Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus

ABSTRACT Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics, representing a serious public health concern. It has been described that one mechanism used by some IAV strains to escape the host innate immune responses and modulate virus pathogenicity involves the ability of the PA-X and NS1 proteins to inhibit the host protein synthesis in infected cells. It was reported that for the 2009 pandemic H1N1 IAV (pH1N1) only the PA-X protein had this inhibiting capability, while the NS1 protein did not. In this work, we have evaluated, for the first time, the combined effect of PA-X- and NS1-mediated inhibition of general gene expression on virus pathogenesis, using a temperature-sensitive, live-attenuated 2009 pandemic H1N1 IAV (pH1N1 LAIV). We found that viruses containing PA-X and NS1 proteins that simultaneously have (PAWT +/NS1MUT +) or do not have (PAMUT −/NS1WT −) the ability to block host gene expression showed reduced pathogenicity in vivo. However, a virus where the ability to inhibit host protein expression was switched between PA-X and NS1 (PAMUT −/NS1MUT +) presented pathogenicity similar to that of a virus containing both wild-type proteins (PAWT +/NS1WT −). Our findings suggest that inhibition of host protein expression is subject to a strict balance, which can determine the successful progression of IAV infection. Importantly, knowledge obtained from our studies could be used for the development of new and more effective vaccine approaches against IAV. IMPORTANCE Influenza A viruses (IAVs) are one of the most common causes of respiratory infections in humans, resulting in thousands of deaths annually. Furthermore, IAVs can cause unpredictable pandemics of great consequence when viruses not previously circulating in humans are introduced into humans. The defense machinery provided by the host innate immune system limits IAV replication; however, to counteract host antiviral activities, IAVs have developed different inhibition mechanisms, including prevention of host gene expression mediated by the viral PA-X and NS1 proteins. Here, we provide evidence demonstrating that optimal control of host protein synthesis by IAV PA-X and/or NS1 proteins is required for efficient IAV replication in the host. Moreover, we demonstrate the feasibility of genetically controlling the ability of IAV PA-X and NS1 proteins to inhibit host immune responses, providing an approach to develop more effective vaccines to combat disease caused by this important respiratory pathogen.

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Modulation of Innate Immune Responses by the Influenza A NS1 and PA-X Proteins

Viruses ◽

10.3390/v10120708 ◽

2018 ◽

Vol 10 (12) ◽

pp. 708 ◽

Cited By ~ 17

Author(s):

Aitor Nogales ◽

Luis Martinez-Sobrido ◽

David Topham ◽

Marta DeDiego

Keyword(s):

Immune Responses ◽

Influenza A ◽

Defense Mechanisms ◽

Synergistic Effects ◽

Viral Proteins ◽

Economic Problem ◽

Innate Immune ◽

Host Protein ◽

Innate Immune Responses ◽

Influenza A Viruses

Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection.

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NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

Journal of Virology ◽

10.1128/jvi.01039-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9693-9711 ◽

Cited By ~ 21

Author(s):

Marta L. DeDiego ◽

Aitor Nogales ◽

Kris Lambert-Emo ◽

Luis Martinez-Sobrido ◽

David J. Topham

Keyword(s):

Immune Responses ◽

Influenza A ◽

Innate Immune ◽

Host Protein ◽

Ns1 Protein ◽

Innate Immune Responses ◽

Virus Surveillance ◽

Dsrna Binding ◽

The Subject ◽

Host Protein Synthesis

ABSTRACT Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. IMPORTANCE Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN sensitivity, and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus.

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NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses

Journal of Virology ◽

10.1128/jvi.01930-16 ◽

2016 ◽

Vol 91 (5) ◽

Cited By ~ 22

Author(s):

Aitor Nogales ◽

Luis Martinez-Sobrido ◽

David J. Topham ◽

Marta L. DeDiego

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Immune Responses ◽

Influenza A ◽

Innate Immune ◽

Temperature Sensitive ◽

Ns1 Protein ◽

Innate Immune Responses ◽

Influenza A Viruses ◽

Virus Surveillance

ABSTRACT Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo.

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Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses

Journal of Virology ◽

10.1128/jvi.00721-17 ◽

2017 ◽

Vol 91 (17) ◽

Cited By ~ 20

Author(s):

Amelia M. Clark ◽

Aitor Nogales ◽

Luis Martinez-Sobrido ◽

David J. Topham ◽

Marta L. DeDiego

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Amino Acid ◽

Proinflammatory Cytokines ◽

Influenza A ◽

Host Gene ◽

Pandemic H1n1 ◽

Ns1 Protein ◽

Host Gene Expression ◽

Mouse Lungs

ABSTRACT In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up to the present. It was previously shown that the NS1 protein from the 2009 pandemic H1N1 (pH1N1) virus is not able to inhibit general gene expression. However, currently circulating pH1N1 viruses have evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) that allow the NS1 protein of contemporary pH1N1 strains to inhibit host gene expression, which correlates with its ability to interact with CPSF30. Infection with a recombinant pH1N1 virus encoding these 6 amino acid changes (pH1N1/NSs-6mut) induced lower levels of proinflammatory cytokines, resulting in viral attenuation in vivo. This might represent an adaptation of pH1N1 virus to humans.

