Structural Stability of Influenza A(H1N1)pdm09 Virus Hemagglutinins

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...

Section: Discussionmentioning

confidence: 66%

mentioning

confidence: 89%

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

Alvarado-Facundo

Gao

Ribas‐Aparicio

et al. 2015

“…Residue 84 of seal11 NA was mutated (Asn84Gln) to remove the potential glycosylation site (NNT to QNT). Secreted proteins were recovered from the culture supernatant and purified by metal affinity chromatography and size exclusion chromatography (SEC) as described previously (29,(32)(33)(34)(35). For structural analyses, the proteins were further subjected to trypsin cleavage and SEC.…”

Section: Methodsmentioning

confidence: 99%

Structural and Functional Analysis of Surface Proteins from an A(H3N8) Influenza Virus Isolated from New England Harbor Seals

Yang

Nguyen

Carney

et al. 2015

Self Cite

In late 2011, an A(H3N8) influenza virus infection resulted in the deaths IMPORTANCECross-species transmission of zoonotic influenza viruses increases public health concerns. Here, we report a molecular and structural study of the major surface proteins from an A(H3N8) influenza virus isolated from New England harbor seals. The results improve our understanding of these viruses as they evolve and provide important information to aid ongoing risk assessment analyses as these zoonotic influenza viruses continue to circulate and adapt to new hosts. Influenza A viruses (IAVs) are classified into subtypes according to the serological reactivity of their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (1, 2). While 16 HA (H1 to H16) and 9 NA (N1 to N9) subtypes have been identified in wild aquatic birds in the last 100 years, only 3 have adapted to the human population, resulting in four pandemics: H1N1 in 1918 and 2009, H2N2 in 1957, and H3N2 in 1968 (3-6). Although aquatic birds are believed to be the natural reservoir for influenza viruses (7), two novel subtypes of influenza A viruses, H17N10 and H18N11, were recently described in New World bats, which thus may be an alternate reservoir of influenza viruses in nature (8,9).Since the first isolation of H1N1 IAV in swine (10) and the subsequent observation that ferrets were susceptible to IAV (11), it became evident that multiple IAV subtypes of either avian or human descent can infect a number of mammalian species (1). Indeed, over the last decade, the perception and impact of influenza virus infections in animals have changed dramatically as a result of a number of recent outbreaks in poultry involving viruses from the H5, H7, and H9 subtypes that resulted in Ͼ1,000 human infections, as well as the swine origin of the recent A(H1N1)pdm09 pandemic virus (4,(12)(13)(14). This has prompted some governments to invest heavily in global surveillance, research, and capacity building. Public health officials now have a keen interest in all IAVs, but particularly in whether animals, such as pigs, that are closely associated with humans can represent a possible pathway for increased interspecies virus adaptation and transmission.Similar to the infection of pigs, IAV (i.e., H7N7, H4N5, H3N3, H13N2, H13N9, and H4N6 subtypes) and influenza B virus infections have been detected in marine mammals, including seals, whales, and sea otters (15)(16)(17)(18)(19)(20)(21)(22). In November 2011, a marine mammal "unusual mortality event" was declared for Maine, New Hampshire, and northern Massachusetts in response to a large number of deaths in the New England harbor seal population. Sequence analysis revealed that an avian-like H3N8 influenza A virus, A/harbor seal/Massachusetts/1/2011 (seal11), was the cause of the outbreak (23). Although A(H3N8), usually found in avian, canine, equine, and swine hosts (24-27), had been isolated from a seal prior to this event (28), the high mortality observed increased interest from public health officials.Here, we focus our...

“…The HA from the pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] has been reported to be less stable than the HAs of other seasonal influenza A virus strains (28)(29)(30)(31), a feature that likely contributes to challenges in the production of A(H1N1)pdm09 vaccines (28,29). Moreover, it was recently reported that the currently circulating A(H1N1)pdm09 virus is acquiring mutations that improve HA stability (31,32) and may therefore improve viral fitness.…”

Section: Influenza Virus Hemagglutinin (Ha) Is the Principal Antigen mentioning

confidence: 99%

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

Wang

DeFeo

Alvarado-Facundo

et al. 2015

Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. IMPORTANCE Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains.Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well as viral adaptation to hosts. HA from the pandemic 2009 H1N1 influenza A virus is less stable than other recent seasonal influenza virus HAs, but the molecular interactions that contribute to HA stability are not fully understood. Here we identify molecular interactions between specific residues in the surface and transmembrane subunits of HA that help regulate the HA conformational changes needed for HA stability and virus entry. These findings contribute to our understanding of the molecular mechanisms controlling HA function and antigen stability. T he influenza virus envelope protein, hemagglutinin (HA), is organized as a noncovalently associated homotrimer on the viral surface. Each monomer of HA is posttranslationally cleaved into HA1 and HA2 subunits that are disulfide linked. The HA trimer consists of a large membrane-distal, globular domain formed only by HA1 and an elongated membrane-proximal stem domain comprised of HA2 and the N-and C-terminal segments of HA1. HA1 mediates virus binding to cell surface sialic acid receptors to initiate viral e...