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.     

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.     

Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein

Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH) libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.     

Analysis of the posttranslational modifications of the influenza virus M2 protein.

The sites of posttranslational modifications of the influenza A virus M2 protein were examined, and the effect of these modifications on the M2 protein ion channel activity was analyzed. Cysteine residues 17 and 19 in the M2 protein ectodomain form disulfide bonds. The cytoplasmic tail is posttranslationally modified by palmitoylation, and mutagenic studies support the view that cysteine residue 50 is the site for fatty acylation. In addition, the cytoplasmic tail of the M2 protein was found to be posttranslationally modified by the addition of phosphate to specific serine residues. Site-directed mutagenesis of serine residues in the M2 protein cytoplasmic tail, combined with phosphoamino acid analysis, indicated that serine residue 64 is the predominant site for phosphorylation but that serine residues 82, 89, and 93 were also phosphorylated but to much lesser extents. Disulfide-bond formation, palmitoylation, and phosphorylation occurred on M2 protein expressed in mammalian cells infected with influenza virus, in mammalian cells in which the M2 protein was expressed from DNA expression vectors, and when the M2 protein was expressed in oocytes of Xenopus laevis. The membrane currents of oocytes of Xenopus laevis expressing wild-type and site-specifically altered forms of the M2 protein, to ablate posttranslational modifications, indicated that none of the posttranslational modifications significantly affected the ion channel activity of the M2 protein in oocytes. Therefore, these data do not indicate a functional role for posttranslational modifications of the M2 protein in its ion channel activity.     

Monoclonal antibody, but not synthetic peptide, targeting the ectodomain of influenza B virus M2 proton channel has antiviral activity

The proton channels of influenza A virus (A/M2) and influenza B virus (BM2) are essential for viral replication. Previously we have shown that monoclonal antibodies targeting the ectodomain of the A/M2 proton channel have antiviral activity in vitro. In this study, we generated both monoclonal antibody and phage displayed peptide against the eight amino acids comprising the ectodomain of the BM2 proton channel and investigated their antiviral activities in vitro. A cytopathic assay showed that the monoclonal antibody potently protected MDCK cells from homologous, but not heterologous, virus infections. A plaque forming assay showed that viral replication was not completely neutralized, but greatly inhibited, by the monoclonal antibody. In contrast, no antiviral activity was observed for the synthetic native or engineered peptides. These results indicate that antibody targeting the M2 proton channel is a promising therapeutic candidate for treating influenza virus infections, and that antibody structure is important for antiviral activity.     

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.     

Permeation and activation of the M2 ion channel of influenza A virus

The M(2) 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, V(rev). We have confirmed the high proton selectivity of the channel (1.5-2.0 x 10(6)) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V(rev) with proton equilibrium potential; 2) measurement of pH(in) and V(rev) while Na(+)(out) was replaced; 3) measurements with limiting external buffer concentration to limit proton currents specifically; and 4) comparison of measurements of M(2)-expressing cells with cells exposed to a protonophore. Increased currents at low pH(out) are due to true activation and not merely increased [H(+)](out) because increased pH(out) 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 NH(4)(+), probably by a different mechanism from H(+).     

eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM)

The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H⁺ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

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.     

UCP2-dependent proton leak in isolated mammalian mitochondria.

A role for uncoupling protein (UCP) homologues in mediating the proton leak in mammalian mitochondria is controversial. We subjected insulinoma (INS-1) cells to adenoviral expression of UCP2 or UCP1 and assessed the proton leak as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential. Cells were infected with different amounts of rat UCP2, and, in other experiments, with either UCP2 or UCP1. The relative molar expression of these subtypes was quantified through comparison with histidine-tagged UCP1 or UCP2 proteins engineered by expression in Escherichia coli. Adenoviral infection with UCP2, compared with beta-galactosidase, resulted in a dose-dependent shift in kinetics indicating increased H(+) flux at any given membrane potential. UCP1 also enhanced H(+) flux, but, on a relative molar basis, the overexpression of the endogenous protein, UCP2, was more potent than UCP1. These results were not due to nonspecific overexpression of mitochondrial protein since UCP1 activity was inhibited by GDP and because overexpression of another membrane carrier protein, the oxoglutarate malate carrier had no effect. UCP2-mediated H(+) conduction was not GDP sensitive. These data suggest that the UCP homologue, UCP2, mediates the proton leak in mitochondria of a mammalian cell wherein UCP2 is the native subtype.     

Multiscale simulation of transmembrane proteins

Multiscale simulation is employed to examine changes in atomistic-level protein structure due to long wavelength membrane undulations and plane stress fields. An ensemble of atomistic-level simulations of a model of a transmembrane influenza A virus M2 proton channel in a dimyristoylphosphatidylcholine (DMPC) bilayer is coupled to a corresponding mesoscopic model of a DMPC bilayer in an explicit mesoscopic solvent. Structural variations in the key proton gating His37 residues of the M2 channel are examined. Small, but distinct variations in the structure of the His37 residues are observed in both the open and closed states of the channel as a result of the coupling to mesoscopic-level membrane motions.     