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994. AS03-Adjuvanted H5N1 Avian Influenza Vaccine Modulates Early Innate Immune Signatures in Peripheral Blood Mononuclear Cells

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.831 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S295-S296

Author(s):

Leigh Howard ◽

Johannes Goll ◽

Travis Jensen ◽

Heather Hill ◽

Casey Gelber ◽

...

Keyword(s):

Gene Expression ◽

Immune Responses ◽

Influenza A ◽

Mononuclear Cells ◽

Immune Cell ◽

Innate Immune ◽

Peripheral Blood Mononuclear ◽

Innate Immune Signaling ◽

Blood Mononuclear Cells ◽

Research Grant

Abstract Background Influenza A/H5N1 vaccines have been poorly immunogenic. Addition of Adjuvant System 03 (AS03) markedly enhances immune responses, but the mechanisms of this enhancement are unclear. Methods We compared gene expression in peripheral blood mononuclear cells (PBMCs) between recipients of AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 vaccine on days 1, 3, 7, and 28 postvaccination. We used a systems vaccinology approach to assess functional classifications of differentially expressed (DE) genes between the two vaccine groups, identify DE genes that correlate with serologic responses, and compare these findings with previous cell-specific assessments. Results AS03-adjuvanted vaccine induced the strongest differential gene expression signals on day 1 after vaccination (Figure 1). Multiple innate immune signaling pathways were activated, including the interferon, JAK-STAT, and TNF pathways, and FC gamma receptor (Fc_R) mediated phagocytosis. Immune pathways specific for antigen processing and presentation and influenza A responses were also enriched. Early differential expression of several signal transduction (day 1) and immunoglobulin (day 7) genes were predictive of peak HAI titer (Figure 2). Compared with cell-specific responses, DE gene, and immunologic pathways of PBMCs were most similar to innate immune cell subsets. However, several pathways were unique to PBMCs, and several cell-type-specific pathways, particularly from neutrophils, were absent in PBMCs (Figure 3). Conclusion Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early differential regulation of multiple innate immune signaling pathways and enrichment of pathways involved in antigen presentation and influenza immune responses. Early expression of several genes was associated with peak HAI responses, suggesting a potential role for application of these signatures in earlier determination of vaccine responses. While PBMC and immune cell-specific results shared key innate immune signals, unique signals were identified by either approach. Disclosures L. Howard, Pfizer: Grant Investigator, Grant recipient. C. B. Creech, Pfizer: Grant Investigator, Research grant. Novartis: Grant Investigator, Research grant. K. Edwards, Novartis: Grant Investigator, Research grant. Novartis: Scientific Advisor, Consulting fee.

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Differential Modulation of Innate Immune Responses in Human Primary Cells by Influenza A Viruses Carrying Human or Avian Nonstructural Protein 1

Journal of Virology ◽

10.1128/jvi.00999-19 ◽

2019 ◽

Vol 94 (1) ◽

Cited By ~ 1

Author(s):

Paula L. Monteagudo ◽

Raquel Muñoz-Moreno ◽

Miguel Fribourg ◽

Uma Potla ◽

Ignacio Mena ◽

...

Keyword(s):

Immune Responses ◽

Influenza A ◽

Nonstructural Protein ◽

Severe Disease ◽

Innate Immune ◽

Innate Immune Responses ◽

Influenza A Viruses ◽

Recombinant Viruses ◽

Nonstructural Protein 1 ◽

Human Primary Cells

ABSTRACT The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-β) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans. IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.

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The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner

Journal of Virology ◽

10.1128/jvi.02312-20 ◽

2021 ◽

Author(s):

Rachel Emily Levene ◽

Shailab D. Shrestha ◽

Marta Maria Gaglia

Keyword(s):

Immune Responses ◽

Influenza A Virus ◽

Half Life ◽

Influenza A ◽

Pandemic H1n1 ◽

Host Gene Expression ◽

X Protein ◽

Protein Levels ◽

H1n1 Strain ◽

Host Shutoff

The influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection. IMPORTANCE The PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

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Influenza A Virus Protein PA-X Contributes to Viral Growth and Suppression of the Host Antiviral and Immune Responses

Journal of Virology ◽

10.1128/jvi.00319-15 ◽

2015 ◽

Vol 89 (12) ◽

pp. 6442-6452 ◽

Cited By ~ 59

Author(s):

