Ion Channel Protein Is Necessary for Filamentous Virion Formation

ABSTRACT Influenza A virus buds from cells as spherical (∼100-nm diameter) and filamentous (∼100 nm × 2 to 20 μm) virions. Previous work has determined that the matrix protein (M1) confers the ability of the virus to form filaments; however, additional work has suggested that the influenza virus M2 integral membrane protein also plays a role in viral filament formation. In examining the role of the M2 protein in filament formation, we observed that the cytoplasmic tail of M2 contains several sites that are essential for filament formation. Additionally, whereas M2 is a nonraft protein, expression of other viral proteins in the context of influenza virus infection leads to the colocalization of M2 with sites of virus budding and lipid raft domains. We found that an amphipathic helix located within the M2 cytoplasmic tail is able to bind cholesterol, and we speculate that M2 cholesterol binding is essential for both filament formation and the stability of existing viral filaments.

Identification of the functional core of the influenza A virus A/M2 proton-selective ion channel

The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21–61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22–46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pKa values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.

Influenza B Virus BM2 Protein Has Ion Channel Activity that Conducts Protons across Membranes

Successful uncoating of the influenza B virus in endosomes is predicted to require acidification of the interior of the virus particle. We report that a virion component, the BM2 integral membrane protein, when expressed in Xenopus oocytes or in mammalian cells, causes acidification of the cells and possesses ion channel activity consistent with proton conduction. Furthermore, coexpression of BM2 with hemagglutinin (HA) glycoprotein prevents HA from adopting its low-pH-induced conformation during transport to the cell surface, and overexpression of BM2 causes a delay in intracellular transport in the exocytic pathway and causes morphological changes in the Golgi. These data are consistent with BM2 equilibrating the pH gradient between the Golgi and the cytoplasm. The transmembrane domain of BM2 protein and the influenza A virus A/M2 ion channel protein both contain the motif HXXXW, and, for both proteins, the His and Trp residues are important for channel function.

Transient calcium current of retinal bipolar cells of the mouse.

1. Isolated bipolar cells were obtained by enzymic (papain) dissociation of the adult mouse retina. The membrane voltage was clamped and the membrane currents were measured by the whole‐cell version of the patch‐clamp technique. Isolated bipolar cells and horizontal cells of the goldfish retina were also studied for comparison. 2. Hyperpolarization from the holding voltage, Vh, of ‐46 mV evoked a slowly activating, Cs+‐sensitive, inward current (probably an h‐current), and depolarization evoked a TEA‐ and Cs+‐sensitive outward current (probably a combination of K+ currents). 3. Depolarization from a more negative Vh (e.g. ‐96 mV) evoked a transient inward current that had maximal amplitude between ‐40 and ‐20 mV. This current was identified as a Ca2+ current (ICa): its amplitude was increased with elevated [Ca2+]o and was decreased with reduced [Ca2+]o, and it was blocked by 4 mM‐Co2+, but not by 5 microM‐TTX. 4. Both the perikaryon and the axon terminal generated ICa with similar properties. 5. The plot of Ca2+ conductance (gCa) against membrane voltage (activation curve) was sigmoidal: in 10 mM [Ca2+]o, gCa increased for membrane voltages more positive than ‐65 mV, was half‐maximal at about ‐25 mV, and reached saturation at about +30 mV. The plot of inactivation of gCa against membrane voltage was also sigmoidal: with 1 s conditioning depolarization in 10 mM [Ca2+]o, gCa decreased for membrane voltages more positive than ‐80 mV, was half‐maximal at about ‐50 mV, and was fully suppressed for voltages greater than ‐30 mV. 6. ICa in the mouse bipolar cells was insensitive to 50 microM‐Cd2+, 10 microM‐nifedipine and 10 microM‐Bay K 8644. In contrast, the calcium currents of bipolar and horizontal cells of the goldfish retina were markedly suppressed by 50 microM‐Cd2+ and 10 microM‐nifedipine, and were augmented several fold by 10 microM‐Bay K 8644. The calcium currents of goldfish bipolar and horizontal cells were sustained, and were activated in a more positive range of potentials than the ICa of mouse bipolar cells. 7. The voltage range at which the ICa of mouse bipolar cells is activated includes the presumed range of membrane potentials spanned during light‐evoked responses; thus, this current may participate in synaptic transmission. The transient character of ICa may also help to shape transient responses of ganglion cells.

Structure and inhibition of the drug-resistant S31N mutant of the M2 ion channel of influenza A virus

The influenza A virus M2 proton channel (A/M2) is the target of the antiviral drugs amantadine and rimantadine, whose use has been discontinued due to widespread drug resistance. Among the handful of drug-resistant mutants, S31N is found in more than 95% of the currently circulating viruses and shows greatly decreased inhibition by amantadine. The discovery of inhibitors of S31N has been hampered by the limited size, polarity, and dynamic nature of its amantadine-binding site. Nevertheless, we have discovered small-molecule drugs that inhibit S31N with potencies greater than amantadine’s potency against WT M2. Drug binding locks the protein into a well-defined conformation, and the NMR structure of the complex shows the drug bound in the homotetrameric channel, threaded between the side chains of Asn31. Unrestrained molecular dynamics simulations predicted the same binding site. This S31N inhibitor, like other potent M2 inhibitors, contains a charged ammonium group. The ammonium binds as a hydrate to one of three sites aligned along the central cavity that appear to be hotspots for inhibition. These sites might stabilize hydronium-like species formed as protons diffuse through the outer channel to the proton-shuttling residue His37 near the cytoplasmic end of the channel.