Functional studies reveal the similarities and differences between AM2 and BM2 proton channels from influenza viruses

AM2 and BM2 proton channels are attractive antiviral drug targets due to their essential roles during influenza virus replication. Although both AM2 and BM2 are proton-selective ion channels, they share little sequence similarity except for the HXXXW sequence, which suggests that their proton conductance properties might differ. To test this hypothesis, we applied two-electrode voltage clamp electrophysiological assays to study the specific conductance, leakage current, channel activation, and inhibition of AM2 and BM2 proton channels. It was found that BM2 channel has a higher specific conductance than AM2 channel at pH 5.5. Unlike AM2 channel, whose proton conductance is asymmetric (from viral exterior to interior), BM2 channel is capable of conducting proton in both directions. Moreover, BM2 requires a more acidic pH for channel activation than AM2, as revealed by its lower pK_a values. Finally, both AM2 and BM2 can be inhibited by Cu(II) and Cu(I). Overall, the results from this side-by-side comparison of AM2 and BM2 channels reveal the structure-function relationships of these two viroporins, and such information might be important for the designing of novel ion channels.     

Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase β1 subunit interacts with M2 proteins of influenza A and B viruses and affects the virus replication

Interplay between the host and influenza virus has a pivotal role for the outcome of infection. The matrix proteins M2/BM2 from influenza (A and B) viruses are small type III integral membrane proteins with a single transmembrane domain, a short amino-terminal ectodomain and a long carboxy-terminal cytoplasmic domain. They function as proton channels, mainly forming a membrane-spanning pore through the transmembrane domain tetramer, and are essential for virus assembly and release of the viral genetic materials in the endosomal fusion process. However, little is known about the host factors which interact with M2/BM2 proteins and the functions of the long cytoplasmic domain are currently unknown. Starting with yeast two-hybrid screening and applying a series of experiments we identified that the β1 subunit of the host Na⁺/K⁺-ATPase β1 subunit (ATP1B1) interacts with the cytoplasmic domain of both the M2 and BM2 proteins. A stable ATP1B1 knockdown MDCK cell line was established and we showed that the ATP1B1 knockdown suppressed influenza virus A/WSN/33 replication, implying that the interaction is crucial for influenza virus replication in the host cell. We propose that influenza virus M2/BM2 cytoplasmic domain has an important role in the virus-host interplay and facilitates virus replication.     

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.     

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.     

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.     

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.     

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.     

Controlling influenza virus replication by inhibiting its proton channel

Influenza A and viruses encode minimalistic proton-selective ion channels known as A/M2 and BM2, respectively. The A/M2 channel is the target of the antiviral drug, amantadine. The structural and mechanistic aspects of proton conductance of the viral ion channels are described and the review makes a case for the development of more effective antivirals.     

Elucidating Relayed Proton Transfer through a His–Trp–His Triad of a Transmembrane Proton Channel by Solid-State NMR

Proton transfer through membrane-bound ion channels is mediated by both water and polar residues of proteins, but the detailed molecular mechanism is challenging to determine. The tetrameric influenza A and B virus M2 proteins form canonical proton channels that use an HxxxW motif for proton selectivity and gating. The BM2 channel also contains a second histidine (His), H27, equidistant from the gating tryptophan, which leads to a symmetric H¹⁹xxxW²³xxxH²⁷ motif. The proton-dissociation constants (pK_a's) of H19 in BM2 were found to be much lower than the pK_a's of H37 in AM2. To determine if the lower pK_a's result from H27-facilitated proton dissociation of H19, we have now investigated a H27A mutant of BM2 using solid-state NMR. ¹⁵N NMR spectra indicate that removal of the second histidine converted the protonation and tautomeric equilibria of H19 to be similar to the H37 behavior in AM2, indicating that the peripheral H27 is indeed the origin of the low pK_a's of H19 in wild-type BM2. Measured interhelical distances between W23 sidechains indicate that the pore constriction at W23 increases with the H19 tetrad charge but is independent of the H27A mutation. These results indicate that H27 both accelerates proton dissociation from H19 to increase the inward proton conductance and causes the small reverse conductance of BM2. The proton relay between H19 and H27 is likely mediated by the intervening gating tryptophan through cation–π interactions. This relayed proton transfer may exist in other ion channels and has implications for the design of imidazole-based synthetic proton channels.     

