Toxicity of Influenza A Virus Matrix Protein 2 for Mammalian Cells is Associated with its Intrinsic Proton-Channeling Activity

Ilyinskii, Petr O.; Gabai, Vladimir L.; Sunyaev, Shamil; Thoidis, Galini; Shneider, Alexander

doi:10.4161/cc.6.16.4564

Cited by 25 publications

(26 citation statements)

References 25 publications

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“…This may explain the unique M1 mRNA splicing kinetics in which M1 production predominates early in infection when NS1 synthesis is low, and then M1 to M2 mRNA splicing is likely enhanced by the high levels of NS1 at late stages of infection 37 . M2 protein has been shown to be cytotoxic to cells when expressed alone 38 , which suggests that M2 levels need to be tightly regulated during infection. Furthermore, M2 is critical for viral budding 39 , indicating that timing of expression is likely important for production of infectious viral particles, as premature synthesis could lead to defective particles.…”

Section: Discussionmentioning

confidence: 99%

Influenza virus mRNA trafficking through host nuclear speckles

et al. 2016

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Influenza A virus is a human pathogen whose genome is comprised of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. Since nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

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Section: Discussionmentioning

confidence: 99%

Influenza virus mRNA trafficking through host nuclear speckles

et al. 2016

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“…The AIV M2 protein was found to be weakly immunogenic and did not induce neutralizing antibodies but reduced virus replication in mice, a nonnatural host (18). Various vaccine approaches have been evaluated for development of an M2 universal vaccine, including passive transfer of M2-specific antibodies (23,41) or immunization with conjugated M2 peptide antigens (7,26), complete M2 protein (16), or the external domain of M2 (5,10,16). Although these studies demonstrated protection against influenza virus challenge in the nonnatural mouse host, the role of M2 protein in immunogenicity and protection in a natural host had not been evaluated.…”

Section: Discussionmentioning

confidence: 99%

Contributions of the Avian Influenza Virus HA, NA, and M2 Surface Proteins to the Induction of Neutralizing Antibodies and Protective Immunity

Nayak

Kumar

DiNapoli

et al. 2010

J Virol

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Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 causes severe disease and mortality in poultry. Increased transmission of H5N1 HPAIV from birds to humans is a serious threat to public health. We evaluated the individual contributions of each of the three HPAIV surface proteins, namely, the hemagglutinin (HA), the neuraminidase (NA), and the M2 proteins, to the induction of HPAIV-neutralizing serum antibodies and protective immunity in chickens. Using reverse genetics, three recombinant Newcastle disease viruses (rNDVs) were engineered, each expressing the HA, NA, or M2 protein of H5N1 HPAIV. Chickens were immunized with NDVs expressing a single antigen (HA, NA, and M2), two antigens (HA؉NA, HA؉M2, and NA؉M2), or three antigens (HA؉NA؉M2). Immunization with HA or NA induced high titers of HPAIVneutralizing serum antibodies, with the response to HA being greater, thus identifying HA and NA as independent neutralization antigens. M2 did not induce a detectable neutralizing serum antibody response, and inclusion of M2 with HA or NA reduced the magnitude of the response. Immunization with HA alone or in combination with NA induced complete protection against HPAIV challenge. Immunization with NA alone or in combination with M2 did not prevent death following challenge, but extended the time period before death. Immunization with M2 alone had no effect on morbidity or mortality. Thus, there was no indication that M2 is immunogenic or protective. Furthermore, inclusion of NA in addition to HA in a vaccine preparation for chickens may not enhance the high level of protection provided by HA.

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“…However, the emergence of widespread highly virulent adamantane-resistant strains such as avian and swine influenza virus strains (9,10) has prompted the need for newer antivirals. Drug discovery efforts targeting M2 have been difficult due to the toxicity of M2 in cells (11), the difficulty of reconstituting M2 in liposomes for largescale application (12,13), and the labor-intensive, low-throughput electrophysiological approaches typically used to study ion channels.…”

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confidence: 99%

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

Sulli

Banik

Schilling

et al. 2013

J Virol

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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.T he M2 gene of influenza virus encodes a 97-amino-acid, homotetrameric, proton-selective ion channel necessary for influenza virus infectivity (1, 2). Activation of M2 is triggered by low pH in host cell endosomes, resulting in an influx of protons into the virus and acidification that releases the viral RNA into the host cell (2-4). M2 also helps to package viral RNA (5, 6) and regulate pH in the Golgi apparatus of infected cells during viral assembly (7). Two FDA-approved adamantane-based drugs, amantadine and rimantadine (2,8), are potent M2 inhibitors that can neutralize influenza virus in humans and other animals. However, the emergence of widespread highly virulent adamantane-resistant strains such as avian and swine influenza virus strains (9, 10) has prompted the need for newer antivirals. Drug discovery efforts targeting M2 have been difficult due to the toxicity of M2 in cells (11), the difficulty of reconstituting M2 in liposomes for largescale application (12, 13), and the labor-intensive, low-throughput electrophysiological approaches typically used to study ion channels.To address these challenges, we developed a rapid, cell-free assay to measure the movement of proton ions directly across the lipid bilayer of virus-like particles (VLPs). High-throughput screening (HTS) of M2-VLPs using Ͼ100,000 compounds identified 19 M2-specific inhibitors, including two novel chemical scaffolds, that inhibited both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) protonophore activity also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane but that is not t...

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Toxicity of Influenza A Virus Matrix Protein 2 for Mammalian Cells is Associated with its Intrinsic Proton-Channeling Activity

Cited by 25 publications

References 25 publications

Influenza virus mRNA trafficking through host nuclear speckles

Influenza virus mRNA trafficking through host nuclear speckles

Contributions of the Avian Influenza Virus HA, NA, and M2 Surface Proteins to the Induction of Neutralizing Antibodies and Protective Immunity

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

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