Virus M2 Protein Research Articles

A Theory for the Proton Transport of the Influenza Virus M2 Protein: Extensive Test against Conductance Data

A theory for calculating the proton flux through the influenza virus M2 channel is tested here against an extensive set of conductance data. The flux is determined by the rate constants for binding to the His 37 tetrad from the two sides of the membrane and the corresponding unbinding rate constants. The rate constants are calculated by explicitly treating the structure and dynamics of the protein. Important features revealed by previous studies, such as a gating role for Val 27 at the entrance to the channel pore, and channel activation by viral exterior pH, are incorporated in this theory. This study demonstrates that the conductance function of the M2 proton channel can now be quantitatively rationalized by the structure and dynamics of the protein.

Expression of a chimerical pcDNA encoding influenza virus M2 protein and HSP70 gene in eukaryotic cell lines

The critical obstacle in developing of an effective human influenza vaccine is continuous antigenic variation of influenza A viruses occurred each year. Conserved antigens are important candidates for vaccines because it is not necessary to match the designed strains with circulating strains. M2 as an ion channel protein induce protective immunity. On the other hand, HSP70 is a molecular chaperon and immunostimulatory component, capable of eliciting effective humoral and cellular immunity against infectious disease. Genetically fusing antigens to HSPs leads to the enrichment of DNA vaccine potency. In the present study, a chimerical DNA plasmid carrying influenza virus M2 protein and HSP70 Gene was constructed and the protein expression in eukaryotic cell line was evaluated. This construct could be used as a potent DNA vaccine. To gain this aim, Influenza A/New Caledonia/20/99 (H1N1) was inoculated into MDCK cell line. The supernatant was collected after 18 hours and total RNA was extracted using Easy-red (iNtRON) solution. Complementary DNA synthesis was carried out by RevertAid First Strand cDNA synthesis kit using uni-12 primer. Full length M2 gene (300bp) was amplified by polymerase chain reactions using designed primers. The PCR product was run on 2% agarose gel following by purification of specific band, cloned into pGEM-T Easy cloning vector (promega) and completely sequenced. The M2 gene was digested from T-vector and subcloned into the pcDNA3.1. Leishmania amazonesis heat shock protein (HSP70) gene was obtained by digestion of pGEM II- HSP70 and subcloned into pCDNA3.1. The digested M2 gene was then subcloned into the N-terminal of HSP70 in pcDNA3.1. Recombinant plasmids were transfected into COS-7 cells to evaluate protein expression. The presence of M2 and HSP70 genes were confirmed by PCR, restriction enzyme analysis and electrophoresis. All the constructs were then verified by DNA sequencing. The vaccine candidates were transfected into COS-7 cells and protein expression was confirmed by indirect Immunofluorescense test, ELISA and western blotting. In ongoing project, the Immunomodulatory effect of HSP70 construct on DNA vaccine efficacy in animal models will be evaluated.

Chicken cyclophilin A is an inhibitory factor to influenza virus replication

BackgroundThe importance of enhancing influenza resistance in domestic flocks is quite clear both scientifically and economically. Chicken is very susceptible to influenza virus. It has been reported that human cellular cyclophilin A (CypA) impaired influenza virus infection in 293T cells. Whether chicken CypA (chCypA) inhibits influenza virus replication is not known. The molecular mechanism of resistance in chicken to influenza virus remains to be studied.ResultsThe chCypA gene was isolated and characterized in the present study. It contained an ORF of 498 bp encoding a polypeptide of 165 amino acids with an estimated molecular mass of 17.8 kDa sharing high identity with mammalian CypA genes. The chCypA demonstrated an anti-influenza activity as expected. ChCypA protein was shown to be able to specifically interact with influenza virus M1 protein. Cell susceptibility to influenza virus was reduced by over-expression of chCypA in CEF cells. The production of recombinant influenza virus A/WSN/33 reduced to one third in chCypA expressing cells comparing to chCypA absent cells. ChCypA was widely distributed in a variety of chicken tissues. It localized in cytoplasm of chicken embryo fibroblast (CEF) cells. Avian influenza virus infection induced its translocation from cytoplasm into nucleus. ChCypA expression was not significantly up-regulated by avian influenza virus infection. The present study indicated that chCypA was an inhibitory protein to influenza virus replication, suggesting a role as an intrinsic immunity factor against influenza virus infection.ConclusionThe present data demonstrates that chCypA possesses anti-influenza virus activity which allows the consideration of genetic improvement for resistance to influenza virus in chickens.

