Histone mimicry and hijacking of host proteins by influenza virus protein NS1

Pathogens can interfere with vital biological processes of their host by mimicking host proteins. The NS1 protein of the influenza A H3N2 subtype possesses a histone H3K4-like sequence at its carboxyl terminus and has been reported to use this mimic to hijack host proteins. However, this mimic lacks a free N-terminus that is essential for binding to many known H3K4 readers. Here we show that the double chromodomains of CHD1adopt an 'open pocket' to interact with the free N-terminal amine of H3K4, and the open pocket permits the NS1 mimic to bind in a distinct conformation. We also explored the possibility that NS1 hijacks other cellular proteins and found that the NS1 mimic has access to only a subset of chromatin-associated factors, such as WDR5. Moreover, methylation of the NS1 mimic can not be reversed by the H3K4 demethylase LSD1. Overall, we thus conclude that the NS1 mimic is an imperfect histone mimic.

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The N-Terminal Extension of the Influenza B Virus Nucleoprotein Is Not Required for Nuclear Accumulation or the Expression and Replication of a Model RNA

Journal of Virology ◽

10.1128/jvi.72.6.5307-5312.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 5307-5312 ◽

Cited By ~ 25

Author(s):

Mark P. Stevens ◽

Wendy S. Barclay

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Viral Rna ◽

Nuclear Accumulation ◽

Influenza B Virus ◽

Virus Protein ◽

Influenza B ◽

Coding Region ◽

B Virus ◽

N Terminus

ABSTRACT The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP. To determine if the N-terminal mutants could support the expression and replication of a model influenza B virus RNA, the genes encoding the subunits of the viral RNA-dependent RNA polymerase (PA, PB1, and PB2) were cloned. Coexpression of NP and the P proteins in 293 cells was found to permit the expression and replication of a transfected model RNA based on segment 4 of B/Maryland/59, in which the hemagglutinin-coding region was replaced by a chloramphenicol acetyltransferase gene. The expression and replication of the synthetic RNA were not affected by the replacement of NP with NP mutants lacking the N-terminal 51 or 69 residues, indicating that the N-terminal extension is not required for transcription or replication of the viral RNA. In addition, we report that the influenza B virus NP cannot be functionally replaced by type A virus NP in this system.

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Host- and Strain-Specific Regulation of Influenza Virus Polymerase Activity by Interacting Cellular Proteins

mBio ◽

10.1128/mbio.00151-11 ◽

2011 ◽

Vol 2 (4) ◽

Cited By ~ 105

Author(s):

Eric Bortz ◽

Liset Westera ