M2 Ion Channel Research Articles

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.

Structural Investigation of Rimantadine Inhibition of the AM2-BM2 Chimera Channel of Influenza Viruses

The M2 channel of influenza A is a target of the adamantane family antiviral drugs. Two different drug-binding sites have been reported: one inside the pore, and the other is a lipid-facing pocket. A previous study showed that a chimera of M2 variants from influenza A and B that contains only the pore-binding site is sensitive to amantadine inhibition, suggesting that the primary site of inhibition is inside the pore. To obtain atomic details of channel-drug interaction, we determined the structures of the chimeric channel with and without rimantadine. Inside the channel and near the N-terminal end, methyl groups of Val27 and Ala30 from four subunits form a hydrophobic pocket around the adamantane, and the drug amino group appears to be in polar contact with the backbone oxygen of Ala30. The structures also reveal differences between the drug-bound and -unbound states of the channel that can explain drug resistance.

Disruption of virus-host cell interactions and cell signaling pathways as an anti-viral approach against influenza virus infections

Influenza is still one of the major plagues worldwide with the threatening potential to cause pandemics. In recent years, increasing levels of resistance to the four FDA approved anti-influenza virus drugs have been described. This situation underlines the urgent need for novel anti-virals in preparation for future influenza epidemics or pandemics. Although the anti-virals currently in use target viral factors such as the neuraminidase or the M2 ion channel, there is an increase in pre-clinical approaches that focus on cellular factors or pathways that directly or indirectly interact with virus replication. This does not only include inhibitors of virus-supportive signaling cascades but also interaction blockers of viral proteins with host cell proteins. This review aims to highlight some of these novel approaches that represent a paradigm change in anti-viral strategies against the influenza virus. Although most of these approaches are still in an early phase of preclinical development they might be very promising particularly with respect to the prevention of viral resistance to potential drugs.

Sex Differences in Neuroprotection Provided by Inhibition of TRPM2 Channels following Experimental Stroke

The calcium-permeable transient receptor potential M2 (TRPM2) ion channel is activated following oxidative stress and has been implicated in ischemic damage; however, little experimental evidence exists linking TRPM2 channel activation to damage following cerebral ischemia. We directly assessed the involvement of TRPM2 channels in ischemic brain injury using pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of TRPM2 expression. Each of the four TRPM2 inhibitors tested provided significant protection to male neurons following in vitro ischemia (oxygen-glucose deprivation, OGD), while having no effect in female neurons. Similarly, TRPM2 knockdown by TRPM2 shRNA resulted in significantly reduced neuronal cell death following OGD only in male neurons. The TRPM2 inhibitor clotrimazole reduced infarct volume in male mice, while having no effect on female infarct volume. Finally, intrastriatal injection of lentivirus expressing shRNA against TRPM2 resulted in significantly smaller striatal infarcts only in male mice following middle cerebral artery occlusion, having no significant effect in female mice. Data presented in the current study demonstrate that TRPM2 inhibition and knockdown preferentially protects male neurons and brain against ischemia in vitro and in vivo, indicating that TRPM2 inhibitors may provide a new therapeutic approach to the treatment of stroke in men.

Identification of human annexin A6 as a novel cellular interactant of influenza A M2 protein: implications for influenza life cycle

Background During its replication, influenza virus utilizes the host cellular machineries for many aspects of its life cycle. Characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. The M2 ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its C-terminal domain, well conserved among influenza A viruses, is accessible to cellular machineries after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. The aim of the study is to identify cellular interactants of M2 that play important regulatory roles during influenza infection.

Antiviral drug resistance in seasonal and pandemic influenza

Two classes of anti-influenza drugs are currently available for the treatment or prophylaxis of influenza. These are the adamantanes (amantadine and rimantadine), which block the activity of the M2 ion channel of influenza A viruses (but not influenza B viruses), and the neuraminidase inhibitors (NAIs), which act by binding to the enzymatic site of the influenza neuraminidase (NA) thereby preventing progeny virions from being released from the host cell during viral replication. Antiviral resistance can occur in influenza viruses and render the drug ineffective for the treatment of patients. Virtually all influenza A viruses currently circulating in the human population are resistant to the adamantanes, while in comparison these viruses remain susceptible to the NAIs. In particular, very low NAI resistance has been observed in pandemic A(H1N1) 2009 viruses, even though unprecedented amounts of these drugs were used.

