The Matrix Gene Segment Destabilizes the Acid and Thermal Stability of the Hemagglutinin of Pandemic Live Attenuated Influenza Virus Vaccines

O’Donnell, Christopher D.; Vogel, Leatrice; Matsuoka, Yumiko; Jin, Hong; Subbarao, Kanta

doi:10.1128/jvi.01107-14

Cited by 33 publications

(35 citation statements)

References 80 publications

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“…As previously reported, CA/09 virus fuses at pH 5.2 to 5.5 ( Fig. 2B) (27,28), while VN/1203 and WSN viruses fuse at pH 5.9 to 6.0 ( Fig. 2B) (29), suggesting that in Raw264.7 macrophages, CA/09 would not encounter an optimal acidic environment with a pH of 5.0 to 5.5 until the lysosomal compartment, where the virus can encounter activated proteases that degrade the virus.…”

supporting

confidence: 52%

Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function

Marvin

Russier

Huerta

et al. 2017

J Virol

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Whether influenza virus replication in macrophages is productive or abortive has been a topic of debate. Utilizing a panel of 28 distinct human, avian, and swine influenza viruses, we found that only a small subset can overcome cellular blocks to productively replicate in murine and primary human macrophages. Murine macrophages have two cellular blocks. The first block is during viral entry, where virions with relatively acid-stable hemagglutinin (HA) proteins are rendered incapable of pH-induced triggering for membrane fusion, resulting in lysosomal degradation. The second block is downstream of viral replication but upstream of late protein synthesis. In contrast, primary human macrophages only have one cellular block that occurs after late protein synthesis. To determine the impact of abortive replication at different stages of the viral life cycle or productive replication on macrophage function, we assessed cytotoxicity, nitric oxide or reactive oxygen species production, and phagocytosis. Intriguingly, productive viral replication decreased phagocytosis of IgG-opsonized bioparticles and Fc receptor CD16 and CD32 surface levels, a function, to our knowledge, never before reported for an RNA virus. These data suggest that replication in macrophages affects cellular function and plays an important role in pathogenesis during infection in vivo.IMPORTANCE Macrophages are a critical first line of defense against respiratory pathogens. Thus, understanding how viruses evade or exploit macrophage function will provide greater insight into viral pathogenicity and antiviral responses. We previously showed that only a subset of highly pathogenic avian (HPAI) H5N1 influenza virus strains could productively replicate in murine macrophages through a hemagglutinin (HA)-mediated mechanism. These studies expand upon this work and demonstrate that productive replication is not specific to unique HPAI H5N1 viruses; an H1N1 strain (A/WSN/33) can also replicate in macrophages. Importantly, we identify two cellular blocks limiting replication that can be overcome by an avian-like pH of activation for nuclear entry and a yet-to-be-identified mechanism(s) to overcome a postnuclear entry block. Overcoming these blocks reduces the cell's ability to phagocytose IgG-opsonized bioparticles by decreasing Fc receptor surface levels, a mechanism previously thought to occur during bacterial and DNA viral infections.KEYWORDS influenza, macrophage, replication M acrophages are one of the first lines of defense against infection. They are poised to secrete large amounts of cytokines, orchestrate the adaptive immune system, and clear infected and dying cells to aid in recovery (1). Further, alveolar macrophages are essential in preventing respiratory failure after infection (2) and for preventing bacterial superinfections during influenza infection (3). However, such responses must be tightly regulated, as excessive cytokine levels contribute to immunopathology and disease severity during infection (4-6).

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supporting

confidence: 52%

Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function

Marvin

Russier

Huerta

et al. 2017

J Virol

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“…The data presented here provide direct evidence that M2 can also protect HA from conformational changes in human H1N1 strains that lack the polybasic cleavage motif. Recently, O'Donnell et al reported that M2 function affects the acid and heat stability of HA proteins of some pandemic strains, including (H1N1)pdm09 (70), but the mechanism was not directly demonstrated. Although M2 enhancement of MX HA function is less pronounced than that of an acid-sensitive H7 HA (9-11), our data with two different H1N1 HA proteins indicate that M2 may promote infection by viruses with non-H7 subtype HA in a strain-dependent manner.…”

Section: Discussionmentioning

confidence: 99%

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

J Virol

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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...

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“…The M gene encodes both the M1 matrix protein and the M2 ion channel. The HA destabilizing property of the M gene in the live attenuated vaccine backbone A/Ann Arbor/60 (H2N2) has been linked to M2 ion channel activity (36), which is required to prevent HA inactivation during trafficking in the mildly acidic secretory pathway (19). The M1 matrix protein contributes to virus assembly, budding, and morphology (37), which may also affect transmissibility (38).…”

Section: Discussionmentioning

confidence: 99%

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

Russier

Yang

Rehg

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

109

210

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Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5–6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2–5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009–2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.

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The Matrix Gene Segment Destabilizes the Acid and Thermal Stability of the Hemagglutinin of Pandemic Live Attenuated Influenza Virus Vaccines

Cited by 33 publications

References 80 publications

Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function

Influenza Virus Overcomes Cellular Blocks To Productively Replicate, Impacting Macrophage Function

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

Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

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