Influenza virus proton channels.

The M2 ion channel proteins of influenza A and B viruses are essential to viral replication. The two ion channels share a common motif, HXXXW, that is responsible for proton selectivity and activation. The ion channel for the influenza A virus, but not influenza B virus, is inhibited by the antiviral drug amantadine and amantadine-resistant escape mutants form in treated influenza A patients. The studies reviewed suggest that an antiviral compound directed against the conserved motif would be more useful than amantadine in inhibiting viral replication.     

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

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-del(29-31)) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del(29-31) 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-del(29-31) virus. We found that the M2-del(29-31) 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-del(29-31) virus in 4 days after cocultivation of a 100:1 ratio of M2-del(29-31) 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.     

Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity

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.     

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

The influenza A virus M2 integral membrane protein has ion channel activity which can be blocked by the antiviral drug amantadine. The M2 protein transmembrane domain is highly conserved in amino acid sequence for all the human, swine, equine, and avian strains of influenza A virus, and thus, known amino acid differences could lead to altered properties of the M2 ion channel. We have expressed in oocytes of Xenopus laevis the M2 protein of human influenza virus A/Udorn/72 and the avian virus A/chicken/Germany/34 (fowl plague virus, Rostock) and derivatives of the Rostock ion channel altered in the presumed pore region. The pH of activation of the M2 ion channels and amantadine block of the M2 ion channels were investigated. The channels were found to be activated by pH in a similar manner but differed in their apparent Kis for amantadine block.     

Flu channel drug resistance: a tale of two sites

The M2 proteins of influenza A and B virus, AM2 and BM2, respectively, are transmembrane proteins that oligomerize in the viral membrane to form proton-selective channels. Proton conductance of the M2 proteins is required for viral replication; it is believed to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. In addition to the role of M2 in proton conductance, recent mutagenesis and structural studies suggest that the cytoplasmic domains of the M2 proteins also play a role in recruiting the matrix proteins to the cell surface during virus budding. As viral ion channels of minimalist architecture, the membrane-embedded channel domain of M2 has been a model system for investigating the mechanism of proton conduction. Moreover, as a proven drug target for the treatment of influenza A infection, M2 has been the subject of intense research for developing new anti-flu therapeutics. AM2 is the target of two anti-influenza A drugs, amantadine and rimantadine, both belonging to the adamantane class of compounds. However, resistance of influenza A to adamantane is now widespread due to mutations in the channel domain of AM2. This review summarizes the structure and function of both AM2 and BM2 channels, the mechanism of drug inhibition and drug resistance of AM2, as well as the development of new M2 inhibitors as potential anti-flu drugs.     

Designing Inhibitors of M2 Proton Channel against H1N1 Swine Influenza Virus

Background

M2 proton channel of H1N1 influenza A virus is the target protein of anti-flu drugs amantadine and rimantadine. However, the two once powerful adamantane-based drugs lost their 90% bioactivity because of mutations of virus in recent twenty years. The NMR structure of the M2 channel protein determined by Schnell and Chou (Nature, 2008, 451, 591–595) may help people to solve the drug-resistant problem and develop more powerful new drugs against H1N1 influenza virus.

Methodology

Docking calculation is performed to build the complex structure between receptor M2 proton channel and ligands, including existing drugs amantadine and rimantadine, and two newly designed inhibitors. The computer-aided drug design methods are used to calculate the binding free energies, with the computational biology techniques to analyze the interactions between M2 proton channel and adamantine-based inhibitors.

Conclusions

1) The NMR structure of M2 proton channel provides a reliable structural basis for rational drug design against influenza virus. 2) The channel gating mechanism and the inhibiting mechanism of M2 proton channel, revealed by the NMR structure of M2 proton channel, provides the new ideas for channel inhibitor design. 3) The newly designed adamantane-based inhibitors based on the modeled structure of H1N1-M2 proton channel have two pharmacophore groups, which act like a “barrel hoop”, holding two adjacent helices of the H1N1-M2 tetramer through the two pharmacophore groups outside the channel. 4) The inhibitors with such binding mechanism may overcome the drug resistance problem of influenza A virus to the adamantane-based drugs.     

