Influenza Virus M2 Protein Ion Channel Activity Helps To Maintain Pandemic 2009 H1N1 Virus Hemagglutinin Fusion Competence during Transport to the Cell Surface

Abstract

The influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability. IMPORTANCEInfluenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage site from pH-induced conformational changes during biosynthesis, but this effect is less well understood for human influenza virus HA proteins that lack polybasic cleavage sites. Using assays that focus on HA entry and fusion, we found that the M2 protein also protects (H1N1)pdm09 influenza A virus HA from premature conformational changes as it transits low-pH compartments during biosynthesis. This work suggests that M2 may play a wider role in preserving HA function in a variety of influenza virus subtypes that infect humans and may be especially important for HA proteins that are less stable. T he influenza virus hemagglutinin (HA) envelope protein mediates virus entry by binding to cell surface receptors, followed by endocytosis and fusion of the viral and endosomal membranes. Cellular proteases cleave the HA precursor (HA0) to generate the HA1 surface subunit, which mediates binding to cell surface sialic acid receptors, and the HA2 transmembrane subunit, which mediates membrane fusion between viral and endosomal membranes during e...

Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

“…Protease sensitivity and Western blotting. The whole viral particles and purified HA proteins have successfully been used to demonstrate the acidic pH-induced HA conformational changes and HA sensitivity to proteolysis (15,38,(42)(43)(44)(45)(46). Following the acidic pH-induced conformational change of HA, L-(tosylamido-2-phenyl ethyl) chloromethyl ketone (TPCK)-treated trypsin digests the HA1 subunit but not the HA2 subunit (42).…”

“…Rabbit antisera against the H1N1 HA2 C helix were produced via immunization with the A/New Caledonia/20/1999 HA2 C-helix peptide conjugated to keyhole limpet hemocyanin (KLH; Pierce) as described previously (38).…”

“…The HA conformational change was assessed in an HA-mediated cell-cell fusion assay on the basis of ␤-Gal complementation (47). As described previously (38), 293T cells were transfected with plasmids expressing HA, HAT, and the ␤-Gal ␣ subunit using the Fugene 6 reagent. At 48 h after transfection, the transfected 293T cells were detached using a nonenzymatic cell dissociation solution (Sigma) and washed with DMEM.…”

Mechanisms of Action of Novel Influenza A/M2 Viroporin Inhibitors Derived from Hexamethylene Amiloride

“…After viral entry into host cells, M2 conducts protons from acidic host-cell endosomes to the virion interior to allow for uncoating and release of viral RNA. M2 on host-cell endosomal membranes is also observed in some cases to conduct protons to elevate secretory vesicle pH, thereby delaying egress of nascent virion particles and preventing viral hemagglutinin from adopting a nonfunctional, low pH conformation (Sugrue et al, 1990;Alvarado-Facundo et al, 2015). The M2 ion channel of influenza B (B/M2) is a functional homolog of A/M2.…”

Influenza vaccines manufacturing in continuous cell lines: problems and solutions

“…It was shown that the M2 protein plays a major role in the preservation of the HA native conformation, preventing the premature HA conformational change to the low pH form during the virus transport to the cell surface [64]. Earlier we demonstrated [65] that the M2 protein of the virus A/Singapore/1/1957 CA (H2N2) has lower ion channel activity than the M2 of A/Switzerland/5389/1995 (H1N1).…”