MARCH8 inhibits influenza A virus infection by targeting viral M2 protein for ubiquitination-dependent degradation in lysosomes

The membrane-associated RING-CH (MARCH) proteins are E3 ligases that regulate the stability of various cellular membrane proteins. MARCH8 has been reported to inhibit the infection of HIV-1 and a few other viruses, thus plays an important role in host antiviral defense. However, the antiviral spectrum and the underlying mechanisms of MARCH8 are incompletely defined. Here, we demonstrate that MARCH8 profoundly inhibits influenza A virus (IAV) replication both in vitro and in mice. Mechanistically, MARCH8 suppresses IAV release through redirecting viral M2 protein from the plasma membrane to lysosomes for degradation. Specifically, MARCH8 catalyzes the K63-linked polyubiquitination of M2 at lysine residue 78 (K78). A recombinant A/Puerto Rico/8/34 virus carrying the K78R M2 protein shows greater replication and more severe pathogenicity in cells and mice. More importantly, we found that the M2 protein of the H1N1 IAV has evolved to acquire non-lysine amino acids at positions 78/79 to resist MARCH8-mediated ubiquitination and degradation. Together, our data support the important role of MARCH8 in host anti-IAV intrinsic immune defense by targeting M2, and suggest the inhibitory pressure of MARCH8 on H1N1 IAV transmission in the human population.

A Novel Mechanism Underlying Antiviral Activity of an Influenza Virus M2-Specific Antibody

ABSTRACT Protective immunity against influenza A viruses (IAVs) generally depends on antibodies to the major envelope glycoprotein, hemagglutinin (HA), whose antigenicity is distinctive among IAV subtypes. On the other hand, the matrix 2 (M2) protein is antigenically highly conserved and has been studied as an attractive vaccine antigen to confer cross-protective immunity against multiple subtypes of IAVs. However, antiviral mechanisms of M2-specific antibodies are not fully understood. Here, we report the molecular basis of antiviral activity of an M2-specific monoclonal antibody (MAb), rM2ss23. We first found that rM2ss23 inhibited A/Aichi/2/1968 (H3N2) (Aichi) but not A/PR/8/1934 (H1N1) (PR8) replication. rM2ss23 altered the cell surface distribution of M2, likely by cross-linking the molecules, and interfered with the colocalization of HA and M2, resulting in reduced budding of progeny viruses. However, these effects were not observed for another strain, PR8, despite the binding capacity of rM2ss23 to PR8 M2. Interestingly, HA was also involved in the resistance of PR8 to rM2ss23. We also found that two amino acid residues at positions 54 and 57 in the M2 cytoplasmic tail were critical for the insensitivity of PR8 to rM2ss2. These findings suggest that the disruption of the M2-HA colocalization on infected cells and subsequent reduction of virus budding is one of the principal mechanisms of antiviral activity of M2-specific antibodies and that anti-M2 antibody-sensitive and -resistant IAVs have different properties in the interaction between M2 and HA. IMPORTANCE Although the IAV HA is the major target of neutralizing antibodies, most of the antibodies are HA subtype specific, restricting the potential of HA-based vaccines. On the contrary, the IAV M2 protein has been studied as a vaccine antigen to confer cross-protective immunity against IAVs with multiple HA subtypes, since M2 is antigenically conserved. Although a number of studies highlight the protective role of anti-HA neutralizing and nonneutralizing antibodies, precise information on the molecular mechanism of action of M2-specific antibodies is still obscure. In this study, we found that an anti-M2 antibody interfered with the HA-M2 association, which is important for efficient budding of progeny virus particles from infected cells. The antiviral activity was IAV strain dependent despite the similar binding capacity of the antibody to M2, and, interestingly, HA was involved in susceptibility to the antibody. Our data provide a novel mechanism underlying antiviral activity of M2-specific antibodies.

Immunogenicity and Protective Efficacy of Influenza A DNA Vaccines Encoding Artificial Antigens Based on Conservative Hemagglutinin Stem Region and M2 Protein in Mice

Background: Development of a universal vaccine capable to induce antibody responses against a broad range of influenza virus strains attracts growing attention. Hemagglutinin stem and the exposed fragment of influenza virus M2 protein are promising targets for induction of cross-protective humoral and cell-mediated response, since they contain conservative epitopes capable to induce antibodies and cytotoxic T lymphocytes (CTLs) to a wide range of influenza virus subtypes. Methods: In this study, we generated DNA vaccine constructs encoding artificial antigens AgH1, AgH3, and AgM2 designed on the basis of conservative hemagglutinin stem fragments of two influenza A virus subtypes, H1N1 and H3N2, and conservative M2 protein, and evaluate their immunogenicity and protective efficacy. To obtain DNA vaccine constructs, genes encoding the designed antigens were cloned into a pcDNA3.1 vector. Expression of the target genes in 293T cells transfected with DNA vaccine constructs has been confirmed by synthesis of specific mRNA. Results: Immunization of BALB/c mice with DNA vaccines encoding these antigens was shown to evoke humoral and T-cell immune responses as well as a moderated statistically significant cross-protective effect against two heterologous viruses A/California/4/2009 (H1N1pdm09) and A/Aichi/2/68 (H3N2). Conclusions: The results demonstrate a potential approach to creating a universal influenza vaccine based on artificial antigens.