Mechanisms of proton conduction and gating in influenza M2 proton channels from solid-state NMR.

The M2 protein of influenza viruses forms an acid-activated tetrameric proton channel. We used solid-state nuclear magnetic resonance spectroscopy to determine the structure and functional dynamics of the pH-sensing and proton-selective histidine-37 in M2 bound to a cholesterol-containing virus-envelope-mimetic membrane so as to better understand the proton conduction mechanism. In the high-pH closed state, the four histidines form an edge-face π-stacked structure, preventing the formation of a hydrogen-bonded water chain to conduct protons. In the low-pH conducting state, the imidazoliums hydrogen-bond extensively with water and undergo microsecond ring reorientations with an energy barrier greater than 59 kilojoules per mole. This barrier is consistent with the temperature dependence of proton conductivity, suggesting that histidine-37 dynamically shuttles protons into the virion. We propose a proton conduction mechanism in which ring-flip-assisted imidazole deprotonation is the rate-limiting step.

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 p58IPK 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 p58IPK by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58IPKin vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58IPK, and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58IPK mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.

Intrinsic Cytoskeleton-Dependent Clustering of Influenza Virus M2 Protein with Hemagglutinin Assessed by FLIM-FRET

The hemagglutinin (HA) of influenza virus organizes the virus bud zone, a domain of the plasma membrane enriched in raft lipids. Using fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET), a technique that detects close colocalization of fluorescent proteins in transfected cells, we show that the viral proton channel M2 clusters with HA but not with a marker for inner leaflet rafts. The FRET signal between M2 and HA depends on the raft-targeting signals in HA and on an intact actin cytoskeleton. We conclude that M2 contains an intrinsic signal that targets the protein to the viral bud zone, which is organized by raft-associated HA and by cortical actin.

The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence

Influenza A virus particles assemble and bud from plasma membrane domains enriched with the viral glycoproteins but only a small fraction of the total M2 protein is incorporated into virus particles when compared to the other viral glycoproteins. A membrane proximal cholesterol recognition/interaction amino acid consensus (CRAC) motif was previously identified in M2 and suggested to play a role in protein function. We investigated the importance of the CRAC motif on virus replication by generating recombinant proteins and viruses containing amino acid substitutions in this motif. Alteration or completion of the M2 CRAC motif in two different virus strains caused no changes in virus replication in vitro. Viruses lacking an M2 CRAC motif had decreased morbidity and mortality in the mouse model of infection, suggesting that this motif is a virulence determinant which may facilitate virus replication in vivo but is not required for basic virus replication in tissue culture.

Tyrosines in the influenza A virus M2 protein cytoplasmic tail are critical for production of infectious virus particles.

The cytoplasmic tail of the influenza A virus M2 protein is required for the production of infectious virions. In this study, critical residues in the M2 cytoplasmic tail were identified by single-alanine scanning mutagenesis. The tyrosine residue at position 76, which is conserved in >99% of influenza virus strains sequenced to date, was identified as being critical for the formation of infectious virus particles using both reverse genetics and a protein trans-complementation assay. Recombinant viruses encoding M2 with the Y76A mutation demonstrated replication defects in MDCK cells as well as in primary differentiated airway epithelial cell cultures, defects in the formation of filamentous virus particles, and reduced packaging of nucleoprotein into virus particles. These defects could all be overcome by a mutation of serine to tyrosine at position 71 of the M2 cytoplasmic tail, which emerged after blind passage of viruses containing the Y76A mutation. These data confirm and extend our understanding of the significance of the M2 protein for infectious virus particle assembly.