Genotypic and phenotypic resistance of pandemic A/H1N1 influenza viruses circulating in Germany

In response to the rapid global spread of an antigenically novel A/H1N1 influenza virus in 2009, the World Heath Organization (WHO) recommended surveillance and monitoring for antiviral resistance of influenza viruses. We designed and evaluated pyrosequencing (PSQ)-based genotypic assays for high-throughput analysis of the susceptibility of pandemic A/H1N1 influenza viruses to neuraminidase (NA) inhibitors. A total of 1570 samples circulating in Germany between April 2009 and April 2010 were tested for determination of molecular markers of resistance to the NA inhibitors oseltamivir and zanamivir, and 635 of them were evaluated by phenotypic fluorescence-based assay with MUNANA substrate. Eight (0.5%) viruses were resistant to oseltamivir due to the H274Y NA substitution (N2 numbering). Six of these oseltamivir-resistant cases were treatment-related; four of them were selected in immunocompromised patients, two in patients suffered from chronic diseases. The two remaining oseltamivir-resistant viruses seem to have evolved in the absence of drug treatment and were isolated from immunocompetent healthy patients. All tested A/H1N1 pandemic viruses were sensitive to zanamivir. In addition, analysis of 1011 pandemic A/H1N1 virus samples by a PSQ-based assay according to the WHO protocol revealed the presence of mutation S31N in the M2 protein that conferred resistance to M2 ion channel inhibitors. Our data demonstrate a low incidence of oseltamivir-resistant pandemic A/H1N1 influenza variants isolated under drug selection pressure as well as community-acquired or naturally evolving viruses.

Emergence of amantadine-resistant avian influenza H5N1 virus in India

This study reports the genetic characterization of highly pathogenic avian influenza (HPAI) virus (subtype H5N1) isolated from poultry in West Bengal, India. We analyzed all the eight genome segments of two viruses isolated from chickens in January 2010 to understand their genetic relationship with other Indian H5N1 isolates and possible connection between different outbreaks. The hemagglutinin (HA) gene of the viruses showed multiple basic amino acids at the cleavage site, a marker for high virulence in chickens. Of greatest concern was that the viruses displayed amino acid substitution from serine-to-asparagine at position 31 of M2 ion channel protein suggesting emergence of amantadine-resistant mutants not previously reported in HPAI H5N1 outbreaks in India. Amino acid lysine at position 627 of the PB2 protein highlights the risk the viruses possess to mammals. In the phylogenetic trees, the viruses clustered within the lineage of avian isolates from India (2008-2009) and avian and human isolates from Bangladesh (2007-2009) in all the genes. Both these viruses were most closely related to the viruses from 2008 in West Bengal within the subclade 2.2.3 of H5N1 viruses.

Combination of peramivir and rimantadine demonstrate synergistic antiviral effects in sub-lethal influenza A (H3N2) virus mouse model

Efficacy of combination of the intramuscularly administered neuraminidase (NA) inhibitor, peramivir, and the orally administered M2 ion channel blocker, rimantadine was evaluated in mouse influenza A/Victoria/3/75 (H3N2) model. Mice were challenged with a sub-lethal virus dose (0-40% mortality in placebo group) and changes in body weights were analyzed by three-dimensional effect analysis to assess mode of drug interactions. Compounds were administered in a 5-day treatment course starting 1h before viral inoculation. The peramivir and rimantadine doses ranged from 0.3-3 mg/kg/d and 5-30 mg/kg/d, respectively. The maximum mean weight loss of 5.19 g was observed in the vehicle-infected group on day 10. In the 1 and 3 mg/kg/d peramivir monotherapy groups, the weight losses were 4.3 and 3.55 g, respectively. In the rimantadine monotherapy group, the weight losses were 3.43, 2.1, and 1.64 g for the 5, 10, and 30 mg/kg/d groups, respectively. Combination of 1mg/kg/d peramivir with 5 and 10 mg/kg/d rimantadine produced weight losses of 1.69 and 0.69 (p<0.05 vs. vehicle and individual agent), respectively, whereas the combination of 3.0 mg/kg/d peramivir with 10 and 30 mg/kg/d rimantadine did not show any weight loss (p<0.05 vs. vehicle and individual agent). The three-dimensional analysis of the weight loss for the majority of the drug combinations of peramivir and rimantadine tested demonstrated synergistic antiviral effects.

NMR Structure and Ion Channel Activity of the p7 Protein from Hepatitis C Virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by (1)H and (13)C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.