Identification of the pore-lining residues of the BM2 ion channel protein of influenza B virus

The influenza B virus BM2 proton-selective ion channel is essential for virus uncoating, a process that occurs in the acidic environment of the endosome. The BM2 channel causes acidification of the interior of the virus particle, which results in dissociation of the viral membrane protein from the ribonucleo-protein core. The BM2 protein is similar to the A/M2 protein ion channel of influenza A virus (A/M2) in that it contains an HXXXW motif. Unlike the A/M2 protein, the BM2 protein is not inhibited by the antiviral drug amantadine. We used mutagenesis to ascertain the pore-lining residues of the BM2 ion channel. The specific activity (relative to wild type), reversal voltage, and susceptibility to modification by (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide of cysteine mutant proteins were measured in oocytes. It was found that mutation of transmembrane domain residues Ser(9), Ser(12), Phe(13), Ser(16), His(19), and Trp(23) to cysteine were most disruptive for ion channel function. These cysteine mutants were also most susceptible to (2-aminoethyl)-methane thiosulfonate and N-ethylmaleimide modification. Furthermore, considerable amounts of dimer were formed in the absence of oxidative reagents when cysteine was introduced at positions Ser(9), Ser(12), Ser(16), or Trp(23). Based on these experimental data, a BM2 transmembrane domain model is proposed. The presence of polar residues in the pore is a probable explanation for the amantadine insensitivity of the BM2 protein and suggests that related but more polar compounds might serve as useful inhibitors of the protein.     

Influenza A M2 Channel Clustering at High Protein/Lipid Ratios: Viral Budding Implications

Protein dynamics in crowded environments is important for understanding protein functions in vivo and is especially relevant for membrane proteins because of the roles of protein-protein interactions in membrane protein functions and their regulation. Here, using solid-state NMR spectroscopy in combination with coarse-grained molecular dynamics simulations, we report that the rotational correlation time for the transmembrane domain of the influenza A M2 proton channel in lipid bilayers increases dramatically at an elevated protein/lipid ratio. This increase is attributable to persistent protein-protein interactions, thus revealing for the first time, to the best of our knowledge, extensive cluster formation of the M2 tetrameric channel. Such clustering appears to have direct biological relevance during budding of the nascent influenza virus, which does not use the endosomal sorting complexes required for transport machinery. Indeed, initial coarse-grained molecular dynamics simulations of the longer M2 construct known as the conductance domain suggest clustering-induced membrane curvature formation.     

Definitive assignment of proton selectivity and attoampere unitary current to the M2 ion channel protein of influenza A virus

The viral ion channel protein M2 supports the transit of influenza virus and its glycoproteins through acidic compartments of the cell. M2 conducts endosomal protons into the virion to initiate uncoating and, by equilibrating the pH at trans-Golgi membranes, preserves the native conformation of acid-sensitive viral hemagglutinin. The exceptionally low conductance of the M2 channel thwarted resolution of single channels by electrophysiological techniques. Assays of liposome-reconstituted M2 yielded the average unitary channel current of the M2 tetramer--1.2 aA (1.2 x 10(-18) A) at neutral pH and 2.7 to 4.1 aA at pH 5.7--which activates the channel. Extrapolation to physiological temperature predicts 4.8 and 40 aA, respectively, and a unitary conductance of 0.03 versus 0.4 fS. This minute activity, below previous estimates, appears sufficient for virus reproduction, but low enough to avert abortive cytotoxicity. The unitary permeability of M2 was within the range reported for other proton channels. To address the ion selectivity of M2, we exploited the coupling of ionic influx and efflux in sealed liposomes. Metal ion fluxes were monitored by proton counterflow, employing a pH probe 1,000 times more sensitive than available Na+ or K+ probes. Even low-pH-activated M2 did not conduct Na+ and K+. The proton selectivity of M2 was estimated to be at least 3 x 10(6) (over sodium or potassium ions), in agreement with electrophysiological studies. The stringent proton selectivity of M2 suggests that the cytopathology of influenza virus does not involve direct perturbation of cellular sodium or potassium gradients.     

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.     

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.     

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.     

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.     

Influenza B virus BM2 protein is transported through the trans-Golgi network as an integral membrane protein

A bicistronic mRNA transcribed from the influenza B virus RNA segment 7 encodes two viral proteins, matrix protein M1 and uncharacterized small protein BM2. In the present study, we focused on the cytoplasmic transport and cellular membrane association of BM2. Immunofluorescence studies of virus-infected cells indicated that BM2 accumulated at the Golgi apparatus immediately after synthesis and then was transported to the plasma membrane through the trans-Golgi network. Localization of a set of BM2 deletion mutants revealed that the N-terminal half of BM2 (residues 2 to 50) was crucial for its transport; in particular, the deletion of residues 2 to 23, deduced to be a transmembrane domain, resulted in diffused distribution of the protein throughout the entire cell. Sucrose gradient flotation and biochemical analyses of the membrane showed that BM2 was tightly associated with cellular membranes as an integral membrane protein. Oligomerization of BM2 was demonstrated by coprecipitation of differentially epitope-tagged BM2 proteins. Taken together, these results strongly suggest that BM2 is integrated into the plasma membrane at the N-terminal hydrophobic domain as fourth membrane protein, in addition to hemagglutinin, neuraminidase, and NB, of the influenza B virus.     

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.