Influenza virus activates inflammasomes via its intracellular M2 ion channel

Influenza virus, a negative stranded RNA virus causing severe illness in humans and animals, stimulates the inflammasome through the NOD-like receptor (NLR), NLRP3. However, the mechanism by which influenza virus activates the NLRP3 inflammasome is unknown. Here, we show that the influenza virus M2 protein, a proton-selective ion channel important in viral pathogenesis, stimulates the NLRP3 inflammasome pathway. M2 channel activity was required for influenza activation of inflammasomes, and was sufficient to activate inflammasomes in primed macrophages and dendritic cells. M2-induced inflammasome activation required its localization to Golgi and was dependent on pH gradient. Our results reveal a mechanism by which influenza virus infection activates inflammasomes, and identifies the sensing of disturbances in intracellular ionic concentrations as a novel pathogen recognition pathway.

Conformational Changes of an Ion Channel Detected Through Water−Protein Interactions Using Solid-State NMR Spectroscopy

The influenza A virus M2 protein is a pH-gated and amantadine-inhibited proton channel important for the virus life cycle. Proton conduction by M2 is known to involve water; however direct experimental evidence of M2-water interaction is scarce. Using (1)H spin diffusion solid-state NMR, we have now determined the water accessibility of the M2 transmembrane domain (M2-TM) in virus-envelope-mimetic lipid membranes and its changes with environment. Site-specific water-protein magnetization transfer indicates that, in the absence of amantadine, the initial spin diffusion rate mainly depends on the radial position of the residues from the pore: pore-lining residues along the helix have similarly high water accessibilities compared to lipid-facing residues. Upon drug binding, the spin diffusion rates become much slower for Gly(34) in the middle of the helix than for the N-terminal residues, indicating that amantadine is bound to the pore lumen between Gly(34) and Val(27). Water-protein spin diffusion buildup curves indicate that spin diffusion is the fastest in the low-pH open state, slower in the high-pH closed state, and the slowest in the high-pH amantadine-bound state. Simulations of the buildup curves using a 3D lattice model yielded quantitative values of the water-accessible surface area and its changes by pH and drug binding. These data provide direct experimental evidence of the pH-induced change of the pore size and the drug-induced dehydration of the pore. This study demonstrates the capability of (1)H spin diffusion NMR for elucidating water interactions with ion channels, water pores, and proton pumps and for probing membrane protein conformational changes that involve significant changes of water-accessible surface areas.

Cholesterol-binding viral proteins in virus entry and morphogenesis.

Up to now less than a handful of viral cholesterol-binding proteins have been characterized, in HIV, influenza virus and Semliki Forest virus. These are proteins with roles in virus entry or morphogenesis. In the case of the HIV fusion protein gp41 cholesterol binding is attributed to a cholesterol recognition consensus (CRAC) motif in a flexible domain of the ectodomain preceding the trans-membrane segment. This specific CRAC sequence mediates gp41 binding to a cholesterol affinity column. Mutations in this motif arrest virus fusion at the hemifusion stage and modify the ability of the isolated CRAC peptide to induce segregation of cholesterol in artificial membranes.Influenza A virus M2 protein co-purifies with cholesterol. Its proton translocation activity, responsible for virus uncoating, is not cholesterol-dependent, and the transmembrane channel appears too short for integral raft insertion. Cholesterol binding may be mediated by CRAC motifs in the flexible post-TM domain, which harbours three determinants of binding to membrane rafts. Mutation of the CRAC motif of the WSN strain attenuates virulence for mice. Its affinity to the raft–non-raft interface is predicted to target M2 protein to the periphery of lipid raft microdomains, the sites of virus assembly. Its influence on the morphology of budding virus implicates M2 as factor in virus fission at the raft boundary. Moreover, M2 is an essential factor in sorting the segmented genome into virus particles, indicating that M2 also has a role in priming the outgrowth of virus buds.SFV E1 protein is the first viral type-II fusion protein demonstrated to directly bind cholesterol when the fusion peptide loop locks into the target membrane. Cholesterol binding is modulated by another, proximal loop, which is also important during virus budding and as a host range determinant, as shown by mutational studies.