Computational Investigation of Drug-Resistant Mutant of M2 Proton Channel (S31N) Against Rimantadine

M2 proton channel is the target for treating the patients who ere suffering from influenza A infection, which facilitates the spread of virions. Amantadine and rimantadine are adamantadine-based drugs, which target M2 proton channel and inhibit the viral replication. Preferably, rimantadine drug is used more than amantadine because of its fewer side effects. However, S31N mutation in the M2 proton channel was highly resistant to the rimantadine drug. Therefore, in the present study, we focused to understand the drug-resistance mechanism of S31N mutation with the aid of molecular docking and dynamics approach. The docking analysis undoubtedly indicates that affinity for rimantadine with mutant-type M2 proton channel is significantly lesser than the native-type M2 proton channel. In addition, RMSD, RMSF, and principal component analysis suggested that the mutation shows increased flexibility. Furthermore, the intermolecular hydrogen bonds analysis showed that there is a complete loss of hydrogen bonds in the mutant complex. On the whole, we conclude that the intermolecular contact was maintained by D-44, a key residue for stable binding of rimantadine. These findings are certainly helpful for better understanding of drug-resistance mechanism and also helpful for designing new drugs for treating influenza infection against drug-resistance target.     

Solution NMR structure of the V27A drug resistant mutant of influenza A M2 channel

The M2 protein of influenza A virus forms a proton-selective channel that is required for viral replication. It is the target of the anti-influenza drugs, amantadine and rimantadine. Widespread drug resistant mutants, however, has greatly compromised the effectiveness of these drugs. Here, we report the solution NMR structure of the highly pathogenic, drug resistant mutant V27A. The structure reveals subtle structural differences from wildtype that maybe linked to drug resistance. The V27A mutation significantly decreases hydrophobic packing between the N-terminal ends of the transmembrane helices, which explains the looser, more dynamic tetrameric assembly. The weakened channel assembly can resist drug binding either by destabilizing the rimantadine-binding pocket at Asp44, in the case of the allosteric inhibition model, or by reducing hydrophobic contacts with amantadine in the pore, in the case of the pore-blocking model. Moreover, the V27A structure shows a substantially increased channel opening at the N-terminal end, which may explain the faster proton conduction observed for this mutant. Furthermore, due to the high quality NMR data recorded for the V27A mutant, we were able to determine the structured region connecting the channel domain to the C-terminal amphipathic helices that was not determined in the wildtype structure. The new structural data show that the amphipathic helices are packed much more closely to the channel domain and provide new insights into the proton transfer pathway.     

Proton conduction through the M2 protein of the influenza A virus; a quantitative,mechanistic analysis of experimental data

The M2 proton channel from influenza A virus forms proton-selective ion channels, which are the target of the drug amantadine. Here, existing experimental data are quantitatively examined for insights into mechanisms to account for the pH- and voltage-dependences of M2 proton conduction. The analysis shows that a model involving protonation equilibria of His37, including pH-dependent changes in the relative rates of diffusion on either side of the pore, is quantitatively able to account for recently reported electrophysiological data examining the pH- and voltage-dependences of Rostock and Weybridge strain M2 proton conduction.     

Kinetics of proton transport into influenza virions by the viral M2 channel

M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m) of ～4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.     

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.     

pH-dependent tetramerization and amantadine binding of the transmembrane helix of M2 from the influenza A virus

The M2 proton channel from the influenza A virus is a small protein with a single transmembrane helix that associates to form a tetramer in vivo. This protein forms proton-selective ion channels, which are the target of the drug amantadine. Here, we propose a mechanism for the pH-dependent association, and amantadine binding of M2, based on studies of a peptide representing the M2 transmembrane segment in dodecylphosphocholine micelles. Using analytical ultracentrifugation, we find that the sedimentation curves for the peptide depend on its concentration in the micellar phase. The data are well-described by a monomer-tetramer equilibrium, and the binding of amantadine shifts the monomer-tetramer equilibrium toward tetrameric species. Both tetramerization and the binding of amantadine lead to increases in the magnitude of the ellipticity at 223 nm in the circular dichroism spectrum of the peptide. The tetramerization and binding of amantadine are more favorable at elevated pH, with a pK(a) that is assigned to a His side chain, the only ionizable residue within the transmembrane helix. Our results, interpreted quantitatively in terms of a reversible monomer and tetramer protonation equilibrium model, suggest that amantadine competes with protons for binding to the deprotonated tetramer, thereby stabilizing the tetramer in a slightly altered conformation. This model accounts for the observed inhibition of proton flux by amantadine. Additionally, our measurements suggest that the M2 tetramer is substantially protonated at neutral pH and that both singly and doubly protonated states could be involved in M2's proton conduction at more acidic pHs.     