Tsuyoshi Hayashi ◽

Leslie A. MacDonald ◽

Toru Takimoto

Keyword(s):

Protein Synthesis ◽

Influenza Virus ◽

Immune Responses ◽

Influenza A Virus ◽

Influenza A ◽

Host Protein ◽

Viral Growth ◽

Host Shutoff ◽

Host Protein Synthesis ◽

Host Antiviral Response

ABSTRACTInfluenza virus infection causes global inhibition of host protein synthesis in infected cells. This host shutoff is thought to allow viruses to escape from the host antiviral response, which restricts virus replication and spread. Although the mechanism of host shutoff is unclear, a novel viral protein expressed by ribosomal frameshifting, PA-X, was found to play a major role in influenza virus-induced host shutoff. However, little is known about the impact of PA-X expression on currently circulating influenza A virus pathogenicity and the host antiviral response. In this study, we rescued a recombinant influenza A virus, A/California/04/09 (H1N1, Cal), containing mutations at the frameshift motif in the polymerase PA gene (Cal PA-XFS). Cal PA-XFS expressed significantly less PA-X than Cal wild type (WT). Cal WT, but not Cal PA-XFS, induced degradation of host β-actin mRNA and suppressed host protein synthesis, supporting the idea that PA-X induces host shutoff via mRNA decay. Moreover, Cal WT inhibited beta interferon (IFN-β) expression and replicated more rapidly than Cal PA-XFS in human respiratory cells. Mice infected with Cal PA-XFS had significantly lower levels of viral growth and greater expression of IFN-β mRNA in their lungs than mice infected with Cal WT. Importantly, more antihemagglutinin and neutralizing antibodies were produced in Cal PA-XFS-infected mice than in Cal WT-infected mice, despite the lower level of virus replication in the lungs. Our data indicate that PA-X of the pandemic H1N1 virus has a strong impact on viral growth and the host innate and acquired immune responses to influenza virus.IMPORTANCEVirus-induced host protein shutoff is considered to be a major factor allowing viruses to evade innate and acquired immune recognition. We provide evidence that the 2009 H1N1 influenza A virus protein PA-X plays a role in virus replication and inhibition of host antiviral response by means of its host protein synthesis shutoff activity bothin vitroandin vivo. We also demonstrated that, while the growth of Cal PA-XFS was attenuated in the lungs of infected animals, this mutant induced a stronger humoral response than Cal WT. Our findings clearly highlight the importance of PA-X in counteracting the host innate and acquired immune responses to influenza virus, an important global pathogen. This work demonstrates that inhibition of PA-X expression in influenza virus vaccine strains may provide a novel way of safely attenuating viral growth while inducing a more robust immune response.

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The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner

10.1101/2020.08.27.271320 ◽

2020 ◽

Author(s):

Rachel Emily Levene ◽

Shailab D. Shrestha ◽

Marta Maria Gaglia

Keyword(s):

Immune Responses ◽

Influenza A Virus ◽

Half Life ◽

Influenza A ◽

Pandemic H1n1 ◽

Host Gene Expression ◽

X Protein ◽

Protein Levels ◽

H1n1 Strain ◽

Host Shutoff

ABSTRACTThe influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection.IMPORTANCEThe PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

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Inhibition of host innate immune responses and pathogenicity of recombinant Newcastle disease viruses expressing NS1 genes of influenza A viruses

Journal of General Virology ◽

10.1099/vir.0.021766-0 ◽

2010 ◽

Vol 91 (8) ◽

pp. 1996-2001 ◽

Cited By ~ 5

Author(s):

Shin-Hee Kim ◽

Siba K. Samal

Keyword(s):

Immune Responses ◽

Newcastle Disease ◽

Influenza A ◽

Rna Binding ◽

Mutational Analysis ◽

Influenza Viruses ◽

Innate Immune ◽

Ns1 Protein ◽

Innate Immune Responses ◽

Influenza A Viruses

The NS1 protein has been associated with the virulence of influenza A viruses. To evaluate the role of the NS1 protein in pathogenicity of pandemic H5N1 avian influenza and H1N1 2009 influenza viruses, recombinant Newcastle disease viruses (rNDVs) expressing NS1 proteins were generated. Expression of the NS1 proteins resulted in inhibition of host innate immune responses (beta interferon and protein kinase R production). In addition, the NS1 proteins were localized predominantly in the nucleus of virus-infected cells. Consequently, expression of the NS1 protein contributed to an increase in pathogenicity of rNDV in chickens. In particular, mutational analysis of H5N1 NS1 protein indicated that both the RNA-binding and effector domains affect virus pathogenicity synergistically. Our study also demonstrated that expression of H1N1/09 NS1 resulted in enhanced replication of rNDV in human cells, indicating that function of the NS1 proteins can be host-species-specific.

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