Proton Channel Activity of Influenza A Virus Matrix Protein 2 Contributes to Autophagy Arrest

Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.     

Influenza B virus BM2 protein is a crucial component for incorporation of viral ribonucleoprotein complex into virions during virus assembly

Influenza B virus contains four integral membrane proteins in its envelope. Of these, BM2 has recently been found to have ion channel activity and is considered to be a functional counterpart to influenza A virus M2, but the role of BM2 in the life cycle of influenza B virus remains unclear. In an effort to explore its function, a number of BM2 mutant viruses were generated by using a reverse genetics technique. The BM2DeltaATG mutant virus synthesized BM2 at markedly lower levels but exhibited similar growth to wild-type (wt) virus. In contrast, the BM2 knockout virus, which did not produce BM2, did not grow substantially but was able to grow normally when BM2 was supplemented in trans by host cells expressing BM2. These results indicate that BM2 is a required component for the production of infectious viruses. In the one-step growth cycle, the BM2 knockout virus produced progeny viruses lacking viral ribonucleoprotein complex (vRNP). The inhibited incorporation of vRNP was regained by trans-supplementation of BM2. An immunofluorescence study of virus-infected cells revealed that distribution of hemagglutinin, nucleoprotein, and matrix (M1) protein of the BM2 knockout virus at the apical membrane did not differ from that of wt virus, whereas the sucrose gradient flotation assay revealed that the membrane association of M1 was greatly affected in the absence of BM2, resulting in a decrease of vRNP in membrane fractions. These results strongly suggest that BM2 functions to capture the M1-vRNP complex at the virion budding site during virus assembly.     

The ion channels coded by viruses

Pathogenicity and virulence are multifactorial traits, depending on interaction of viruses with susceptible cells and organisms. The ion channels coded by viruses, viroporins, represent only one factor taking part in the cascade of interactions between virus and cell, leading to the entry of virus, replication and to profound changes in membrane permeability. The M2 protein from influenza A virus forms proton-selective, pH-regulated channel involved in regulating vesicular pH, a function important for the correct maturation of HA glycoprotein. The NB glycoprotein of influenza B viruses is an integral membrane protein with an ion channel activity. The CM2 protein of influenza C virus is an integral membrane glycoprotein structurally analogous to influenza A virus M2 and influenza B virus NB proteins. The picornavirus 3A protein is involved in cell lysis and shows homology with other lytic proteins. Vpu is an oligomeric integral membrane protein encoded by HIV-1, which forms ion channels. The togavirus 6K protein shows structural similarities with other viroporins.     

Inhibition of influenza M2-induced cell death alleviates its negative contribution to vaccination efficiency

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed.     

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.     

Design of an Efficient Inhibitor for the Influenza A Virus M2 Ion Channel

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H+ proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.     

Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells

Bax is a Bcl-2 family protein with proapoptotic activity, which has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. In control HeLa cells, Bax is present in the cytosol and weakly associated with mitochondria as a monomer with an apparent molecular mass of 20,000 Da. After treatment of the HeLa cells with the apoptosis inducer staurosporine or UV irradiation, Bax associated with mitochondria is present as two large molecular weight oligomers/complexes of 96,000 and 260,000 Da, which are integrated into the mitochondrial membrane. Bcl-2 prevents Bax oligomerization and insertion into the mitochondrial membrane. The outer mitochondrial membrane protein voltage-dependent anion channel and the inner mitochondrial membrane protein adenosine nucleotide translocator do not coelute with the large molecular weight Bax oligomers/complexes on gel filtration. Bax oligomerization appears to be required for its proapoptotic activity, and the Bax oligomer/complex might constitute the structural entirety of the cytochrome c-conducting channel in the outer mitochondrial membrane.     

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.     

Ion channels as antivirus targets

Ion channels are membrane proteins that are found in a number of viruses and which are of crucial physiological importance in the viral life cycle.They have one common feature in that their action mode involves a change of electrochemical or proton gradient across the bilayer lipid membrane which modulates viral or cellular activity.We will discuss a group of viral channel proteins that belong to the viroproin family,and which participate in a number of viral functions including promoting the release of viral particles from cells.Blocking these channel-forming proteins may be"lethal",which can be a suitable and potential therapeutic strategy.In this review we discuss seven ion channels of viruses which can lead serious infections in human beings: M2 of influenza A,NB and BM2 of influenza B,CM2 of influenza C,Vpu of HIV-1,p7 of HCV and 2B of picornaviruses.