A Novel Role for PDZ-Binding Motif of Influenza A Virus Nonstructural Protein 1 in Regulation of Host Susceptibility to Postinfluenza Bacterial Superinfections

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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Modification of Nonstructural Protein 1 of Influenza A Virus by SUMO1

Journal of Virology ◽

10.1128/jvi.00877-10 ◽

2010 ◽

Vol 85 (2) ◽

pp. 1086-1098 ◽

Cited By ~ 50

Author(s):

K. Xu ◽

C. Klenk ◽

B. Liu ◽

B. Keiner ◽

J. Cheng ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Nonstructural Protein ◽

Nonstructural Protein 1

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Influenza A Virus Protein NS1 Exhibits Strain-Independent Conformational Plasticity

Journal of Virology ◽

10.1128/jvi.00917-19 ◽

2019 ◽

Vol 93 (21) ◽

Cited By ~ 2

Author(s):

Sayantan Mitra ◽

Dilip Kumar ◽

Liya Hu ◽

Banumathi Sankaran ◽

Mahdi Muhammad Moosa ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Nonstructural Protein ◽

Major Group ◽

Cellular Proteins ◽

Conformational Preference ◽

Nonstructural Protein 1 ◽

Open Conformation ◽

Cellular Factors ◽

Strain Dependent

ABSTRACT Influenza A virus (IAV) nonstructural protein 1 (NS1), a potent antagonist of the host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR, with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of the ED with respect to the RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 [H6N6-NS1(E71)], and the LR deletion mutant H6N6-NS1(Δ80-84/E71) mimicking the major group H5N1-NS1 exhibit “open,” “semiopen,” and “closed” conformations, respectively, suggesting that NS1 exhibits a strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an open conformation similar to that of the minor group of H5N1-NS1 [H5N1-NS1(G71)]. We also show that H6N6-NS1(Δ80-84/E71) under a different crystallization condition and H6N6-NS1(Δ80-84/G71) also exhibit open conformations, suggesting that NS1 can adopt an open conformation irrespective of E or G at the 71st position. Our single-molecule fluorescence resonance energy transfer (FRET) analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in an open conformation. Further, our coimmunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinities. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection. IMPORTANCE IAV is responsible for several pandemics over the last century and continues to infect millions annually. The frequent rise in drug-resistant strains necessitates exploring novel targets for developing antiviral drugs that can reduce the global burden of influenza infection. Because of its critical role in the replication and pathogenesis of IAV, nonstructural protein 1 (NS1) is a potential target for developing antivirals. Previous studies suggested that NS1 adopts strain-dependent “open,” “semiopen,” and “closed” conformations. Here we show, based on three crystal structures, that NS1 irrespective of strain differences can adopt an open conformation. We further show that NS1 from different strains primarily exists in an open conformation in solution and binds to cellular proteins with a similar affinity. Together, our findings suggest that conformational polymorphism facilitated by a flexible linker is intrinsic to NS1, and this may be the underlying factor allowing NS1 to bind several cellular factors during IAV replication.

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Epigenetic Modification Is Regulated by the Interaction of Influenza A Virus Nonstructural Protein 1 with the De Novo DNA Methyltransferase DNMT3B and Subsequent Transport to the Cytoplasm for K48-Linked Polyubiquitination

Journal of Virology ◽

10.1128/jvi.01587-18 ◽

2019 ◽

Vol 93 (7) ◽

Cited By ~ 2

Author(s):

Shi Liu ◽

Li Liu ◽

Gang Xu ◽

Zhongying Cao ◽

Qing Wang ◽

...