Maternal and Neonatal Outcomes After Antepartum Treatment of Influenza With Antiviral Medications

Pregnant women are at increased risk of severe morbidity and mortality, secondary to influenza infection. Vaccination programs are the cornerstone of influenza preventive efforts and when they fail, 2 classes of antiviral drugs are important for both postexposure prophylaxis and treatment. The first class, the M2 ion channel inhibitors, includes amantadine and rimantadine, and is effective for the prophylaxis and treatment of influenza A. The second class, the neuraminidase inhibitors, includes oseltamivir, which is effective against both influenza A (including novel H1N1) and B. Because of increasing resistance to oseltamivir, this drug has been used with M2 ion channel inhibitors as 2-drug therapy for influenza A. Little data are not available on the safety of these 2 drug classes when used for treatment of influenza in pregnant women. This retrospective cohort study compared the maternal and neonatal outcomes of pregnant women treated antepartum for influenza with M2 ion channel inhibitors (amantadine, rimantadine), oseltamivir, or a control group. The study was conducted during 5 influenza seasons at a hospital in Texas. The comparative data showed that antepartum treatment with M2 ion channel inhibitors (n = 104), oseltamivir (n = 135), and a control group (n = 82,097) were not associated with increased rates of the following outcomes: gestational diabetes (P = 0.388), preterm birth (P = 0.190), premature rupture of the membranes (P = 0.154), or preeclampsia (P = 0.209). No differences were found among the 3 groups regarding the duration of hospital stay for the mother or the infant, the incidence of fever in labor, stillbirth, or major or minor malformations. Moreover, there were no significant differences among singleton live-born neonates without major malformations in fetal weight (P = 0.186), number of neonates who required intubation (P = 0.552) or intensive care nursery admission (P = 0.418), or the incidence of hyperbilirubinemia (P = 0.282). There were no neonatal deaths among live-born singletons and none had grade 3 or 4 intraventricular hemorrhages or seizures. Two preterm neonates exposed to antivirals in the second trimester developed necrotizing enterocolitis. One was exposed to amantadine and the other to oseltamivir. These findings show that antepartum-antiviral exposure does not increase the risk of adverse maternal or neonatal outcomes.

Combination Chemotherapy for Influenza

The emergence of pandemic H1N1 influenza viruses in April 2009 and the continuous evolution of highly pathogenic H5N1 influenza viruses underscore the urgency of novel approaches to chemotherapy for human influenza infection. Anti-influenza drugs are currently limited to the neuraminidase inhibitors (oseltamivir and zanamivir) and to M2 ion channel blockers (amantadine and rimantadine), although resistance to the latter class develops rapidly. Potential targets for the development of new anti-influenza agents include the viral polymerase (and endonuclease), the hemagglutinin, and the non-structural protein NS1. The limitations of monotherapy and the emergence of drug-resistant variants make combination chemotherapy the logical therapeutic option. Here we review the experimental data on combination chemotherapy with currently available agents and the development of new agents and therapy targets.

Coexistence of two adamantane binding sites in the influenza A M2 ion channel

The influenza A virus contains a proton-selective ion channel (M2) that is the target of the adamantane family of drug inhibitors. Two recently published studies relating to adamantane binding of the M2 ion channel using X-ray crystallography and solution NMR have reignited interest in the potential use of adamantanes in combating the spread of influenza A. However, these two studies propose different binding sites for the adamantane drugs with the X-ray M2/amantadine structure favoring an ion channel pore-binding model and the solution NMR M2/rimantadine structure suggesting the existence of a lipid-facing binding pocket. We conducted a series of surface plasmon resonance (SPR) experiments designed to accurately measure the affinity of amantadine and rimantadine to M2 ion channels embedded in 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC) liposomes. We find that this class of drug is capable of binding M2 with two different affinities in the order of 10(-4) and 10(-7) M, suggesting that both proposed binding sites are feasible. Furthermore, by examining drug binding to M2 mutant constructs (V27A, S31N, and D44A), it was possible to probe the location of the two binding sites. We show that a high-affinity binding site corresponds to the M2 ion channel pore whereas the secondary, low-affinity binding site can be attributed to the lipid face of the pore. These SPR results are in excellent agreement with the most recent solid-state NMR study of amantadine-bound M2 in lipid bilayers and provide independent support that the ion channel pore-binding site is responsible for the pharmacological activity elicited by the adamantane drugs.

Drugs in Development for Influenza

The emergence and global spread of the 2009 pandemic H1N1 influenza virus reminds us that we are limited in the strategies available to control influenza infection. Vaccines are the best option for the prophylaxis and control of a pandemic; however, the lag time between virus identification and vaccine distribution exceeds 6 months and concerns regarding vaccine safety are a growing issue leading to vaccination refusal. In the short-term, antiviral therapy is vital to control the spread of influenza. However, we are currently limited to four licensed anti-influenza drugs: the neuraminidase inhibitors oseltamivir and zanamivir, and the M2 ion-channel inhibitors amantadine and rimantadine. The value of neuraminidase inhibitors was clearly established during the initial phases of the 2009 pandemic when vaccines were not available, i.e. stockpiles of antivirals are valuable. Unfortunately, as drug-resistant variants continue to emerge naturally and through selective pressure applied by use of antiviral drugs, the efficacy of these drugs declines. Because we cannot predict the strain of influenza virus that will cause the next epidemic or pandemic, it is important that we develop novel anti-influenza drugs with broad reactivity against all strains and subtypes, and consider moving to multiple drug therapy in the future. In this article we review the experimental data on investigational antiviral agents undergoing clinical trials (parenteral zanamivir and peramivir, long-acting neuraminidase inhibitors and the polymerase inhibitor favipiravir [T-705]) and experimental antiviral agents that target either the virus (the haemagglutinin inhibitor cyanovirin-N and thiazolides) or the host (fusion protein inhibitors [DAS181], cyclo-oxygenase-2 inhibitors and peroxisome proliferator-activated receptor agonists).

Molecular dynamics simulation of oseltamivir resistance in neuraminidase of avian influenza H5N1 virus

The outbreak of avian influenza virus H5N1 has raised a global concern because of its high virulence and mutation rate. Although two classes of antiviral drugs, M2 ion channel protein inhibitors and neuraminidase inhibitors, are expected to be important in controlling the early stages of a potential pandemic. Different strains of influenza viruses have differing degrees of resistance against the antivirals. In order to analyze the detailed information on the viral resistance, molecular dynamics simulations were carried out for the neuraminidase (NA) complex with oseltamivir. The carboxylate of Glu276 of H252Y NA faces toward the O-ethyl-propyl group of oseltamivir, Glu276 of wild-type NA adopts a conformation pointing away from the oseltamivir. τ2 and τ3 torsional angles fluctuation of the oseltamivir are relatively high for the H252Y mutant NA complex. In addition, there are fewer hydrogen bonds between the oseltamivir and H252Y mutation NA. The results show that H252Y mutation NA has high resistance against the drug.

5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

BackgroundEmergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.ResultsIn the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice.ConclusionsOur findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.

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.

Maternal and Neonatal Outcomes After Antepartum Treatment of Influenza With Antiviral Medications

To review the maternal and neonatal outcomes after antepartum exposure to M2 ion channel inhibitors or oseltamivir to provide some guidance on the risk, if any, of antiviral medication during pregnancy. This was a retrospective cohort study examining maternal and neonatal outcomes after antepartum exposure to antiviral therapy for influenza. We evaluated maternal characteristics, pregnancy outcomes, and fetal outcomes and compared them with our overall obstetric population. Exposure to antiviral therapies (M2 ion channel inhibitors [n=104] compared with oseltamivir [n=135] compared with the control group [n=82,097]) during pregnancy was not associated with increased rates of preterm birth (7% compared with 10% compared with 6%, P=.190), premature rupture of membranes (23% compared with 16% compared with 22%, P=.154), gestational diabetes (4% compared with 8% compared with 6%, P=.388), or preeclampsia (6% compared with 1% compared with 4%, P=.209). Exposure was not associated with increased duration of hospital stay for mother or neonate. There were no differences in the incidence of minor malformations (19% compared with 15% compared with 22%, P=.101). Liveborn singletons without major malformations did not have differences in fetal weight (3,238+/-586 g compared with 3,281+/-642 g compared with 3,336+/-571 g, P=.186), need for intubation (2% compared with 0.8% compared with 1%, P=.552), intensive care nursery admission (3% compared with 3% compared with 2%, P=.418), or hyperbilirubinemia (12% compared with 9% compared with 8%, P=.282). Liveborn singletons had no grade 3 or 4 intraventricular hemorrhages, seizures, or neonatal deaths. Two preterm neonates exposed to different classes of medications had necrotizing enterocolitis (1.0% compared with 0.8% compared with 0.02%, P<.001). We found no evidence of an association between antepartum antiviral exposure and adverse outcomes. II.

Advances in the Structure-Based Design of the Influenza A Neuraminidase Inhibitors

Since 2003, highly pathogenic H5N1 influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. At present, there are only three licensed anti-Influenza drugs namely Relenza (Zanamivir - ZMV), Tamiflu (Oseltamivir - OTV) and Amantadine/Rimantadine. The latter targets the M2 ion channel whereas the other compounds target neuraminidase (NA) and were designed through structure-based enzyme inhibitor programmes. Some structural knowledge of the Influenza neuraminidase is now known, due to remarkable advances in crystallographic techniques. The structure of H5N1 NA is particularly attractive because it offers new opportunities for drug design. Besides a profound impact that structural biology has had on understanding the Influenza virus and the rational design of antivirals, computational methods are now a viable partner to experiment in designing NA inhibitors. We herein discuss the development of current neuraminidase inhibitors, the emergence of resistance to them, and recent research progress towards the development of new inhibitors.

Architecture of a nascent viral fusion pore

Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three-dimensional architecture of influenza virus–liposome complexes at pH 5.5 was investigated by electron cryo-tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane-centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of ‘leaky' fusion to the observed prefusion structures is discussed.