Pharmacological Relevant Amantadine Binding Site is in the Pore of Influenza a Virus M2 Proton Channel

The influenza A virus M2 protein (A/M2) and the influenza B virus BM2 protein are both homotetrameric pH-activated proton channel that facilitates viral uncoating by acidification the interior of endosomally encapsulated virus. Anti-viral drugs amantadine and its derivative rimantadine inhibit A/M2 channel of influenza A virus, but not BM2 channel of influenza B virus. The atomic structure of the pore-transmembrane (TM) domain peptide has been determined by X-ray crystallography (Stouffer et al., Nature 451, 596-599 [2008]) and of a larger M2 peptide by NMR methods (Schnell and Chou, Nature 451, 591-595 [2008]). The crystallographic data shows electron density (at 3.5 A resolution) in the channel pore, consistent with amantadine blocking the pore of the channel. In contrast, the NMR data show four rimantadine molecules bound on the outside of the helices towards the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 and a hydrogen bond between rimantadine and Asp44. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. The cytoplasmic binding site predicts that D44 and R45 to alanine mutations would interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by TEVC recordings in oocytes of Xenopus laevis, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, two chimeras containing 5 residues of the A/M2 ectodomain and residues 24-36 of the A/M2 TM domain show 85% amantadine/rimantadine sensitivity and specific activity comparable to wt BM2. These functional data suggest the pharmacological relevant amantadine/rimantadine binding site is in the pore of the M2 channel.

Study on preparation and identification of monoclonal antibodies immunized with H5N1 influenza virus M1 protein

To prepare monoclonal antibodies specific for M1 protein of H5N1 subtype human influenza virus, this work may provide new tool in rapid diagnosis and study of type A influenza virus. BALB/c mice were immunized with purified recombinant H5N1 (A/Anhui/1/2005)/M1 protein expressed in E. coli. Spleen cells of the immunized mice were fused with sp2/0 cells to produce hybridoma cell lines. ELISA was performed to identify the monoclonal antibody against M1 protein of H5N1. Immunofluorescence assay (IFA) were applied to identify the specificity of these antibodies. Three hybridoma cell lines steadily secreting anti-H5N1/M1 McAb were obtained, and their cross reactivity was confirmed by cross-reaction test and IFA. Monoclonal antibodies immunized with H5N1 subtype influenza virus M1 protein are cross-reactive, which can be used to detect different subtype of influenze virus type A.

Attacking the flu: New prospects for the rational design of antivirals

Attacking the flu: New prospects for the rational design of antivirals

Discovery of multiple lead compounds as M2 inhibitors through the focused screening of a small primary amine library.

AbstractThe discovery of new anti-influenza drugs is urgent, particularly considering the recent threat of swine flu. In this study, the influenza virus M2 protein was expressed in HEK293 cells and shown to have selective ion channel activity for monovalent ions. The anti-influenza virus drug amantadine hydrochloride significantly attenuated the inward current induced by hyperpolarization of HEK293 cell membranes. Although adamantine derivatives are the only M2 drugs for influenza virus A, their use is limited in the US due to drug resistance. Here we report the discovery of multiple M2 inhibitor lead compounds that were rapidly generated through focused screening of a small primary amine library. The screen was designed using a scaffold-hopping strategy based on amantadine. This study suggests that an antiviral compound directed against a conserved motif may be more useful than amantadine in inhibiting viral replication.

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

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

Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species

The mechanisms by which replicating influenza viruses decrease the expression and function of amiloride-sensitive epithelial sodium channels (ENaCs) have not been elucidated. We show that expression of M2, a transmembrane influenza protein, decreases ENaC membrane levels and amiloride-sensitive currents in both Xenopus oocytes, injected with human alpha-, beta-, and gamma-ENaCs, and human airway cells (H441 and A549), which express native ENaCs. Deletion of a 10-aa region within the M2 C terminus prevented 70% of this effect. The M2 ENaC down-regulation occurred at normal pH and was prevented by MG-132, a proteasome and lysosome inhibitor. M2 had no effect on Liddle ENaCs, which have decreased affinity for Nedd4-2. H441 and A549 cells transfected with M2 showed higher levels of reactive oxygen species, as shown by the activation of redox-sensitive dyes. Pretreatment with glutathione ester, which increases intracellular reduced thiol concentrations, or protein kinase C (PKC) inhibitors prevented the deleterious effects of M2 on ENaCs. The data suggest that M2 protein increases steady-state concentrations of reactive oxygen intermediates that simulate PKC and decrease ENaCs by enhancing endocytosis and its subsequent destruction by the proteasome. These novel findings suggest a mechanism for the influenza-induced rhinorrhea and life-threatening alveolar edema in humans.

