Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses

Abstract Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.

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Influenza A viruses balance ER stress with host protein synthesis shutoff

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024681118 ◽

2021 ◽

Vol 118 (36) ◽

pp. e2024681118

Author(s):

Beryl Mazel-Sanchez ◽

Justyna Iwaszkiewicz ◽

Joao P. P. Bonifacio ◽

Filo Silva ◽

Chengyue Niu ◽

...

Keyword(s):

Viral Replication ◽

Er Stress ◽

Host Cell ◽

Messenger Rna ◽

Influenza A ◽

Nonstructural Protein ◽

Host Protein ◽

Stress Induction ◽

Nonstructural Protein 1

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

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Role of Initiating Nucleoside Triphosphate Concentrations in the Regulation of Influenza Virus Replication and Transcription

Journal of Virology ◽

10.1128/jvi.00627-08 ◽

2008 ◽

Vol 82 (14) ◽

pp. 6902-6910 ◽

Cited By ~ 27

Author(s):

Frank T. Vreede ◽

Hugh Gifford ◽

George G. Brownlee

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

De Novo ◽

Nucleoside Triphosphate ◽

Ribonucleoprotein Complexes ◽

Influenza Virus Replication ◽

High Concentrations

ABSTRACT The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3′-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

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Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses

Journal of Virology ◽

10.1128/jvi.02082-06 ◽

2007 ◽

Vol 81 (7) ◽

pp. 3058-3067 ◽

Cited By ~ 217

Author(s):

Christina Ehrhardt ◽

Thorsten Wolff ◽

Stephan Pleschka ◽

Oliver Planz ◽

Wiebke Beermann ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Glycogen Synthase ◽

Nonstructural Protein ◽

Akt Pathway ◽

Ns1 Protein ◽

Nonstructural Protein 1 ◽

Cell Death Program ◽

Pi3k Activation

ABSTRACT Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3β and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.

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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 RNA- and TRIM25-Binding Domains of Influenza Virus NS1 Protein Are Essential for Suppression of NLRP3 Inflammasome-Mediated Interleukin-1β Secretion

Journal of Virology ◽

10.1128/jvi.00120-16 ◽

2016 ◽

Vol 90 (8) ◽

pp. 4105-4114 ◽

Cited By ~ 45

Author(s):

Miyu Moriyama ◽

I-Yin Chen ◽

Atsushi Kawaguchi ◽

Takumi Koshiba ◽

Kyosuke Nagata ◽

...

Keyword(s):

Influenza Virus ◽

Nlrp3 Inflammasome ◽

Influenza A ◽

Rna Binding ◽

Nonstructural Protein ◽

Binding Domain ◽

Interleukin 1Β ◽

Ns1 Protein ◽

Caspase 1 ◽

Nonstructural Protein 1

ABSTRACTInflammasomes are cytosolic multimolecular protein complexes that stimulate the activation of caspase-1 and the release of mature forms of interleukin-1β (IL-1β) and IL-18. We previously demonstrated that the influenza A virus M2 protein stimulates IL-1β secretion following activation of the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. The nonstructural protein 1 (NS1) of influenza virus inhibits caspase-1 activation and IL-1β secretion. However, the precise mechanism by which NS1 inhibits IL-1β secretion remains unknown. Here, we showed that J774A.1 macrophages stably expressing the NS1 protein inhibited IL-1β secretion after infection with recombinant influenza virus lacking the NS1 gene. Coimmunoprecipitation assay revealed that the NS1 protein interacts with NLRP3. Importantly, the NS1 protein inhibited the NLRP3/ASC-induced single-speck formation required for full activation of inflammasomes. The NS1 protein of other influenza virus strains, including a recent pandemic strain, also inhibited inflammasome-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) were required for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results shed light on a mechanism by which the NS1 protein of influenza virus suppresses NLRP3 inflammasome-mediated IL-1β secretion.IMPORTANCEInnate immune sensing of influenza virus via pattern recognition receptors not only plays a key role in generating type I interferons but also triggers inflammatory responses. We previously demonstrated that the influenza A virus M2 protein activates the NLRP3 inflammasome, leading to the secretion of interleukin-1β (IL-1β) and IL-18 following the activation of caspase-1. Although the nonstructural protein 1 (NS1) of influenza virus inhibits IL-1β secretion, the precise mechanism by which it achieves this remains to be defined. Here, we demonstrate that the NS1 protein interacts with NLRP3 to suppress NLRP3 inflammasome activation. J774A.1 macrophages stably expressing the NS1 protein suppressed NLRP3-mediated IL-1β secretion. The NS1 RNA-binding domain (basic residues 38 and 41) and TRIM25-binding domain (acidic residues 96 and 97) are important for suppression of NLRP3 inflammasome-mediated IL-1β secretion. These results will facilitate the development of new anti-inflammatory drugs.

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Dual R108K and G189D Mutations in the NS1 Protein of A/H1N1 Influenza Virus Counteract Host Innate Immune Responses

Viruses ◽

10.3390/v13050905 ◽

2021 ◽

Vol 13 (5) ◽

pp. 905

Author(s):

Meng-Ting Huang ◽

Sen Zhang ◽

Ya-Nan Wu ◽

Wei Li ◽

Yu-Chang Li ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Nonstructural Protein ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Innate Immune ◽

Ns1 Protein ◽

H1n1 Influenza Virus ◽

Functional Sites ◽

Nonstructural Protein 1

Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV (A/California/07/2009 pH1N1) emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites; however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host–influenza NS1 protein interactions.

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In Vivo Imaging of Influenza Virus Infection in Immunized Mice

mBio ◽

10.1128/mbio.00714-17 ◽

2017 ◽

Vol 8 (3) ◽

Cited By ~ 11

Author(s):

Rita Czakó ◽

Leatrice Vogel ◽

Elaine W. Lamirande ◽

Kevin W. Bock ◽

Ian N. Moore ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Influenza A ◽

Imaging System ◽

Influenza Virus Infection ◽

Antigenic Drift ◽

Efficacy Evaluation ◽

Imaging Results ◽

Preclinical Efficacy

ABSTRACT Immunization is the cornerstone of seasonal influenza control and represents an important component of pandemic preparedness strategies. Using a bioluminescent reporter virus, we demonstrate the application of noninvasive in vivo imaging system (IVIS) technology to evaluate the preclinical efficacy of candidate vaccines and immunotherapy in a mouse model of influenza. Sequential imaging revealed distinct spatiotemporal kinetics of bioluminescence in groups of mice passively or actively immunized by various strategies that accelerated the clearance of the challenge virus at different rates and by distinct mechanisms. Imaging findings were consistent with conclusions derived from virus titers in the lungs and, notably, were more informative than conventional efficacy endpoints in some cases. Our findings demonstrate the reliability of IVIS as a qualitative approach to support preclinical evaluation of candidate medical countermeasures for influenza in mice. IMPORTANCE Influenza A viruses remain a persistent threat to public health. Vaccination and immunotherapy are effective countermeasures for the control of influenza but must contend with antigenic drift and the risk of resistance to antivirals. Traditional preclinical efficacy studies for novel vaccine and pharmaceutical candidates can be time-consuming and expensive and are inherently limited in scope. In vivo imaging approaches offer the potential to noninvasively track virus replication in real time in animal models. In this study, we demonstrate the utility of bioluminescent imaging for tracking influenza virus replication in the lungs of immunized mice and also identify important factors that may influence the accurate interpretation of imaging results. Our findings support the potential of IVIS approaches to enhance traditional preclinical efficacy evaluation of candidate vaccines and human monoclonal antibodies for the prevention and treatment of influenza.

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Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1

Journal of Virology ◽

10.1128/jvi.01875-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 9

Author(s):

C. Chauché ◽

A. Nogales ◽

H. Zhu ◽

D. Goldfarb ◽

A. I. Ahmad Shanizza ◽

...

Keyword(s):

Influenza Virus ◽

Immune Evasion ◽

Influenza A ◽

Nonstructural Protein ◽

Ns1 Protein ◽

Equine Influenza Virus ◽

Influenza A Viruses ◽

Equine Influenza ◽

Nonstructural Protein 1 ◽

Avian Origin

ABSTRACT Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstructural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually exclusive mechanisms: while the NS1 proteins of early EIVs block general gene expression by binding to cellular polyadenylation-specific factor 30 (CPSF30), NS1 proteins from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation. IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remain unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the molecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

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Broad influenza-specific CD8+ T-cell responses in humanized mice vaccinated with influenza virus vaccines

Blood ◽

10.1182/blood-2008-05-157016 ◽

2008 ◽

Vol 112 (9) ◽

pp. 3671-3678 ◽

Cited By ~ 43

Author(s):

Chun I. Yu ◽

Michael Gallegos ◽

Florentina Marches ◽

Gerard Zurawski ◽

Octavio Ramilo ◽

...

Keyword(s):

T Cells ◽

Influenza Virus ◽

Immune Responses ◽

Virus Vaccine ◽

Cd8 T Cells ◽

Influenza Virus Vaccine ◽

Preclinical Testing ◽

In Vivo Models ◽

Nonstructural Protein 1

The development of novel human vaccines would be greatly facilitated by the development of in vivo models that permit preclinical analysis of human immune responses. Here, we show that nonobese diabetic severe combined immunodeficiency (NOD/SCID) β2 microglobulin−/− mice, engrafted with human CD34+ hematopoietic progenitors and further reconstituted with T cells, can mount specific immune responses against influenza virus vaccines. Live attenuated trivalent influenza virus vaccine induces expansion of CD8+ T cells specific to influenza matrix protein (FluM1) and nonstructural protein 1 in blood, spleen, and lungs. On ex vivo exposure to influenza antigens, antigen-specific CD8+ T cells produce IFN-γ and express cell-surface CD107a. FluM1-specific CD8+ T cells can be also expanded in mice vaccinated with inactivated trivalent influenza virus vaccine. Expansion of antigen-specific CD8+ T cells is dependent on reconstitution of the human myeloid compartment. Thus, this humanized mouse model permits preclinical testing of vaccines designed to induce cellular immunity, including those against influenza virus. Furthermore, this work sets the stage for systematic analysis of the in vivo functions of human DCs. This, in turn, will allow a new approach to the rational design and preclinical testing of vaccines that cannot be tested in human volunteers.

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Impact of influenza virus during pregnancy: from disease severity to vaccine efficacy

Future Virology ◽

10.2217/fvl-2020-0024 ◽

2020 ◽

Vol 15 (7) ◽

pp. 441-453

Author(s):

Ana Vazquez-Pagan ◽

Rebekah Honce ◽

Stacey Schultz-Cherry

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Immune Responses ◽

Pregnant Women ◽

Disease Severity ◽

Influenza A ◽

Antiviral Treatment ◽

Influenza Virus Infection ◽

Severe Influenza ◽

The Impact

Pregnant women are among the individuals at the highest risk for severe influenza virus infection. Infection of the mother during pregnancy increases the probability of adverse fetal outcomes such as small for gestational age, preterm birth and fetal death. Animal models of syngeneic and allogeneic mating can recapitulate the increased disease severity observed in pregnant women and are used to define the mechanism(s) of that increased severity. This review focuses on influenza A virus pathogenesis, the unique immunological landscape during pregnancy, the impact of maternal influenza virus infection on the fetus and the immune responses at the maternal–fetal interface. Finally, we summarize the importance of immunization and antiviral treatment in this population and highlight issues that warrant further investigation.

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