Comparative Analyses of the Antiviral Activities of IgG and IgA Antibodies to Influenza A Virus M2 Protein

The influenza A virus (IAV) matrix-2 (M2) protein is an antigenically conserved viral envelope protein that plays an important role in virus budding together with another envelope protein, hemagglutinin (HA). An M2-specific mouse monoclonal IgG antibody, rM2ss23, which binds to the ectodomain of the M2 protein, has been shown to be a non-neutralizing antibody, but inhibits plaque formation of IAV strains. In this study, we generated chimeric rM2ss23 (ch-rM2ss23) IgG and IgA antibodies with the same variable region and compared their antiviral activities. Using gel chromatography, ch-rM2ss23 IgA were divided into three antibody subsets: monomeric IgA (m-IgA), dimeric IgA (d-IgA), and trimeric and tetrameric IgA (t/q-IgA). We found that t/q-IgA had a significantly higher capacity to reduce the plaque size of IAVs than IgG and m-IgA, most likely due to the decreased number of progeny virus particles produced from infected cells. Interestingly, HA-M2 colocalization was remarkably reduced on the infected cell surface in the presence of ch-rM2ss23 antibodies. These results indicate that anti-M2 polymeric IgA restricts IAV budding more efficiently than IgG and suggest a role of anti-M2 IgA in cross-protective immunity to IAVs.

A Glu-Glu-Tyr sequence in the cytoplasmic tail of the M2 protein renders IAV susceptible to restriction of HA-M2 association in primary human macrophages

Influenza A virus (IAV) assembly at the plasma membrane is orchestrated by at least five viral components including hemagglutinin (HA), neuraminidase (NA), matrix (M1), the ion channel M2, and viral ribonucleoprotein (vRNP) complexes although particle formation itself requires only HA and/or NA. While these five viral components are expressed efficiently in primary human monocyte-derived macrophages (MDM) upon IAV infection, this cell type does not support efficient HA-M2 association and IAV particle assembly at the plasma membrane. The defects in HA-M2 association and particle assembly are specific to MDM and not observed in a monocytic cell line differentiated into macrophage-like cells. Notably, both these defects can be reversed upon disruption of the actin cytoskeleton. In the current study, we sought to examine whether M2 contributes to particle assembly in MDM and to identify a viral determinant involved in the MDM-specific and actin-dependent suppression of IAV assembly. An analysis using correlative fluorescence and scanning electron microscopy showed that an M2-deficient virus fails to form budding structures at the cell surface even after F-actin is disrupted, indicating that M2 is essential for virus particle formation at the MDM surface. Notably, proximity ligation analysis revealed that single amino acid substitution in a Glu-Glu-Tyr sequence (residues 74-76) in the M2 cytoplasmic tail allows HA-M2 association to occur efficiently even in MDM with intact actin cytoskeleton. This phenotype did not correlate with known phenotypes of the M2 substitution mutants regarding M1 interaction or vRNP packaging in epithelial cells. Overall, our study identifies a viral determinant for susceptibility to cytoskeleton-dependent regulation in MDM and hence, sheds light on the molecular mechanism behind the MDM-specific restriction of IAV assembly.ImportanceNon-permissive cell types that are unable to support viral replication serve as important tools for identification of host factors that either block viral replication (restriction factors) or support viral replication in permissive cell types (co-factors). We previously identified the MDM as a cell type that is non-permissive to IAV assembly, likely due to a block in HA-M2 association. In the current study, we determined that the IAV M2 protein is necessary for virus particle formation in MDM but also renders the virus susceptible to the MDM-specific suppression of virus assembly. We identified a specific amino acid motif in the M2 cytoplasmic tail, disruption of which allows M2 to associate with HA even in MDM. Our findings strongly support the possibility that the MDM-specific defect in HA-M2 association is due to the presence of a restriction factor(s) in MDM, which likely interacts directly with the M2 cytoplasmic domain, rather than indirectly through other internal viral components, and thereby prevents M2 from associating with HA.

Virtual Screening Identifies Chebulagic Acid as an Inhibitor of the M2(S31N) Viral Ion Channel and Influenza A Virus

The increasing prevalence of drug-resistant influenza viruses emphasizes the need for new antiviral countermeasures. The M2 protein of influenza A is a proton-gated, proton-selective ion channel, which is essential for influenza replication and an established antiviral target. However, all currently circulating influenza A virus strains are now resistant to licensed M2-targeting adamantane drugs, primarily due to the widespread prevalence of an M2 variant encoding a serine to asparagine 31 mutation (S31N). To identify new chemical leads that may target M2(S31N), we performed a virtual screen of molecules from two natural product libraries and identified chebulagic acid as a candidate M2(S31N) inhibitor and influenza antiviral. Chebulagic acid selectively restores growth of M2(S31N)-expressing yeast. Molecular modeling also suggests that chebulagic acid hydrolysis fragments preferentially interact with the highly-conserved histidine residue within the pore of M2(S31N) but not adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development.