Palmitoylation of the Influenza A Virus M2 Protein Is Not Required for Virus Replication In Vitro but Contributes to Virus Virulence

The influenza A virus M2 protein has important roles during virus entry and in the assembly of infectious virus particles. The cytoplasmic tail of the protein can be palmitoylated at a cysteine residue, but this residue is not conserved in a number of human influenza A virus isolates. Recombinant viruses encoding M2 proteins with a serine substituted for the cysteine at position 50 were generated in the A/WSN/33 (H1N1) and A/Udorn/72 (H3N2) genetic backgrounds. The recombinant viruses were not attenuated for replication in MDCK cells, Calu-3 cells, or in primary differentiated murine trachea epithelial cell cultures, indicating there was no significant contribution of M2 palmitoylation to virus replication in vitro. The A/WSN/33 M2C50S virus displayed a slightly reduced virulence after infection of mice, suggesting that there may be novel functions for M2 palmitoylation during in vivo infection.

Discovery of spiro-piperidine inhibitors and their modulation of the dynamics of the M2 proton channel from influenza A virus.

Amantadine has been used for decades as an inhibitor of the influenza A virus M2 protein (AM2) in the prophylaxis and treatment of influenza A infections, but its clinical use has been limited by its central nervous system (CNS) side effects as well as emerging drug-resistant strains of the virus. With the goal of searching for new classes of M2 inhibitors, a structure-activity relation study based on 2-[3-azaspiro(5,5)undecanol]-2-imidazoline (BL-1743) was initiated. The first generation BL-1743 series of compounds has been synthesized and tested by two-electrode voltage-clamp (TEV) assays. The most active compound from this library, 3-azaspiro[5,5]undecane hydrochloride (9), showed an IC(50) as low as 0.92 +/- 0.11 microM against AM2, more than an order of magnitude more potent than amantadine (IC(50) = 16 microM). (15)N and (13)C solid-state NMR was employed to determine the effect of compound 9 on the structure and dynamics of the transmembrane domain of AM2 (AM2-TM) in phospholipid bilayers. Compared to amantadine, spiro-piperidine 9 (1) induces a more homogeneous conformation of the peptide, (2) reduces the dynamic disorder of the G34-I35 backbone near the water-filled central cavity of the helical bundle, and (3) influences the dynamics and magnetic environment of more residues within the transmembrane helices. These data suggest that spiro-piperidine 9 binds more extensively with the AM2 channel, thus leading to stronger inhibitory potency.

Monoclonal antibody recognizing SLLTEVET epitope of M2 protein potently inhibited the replication of influenza A viruses in MDCK cells

The ectodomain of influenza A virus M2 protein (M2e) is composed of 24 amino acids and induces antibodies with inhibitory effect against a broad spectrum of influenza A subtypes in vitro and in vivo. Although relatively conserved, 21 M2e variants emerged in recent influenza A strains, most of the mutations appeared in the middle part of M2e domain. In this study, we characterized the in vitro inhibition efficacy of a monoclonal antibody (mAb) M2e8-7 recognizing the N terminus highly conserved epitope SLLTEVET (aa 2–9) which is common for both M1 and M2 proteins. Peptide binding assay showed that mAb M2e8-7 reacted strongly with M2e and 19 M2e variant peptides. The mAb M2e8-7 potently inhibited the replication of influenza A virus H1 and H3 subtypes in MDCK cells. Two important amino acids in M2e epitope, Threonine at position five and the Glutamic acid at position six, were identified to lead antibody-escaping variants. These results brought new insight in developing vaccine and therapeutic agents against influenza A virus infections.