Keyword(s):

Dna Methylation ◽

Influenza A Virus ◽

Influenza A ◽

Dna Methyltransferase ◽

De Novo ◽

Epigenetic Modification ◽

Nonstructural Protein ◽

Ns1 Protein ◽

Nonstructural Protein 1 ◽

Stat Signaling

ABSTRACT The influenza virus nonstructural protein 1 (NS1) is a nonstructural protein that plays a major role in antagonizing host interferon responses during infection. However, a clear role for the NS1 protein in epigenetic modification has not been established. In this study, NS1 was found to regulate the expression of some key regulators of JAK-STAT signaling by inhibiting the DNA methylation of their promoters. Furthermore, DNA methyltransferase 3B (DNMT3B) is responsible for this process. Upon investigating the mechanisms underlying this event, NS1 was found to interact with DNMT3B but not DNMT3A, leading to the dissociation of DNMT3B from the promoters of the corresponding genes. In addition, the interaction between NS1 and DNMT3B changed the localization of DNMT3B from the nucleus to the cytosol, resulting in K48-linked ubiquitination and degradation of DNMT3B in the cytosol. We conclude that NS1 interacts with DNMT3B and changes its localization to mediate K48-linked polyubiquitination, subsequently contributing to the modulation of the expression of JAK-STAT signaling suppressors. IMPORTANCE The nonstructural protein 1 (NS1) of the influenza A virus (IAV) is a multifunctional protein that counters cellular antiviral activities and is a virulence factor. However, the involvement of NS1 in DNA methylation during IAV infection has not been established. Here, we reveal that the NS1 protein binds the cellular DNMT3B DNA methyltransferase, thereby inhibiting the methylation of the promoters of genes encoding suppressors of JAK-STAT signaling. As a result, these suppressor genes are induced, and JAK-STAT signaling is inhibited. Furthermore, we demonstrate that the NS1 protein transports DNMT3B to the cytoplasm for ubiquitination and degradation. Thus, we identify the NS1 protein as a potential trigger of the epigenetic deregulation of JAK-STAT signaling suppressors and illustrate a novel mechanism underlying the regulation of host immunity during IAV infection.

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Sublingual Immunization with a Live Attenuated Influenza A Virus Lacking the Nonstructural Protein 1 Induces Broad Protective Immunity in Mice

PLoS ONE ◽

10.1371/journal.pone.0039921 ◽

2012 ◽

Vol 7 (6) ◽

pp. e39921 ◽

Cited By ~ 29

Author(s):

Hae-Jung Park ◽

Boris Ferko ◽

Young-Ho Byun ◽

Joo-Hye Song ◽

Gye-Yeong Han ◽

...

Keyword(s):

Influenza A Virus ◽

Protective Immunity ◽

Influenza A ◽

Nonstructural Protein ◽

Nonstructural Protein 1

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G45R on nonstructural protein 1 of influenza A virus contributes to virulence by increasing the expression of proinflammatory cytokines in mice

Archives of Virology ◽

10.1007/s00705-016-3072-8 ◽

2016 ◽

Vol 162 (1) ◽

pp. 45-55 ◽

Cited By ~ 2

Author(s):

Challika Kaewborisuth ◽

Bryan Kaplan ◽

Mark Zanin ◽

David Finkelstein ◽

Richard J. Webby ◽

...

Keyword(s):

Influenza A Virus ◽

Proinflammatory Cytokines ◽

Influenza A ◽

Nonstructural Protein ◽

Nonstructural Protein 1

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The Effector Domain of the Influenza A Virus Nonstructural Protein NS1 Triggers Host Shutoff by Mediating Inhibition and Global Deregulation of Host Transcription When Associated with Specific Structures in the Nucleus

mBio ◽

10.1128/mbio.02196-21 ◽

2021 ◽

Author(s):

Wolfgang Nacken ◽

André Schreiber ◽

Dörthe Masemann ◽

Stephan Ludwig

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Nonstructural Protein ◽

Host Gene ◽

Central Feature ◽

Effector Domain ◽

Polya Signal ◽

Nonstructural Protein 1 ◽

Host Shutoff

We investigated the role of IAV nonstructural protein 1 NS1 in host gene shutoff—a central feature of IAV replication. We demonstrate that the effector domain of NS1 alone mediates host gene shutoff by inhibition of host transcription and by deregulation of the polyadenylation (polyA) signal-mediated 3′ termination of host transcription.

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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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