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.     

The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging

The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.     

Ion selectivity and activation of the M2 ion channel of influenza virus.

The influenza A virus-associated M2 ion channel is generally believed to function during uncoating of virions in infected cells. On endocytosis of a virion into the lumen of endosomes, the M2 ion channel is thought to cause acidification of the virion interior. In addition, the influenza virus M2 ion channel is thought to function in the exocytic pathway by equilibrating the pH gradient between the acidic lumen of the trans-Golgi network and the neutral cytoplasm. A necessary test of the proposed roles of the influenza virus M2 ion channel in the virus life cycle is to show directly that the M2 ion channel conducts protons. We have measured the ionic selectivity and activation of three subtypes (Udorn, Weybridge, and Rostock) of the M2 ion channel in oocytes of Xenopus laevis by measurement of 1) the intracellular pH (pHin) of voltage-clamped oocytes, 2) the current-voltage relationship in solutions of various pH and ionic composition, and 3) the flux of 86Rb. We took advantage of the low pHin achieved during incubation in low pH medium to study the effects of low pHin on M2 activation. Oocytes expressing each of the three subtypes of the M2 protein a) underwent a slow acidification when incubated in medium of low pH (acidification was blocked by the M2 ion channel inhibitor, amantadine); b) had current-voltage relationships that shifted to more positive values and had greater conductance when the pHout was lowered (this relationship was modified when Na- was replaced by NH4+ or Li+); c) had an amantadine-sensitive influx of Rb+. The effects on the current-voltage relationship of reduced pHin were opposed to the increased conductance found with reduced pHout. We interpret these results to indicate that the M2 ion channel is capable of conducting H+ and that other ions may also be conducted. Moreover, the channel conductance is reduced by decreased pHin. These findings are consistent with the proposed roles of the M2 protein in the life cycle of influenza A virus.     

A型流感病毒H5N1的M2离子通道稳定细胞株的建立

A型流感病毒H5N1的M2离子通道(H5M2)基因经优化后由人工合成,适合于哺乳动物细胞中表达.通过酶切克隆于pcDNA4质粒,并在HEK293细胞中建立稳定细胞株.Western blotting和免疫荧光证实H5M2在稳定细胞中只有在四环素诱导下才能表达,并经膜片钳证实在HEK293细胞中表达的H5M2具有H 通道活性,为M2离子通道功能的研究和M2离子通道阻断剂筛选方法的建立提供了参考.     

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.     

Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport.

The influenza A/fowl plague virus/Rostock/34 hemagglutinin (HA), which is cleaved intracellularly and has a high pH threshold (pH 5.9) for undergoing its conformational change to the low-pH form, was expressed from cDNA in CV-1 and HeLa T4 cells in the absence of other influenza virus proteins. It was found, by biochemical assays, that the majority of the HA molecules were in a form indistinguishable from the low-pH form of HA. The acidotropic agent, ammonium chloride, stabilized the accumulation of HA in its native form. Coexpression of HA and the homotypic influenza virus M2 protein, which has ion channel activity, stabilized the accumulation of HA in its pH neutral (native) form, and the M2 protein ion channel blocker, amantadine, prevented the rescue of HA in its native form. These data provide direct evidence that the influenza virus M2 protein ion channel activity can affect the status of the conformational form of cleaved HA during intracellular transport.     

Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^IPK that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58^IPK by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58^IPKin vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^IPK, and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58^IPK-mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.     

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.     

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.     

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.     

Reconstitution of the influenza virus M2 ion channel in lipid bilayers

M₂, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M₂ baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25–500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25–90 pS). (3) The channels were selective for cations; t _Na = 0.75 when 150 mm NaCl bathed both sides of the membrane. (4) Amantadine reduced the probability of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs ～ Na > Li. The pH activation, amantadine block and ion selectivity of the M₂ protein ion channel in bilayers are consistent with those observed on expression of the M₂ protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M₂ protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

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.     

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.     

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.     

Viral ion channels: structure and function

Viral ion channels are short auxiliary membrane proteins with a length of ca. 100 amino acids. They are found in enveloped viruses from influenza A, influenza B and influenza C (Orthomyxoviridae), and the human immunodeficiency virus type 1 (HIV-1, Retroviridae). The channels are called M2 (influenza A), NB (influenza B), CM2 (influenza C) and Vpu (HIV-1). Recently, in Paramecium bursaria chlorella virus (PBCV-1, Phycodnaviridae), a K+ selective ion channel has been discovered. The viral channels form homo oligomers to allow an ion flux and represent miniaturised systems. Proton conductivity of M2 is established; NB, Vpu and the potassium channel from PBC-1 conduct ions; for CM2 ion conductivity is still under proof. This review summarises the current knowledge of these short viral membrane proteins. Their discovery is outlined and experimental evidence for their structure and function is discussed. Studies using computational methods are presented as well as investigations of drug-protein interactions.     

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.     

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.     

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

The influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.     

Viroporins: structure and biological functions

Viroporins are small, hydrophobic proteins that are encoded by a wide range of clinically relevant animal viruses. When these proteins oligomerize in host cell membranes, they form hydrophilic pores that disrupt a number of physiological properties of the cell. Viroporins are crucial for viral pathogenicity owing to their involvement in several diverse steps of the viral life cycle. Thus, these viral proteins, which include influenza A virus matrix protein 2 (M2), HIV-1 viral protein U (Vpu) and hepatitis C virus p7, represent ideal targets for therapeutic intervention, and several compounds that block their pore-forming activity have been identified. Here, we review recent studies in the field that have advanced our knowledge of the structure and function of this expanding family of viral proteins.