Influenza A viruses balance ER stress with host protein synthesis shutoff

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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Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1920582117 ◽

2020 ◽

Vol 117 (12) ◽

pp. 6550-6558 ◽

Cited By ~ 1

Author(s):

Jae-Hyun Cho ◽

Baoyu Zhao ◽

Jie Shi ◽

Nowlan Savage ◽

Qingliang Shen ◽

...

Keyword(s):

Molecular Recognition ◽

Influenza A ◽

Nonstructural Protein ◽

Conformational Dynamics ◽

Relaxation Dispersion ◽

Binding Kinetics ◽

Host Protein ◽

Dynamics Simulation ◽

Nonstructural Protein 1 ◽

Strain Dependent

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85β subunit. Here we present the mechanism underlying the molecular recognition of the p85β subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85β of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85β. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1EDexists in a dynamic equilibrium between p85β-binding–competent and –incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1EDproteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85β. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1EDcan result in the faster hijacking of p85β compared to Ud NS1ED, although the former has a lower affinity to p85β than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

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Repurposing Sigma-1 Receptor Ligands for COVID-19 Therapy?

Frontiers in Pharmacology ◽

10.3389/fphar.2020.582310 ◽

2020 ◽

Vol 11 ◽

Author(s):

José Miguel Vela

Keyword(s):

Stress Response ◽

Viral Replication ◽

Er Stress ◽

Host Cell ◽

Drug Repurposing ◽

Host Protein ◽

Unique Challenge ◽

Sigma 1 Receptor ◽

Sigma 1

Outbreaks of emerging infections, such as COVID-19 pandemic especially, confront health professionals with the unique challenge of treating patients. With no time to discover new drugs, repurposing of approved drugs or in clinical development is likely the only solution. Replication of coronaviruses (CoVs) occurs in a modified membranous compartment derived from the endoplasmic reticulum (ER), causes host cell ER stress and activates pathways to facilitate adaptation of the host cell machinery to viral needs. Accordingly, modulation of ER remodeling and ER stress response might be pivotal in elucidating CoV-host interactions and provide a rationale for new therapeutic, host-based antiviral approaches. The sigma-1 receptor (Sig-1R) is a ligand-operated, ER membrane-bound chaperone that acts as an upstream modulator of ER stress and thus a candidate host protein for host-based repurposing approaches to treat COVID-19 patients. Sig-1R ligands are frequently identified in in vitro drug repurposing screens aiming to identify antiviral compounds against CoVs, including severe acute respiratory syndrome CoV-2 (SARS-CoV-2). Sig-1R regulates key mechanisms of the adaptive host cell stress response and takes part in early steps of viral replication. It is enriched in lipid rafts and detergent-resistant ER membranes, where it colocalizes with viral replicase proteins. Indeed, the non-structural SARS-CoV-2 protein Nsp6 interacts with Sig-1R. The activity of Sig-1R ligands against COVID-19 remains to be specifically assessed in clinical trials. This review provides a rationale for targeting Sig-1R as a host-based drug repurposing approach to treat COVID-19 patients. Evidence gained using Sig-1R ligands in unbiased in vitro antiviral drug screens and the potential mechanisms underlying the modulatory effect of Sig-1R on the host cell response are discussed. Targeting Sig-1R is not expected to reduce dramatically established viral replication, but it might interfere with early steps of virus-induced host cell reprogramming, aid to slow down the course of infection, prevent the aggravation of the disease and/or allow a time window to mature a protective immune response. Sig-1R-based medicines could provide benefit not only as early intervention, preventive but also as adjuvant therapy.

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

Nature Communications ◽

10.1038/s41467-019-12632-5 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Miyu Moriyama ◽

Takumi Koshiba ◽

Takeshi Ichinohe

Keyword(s):

Mitochondrial Dna ◽

Influenza Virus ◽

Immune Responses ◽

Virus Replication ◽

Influenza A ◽

Nonstructural Protein ◽

Encephalomyocarditis Virus ◽

Dependent Manner ◽

Nonstructural Protein 1

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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Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein: Striking Differences between Different Virus Subtypes

Journal of Virology ◽

10.1128/jvi.01714-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5995-6006 ◽

Cited By ~ 134

Author(s):

Krister Melén ◽

Leena Kinnunen ◽

Riku Fagerlund ◽

Niina Ikonen ◽

Karen Y. Twu ◽

...

Keyword(s):

Host Cell ◽

Influenza A Virus ◽

Influenza A ◽

Rna Binding ◽

Nonstructural Protein ◽

Ns1 Protein ◽

Nonstructural Protein 1 ◽

Importin Α ◽

Localization Signal ◽

Ns1a Protein

ABSTRACT Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin α isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin α/β pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.

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Relative contribution of nonstructural protein 1 in dengue pathogenesis

Journal of Experimental Medicine ◽

10.1084/jem.20191548 ◽

2020 ◽

Vol 217 (9) ◽

Cited By ~ 2

Author(s):

Pei Xuan Lee ◽

Donald Heng Rong Ting ◽

Clement Peng Hee Boey ◽

Eunice Tze Xin Tan ◽

Janice Zuo Hui Chia ◽

...

Keyword(s):

Nonstructural Protein ◽

Critical Role ◽

Health Concern ◽

Relative Contribution ◽

Nonstructural Protein 1 ◽

Structural Region ◽

Strain Dependent ◽

Highly Virulent

Dengue is a major public health concern in the tropical and subtropical world, with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of subunit vaccine approaches, and nonstructural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non–mouse-adapted DENV2 strains that are highly virulent. Exogenous administration of purified NS1 also failed to worsen in vivo vascular leakage in sublethally infected mice. Neither method of NS1 immune neutralization changed the disease outcome of a chimeric strain expressing a vascular leak-potent NS1. Instead, virus chimerization involving the prME structural region indicated that these proteins play a critical role in driving in vivo fitness and virulence of the virus, through induction of key proinflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain dependent, which warrants reevaluation of NS1 as a universal dengue vaccine candidate.

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Identification and in vivo Efficacy Assessment of Approved Orally Bioavailable Human Host Protein-Targeting Drugs With Broad Anti-influenza A Activity

Frontiers in Immunology ◽

10.3389/fimmu.2019.01097 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 6

Author(s):

Theresa Enkirch ◽

Svenja Sauber ◽

Danielle E. Anderson ◽

Esther S. Gan ◽

Dimitar Kenanov ◽

...

Keyword(s):

Influenza A ◽

Protein Targeting ◽

Host Protein ◽

In Vivo Efficacy ◽

Human Host ◽

Efficacy Assessment ◽

Orally Bioavailable

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Viruses harness YxxØ motif to interact with host AP2M1 for replication: A vulnerable broad-spectrum antiviral target

Science Advances ◽

10.1126/sciadv.aba7910 ◽

2020 ◽

Vol 6 (35) ◽

pp. eaba7910

Author(s):

Shuofeng Yuan ◽

Hin Chu ◽

Jingjing Huang ◽

Xiaoyu Zhao ◽

Zi-Wei Ye ◽

...

Keyword(s):

Broad Spectrum ◽

Influenza A ◽

Host Protein ◽

Influenza A Viruses ◽

Protein Protein Interaction ◽

Enterovirus A71 ◽

Evolutionarily Conserved ◽

Immunodeficiency Virus

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.

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