Inhibitors of the Influenza A Virus M2 Proton Channel Discovered Using a High-Throughput Yeast Growth Restoration Assay

The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.     

Characterization of inhibition of M2 ion channel activity by BL-1743, an inhibitor of influenza A virus.

The influenza A virus M2 integral membrane protein has ion channel activity that can be inhibited by the antiviral drug amantadine. Recently, a spirene-containing compound, BL-1743 (2-[3-azaspiro (5,5)undecanol]-2-imidazoline), that inhibits influenza virus growth was identified (S. Kurtz, G. Lao, K. M. Hahnenberger, C. Brooks, O. Gecha, K. Ingalls, K.-I. Numata, and M. Krystal, Antimicrob. Agents Chemother. 39:2204-2209, 1995). We have examined the ability of BL-1743 to inhibit the M2 ion channel when expressed in oocytes of Xenopus laevis. BL-1743 inhibition is complete as far as can be measured by electrophysiological methods and is reversible, with a reverse reaction rate constant of 4.0 x 10(-3) s(-1). In contrast, amantadine inhibition is irreversible within the time frame of the experiment. However, BL-1743 inhibition and amantadine inhibition have similar properties. The majority of isolated influenza viruses resistant to BL-1743 are also amantadine resistant. In addition, all known amino acid changes which result in amantadine resistance also confer BL-1743 resistance. However, one BL-1743-resistant virus isolated, designated M2-I35T, contained the change Ile-35-->Thr. This virus is >70-fold more resistant to BL-1743 and only 10-fold more resistant to amantadine than the wild-type virus. When the ion channel activity of M2-I35T was examined in oocytes, it was found that M2-I35T is BL-1743 resistant but is reversibly inhibited by amantadine. These findings suggest that these two drugs interact differently with the M2 protein transmembrane pore region.     

Insights from investigating the interactions of adamantane-based drugs with the M2 proton channel from the H1N1 swine virus

The M2 proton channel is one of indispensable components for the influenza A virus that plays a vital role in its life cycle and hence is an important target for drug design against the virus. In view of this, the three-dimensional structure of the H1N1-M2 channel was developed based on the primary sequence taken from a patient recently infected by the H1N1 (swine flu) virus. With an explicit water-membrane environment, molecular docking studies were performed for amantadine and rimantadine, the two commercial drugs generally used to treat influenza A infection. It was found that their binding affinity to the H1N1-M2 channel is significantly lower than that to the H5N1-M2 channel, fully consistent with the recent report that the H1N1 swine virus was resistant to the two drugs. The findings and the relevant analysis reported here might provide useful structural insights for developing effective drugs against the new swine flu virus.     

Structure of Amantadine-Bound M2 Transmembrane Peptide of Influenza A in Lipid Bilayers from Magic-Angle-Spinning Solid-State NMR: The Role of Ser31 in Amantadine Binding

The M2 proton channel of influenza A is the target of the antiviral drugs amantadine and rimantadine, whose effectiveness has been abolished by a single-site mutation of Ser31 to Asn in the transmembrane domain of the protein. Recent high-resolution structures of the M2 transmembrane domain obtained from detergent-solubilized protein in solution and crystal environments gave conflicting drug binding sites. We present magic-angle-spinning solid-state NMR results of Ser31 and a number of other residues in the M2 transmembrane peptide (M2TMP) bound to lipid bilayers. Comparison of the spectra of the membrane-bound apo and complexed M2TMP indicates that Ser31 is the site of the largest chemical shift perturbation by amantadine. The chemical shift constraints lead to a monomer structure with a small kink of the helical axis at Gly34. A tetramer model is then constructed using the helix tilt angle and several interhelical distances previously measured on unoriented bilayer samples. This tetramer model differs from the solution and crystal structures in terms of the openness of the N-terminus of the channel, the constriction at Ser31, and the side-chain conformations of Trp41, a residue important for channel gating. Moreover, the tetramer model suggests that Ser31 may interact with amantadine amine via hydrogen bonding. While the apo and drug-bound M2TMP have similar average structures, the complexed peptide has much narrower linewidths at physiological temperature, indicating drug-induced changes of the protein dynamics in the membrane. Further, at low temperature, several residues show narrower lines in the complexed peptide than the apo peptide, indicating that amantadine binding reduces the conformational heterogeneity of specific residues. The differences of the current solid-state NMR structure of the bilayer-bound M2TMP from the detergent-based M2 structures suggest that the M2 conformation is sensitive to the environment, and care must be taken when interpreting structural findings from non-bilayer samples.     

The chemical and dynamical influence of the anti-viral drug amantadine on the M2 proton channel transmembrane domain

The M(2) proton channel plays a vital role in the life cycle of the influenza A virus. His(37), the key residue in the M(2) transmembrane domain (M(2)-TMD), plays a central role in the proton conductance mechanism. The anti-influenza drug, amantadine, inhibits the channel activity through binding to the pore of the M(2) channel. The nuclear spin relaxation data and polarization inversion spin exchange at the magic angle spectra show that both the polypeptide backbone and His(37) side chain are more constrained in the presence of amantadine. Using (15)N cross polarization magic-angle spinning NMR spectroscopy, the protonation of His(37) of M(2)-TMD in lipid bilayers was monitored in the absence and presence of amantadine as a function of pH. Binding amantadine lowers the His(37) pK(a) values by approximately three orders of magnitude compared with the first pK(a) of histidine in amantadine-free M(2)-TMD. Amantadine's influence on the His(37) chemical properties suggests a novel mechanism by which amantadine may inhibit proton conductance.     

Peptide carbocyclic derivatives as inhibitors of the viroporin function of RNA-containing viruses

New carbocyclic derivatives of amino acids, peptides, and other compounds have been synthesized, and their antiviral activity toward the influenza A/H5N1 and hepatitis C viruses has been studied in vitro. It has been shown that the aminoacyl derivatives of aminoadamantane are capable of inhibiting the replication of the highly virulent strain of the avian influenza virus (H5N1), which is resistant to aminoadamantanes amantadine and rimantadine. The effect of the configuration of the carbocyclic moiety of the dipeptide H-Pro-Trp-OH on the antiviral properties toward the hepatitis C virus has been studied. The cyclohexyloxycarbonyl derivative of the H-Pro-Trp-OH dipeptide strongly inhibited the replication of HCV in vitro. Some compounds have been found to exhibit a high virucidal activity toward influenza A/H5N1 and HCV virions.     

A Drosophila model for genetic analysis of influenza viral/host interactions

Influenza viruses impose a constant threat to vertebrates susceptible to this family of viruses. We have developed a new tool to study virus-host interactions that play key roles in viral replication and to help identify novel anti-influenza drug targets. Via the UAS/Gal4 system we ectopically expressed the influenza virus M2 gene in Drosophila melanogaster and generated dose-sensitive phenotypes in the eye and wing. We have confirmed that the M2 proton channel is properly targeted to cell membranes in Drosophila tissues and functions as a proton channel by altering intracellular pH. As part of the efficacy for potential anti-influenza drug screens, we have also demonstrated that the anti-influenza drug amantadine, which targets the M2 proton channel, suppressed the UAS-M2 mutant phenotype when fed to larvae. In a candidate gene screen we identified mutations in components of the vacuolar V1V0 ATPase that modify the UAS-M2 phenotype. Importantly, in this study we demonstrate that Drosophila genetic interactions translate directly to physiological requirements of the influenza A virus for these components in mammalian cells. Overexpressing specific V1 subunits altered the replication capacity of influenza virus in cell culture and suggests that drugs targeting the enzyme complex via these subunits may be useful in anti-influenza drug therapies. Moreover, this study adds credence to the idea of using the M2 "flu fly" to identify new and previously unconsidered cellular genes as potential drug targets and to provide insight into basic mechanisms of influenza virus biology.     

The oligomeric state of the active BM2 ion channel protein of influenza B virus

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.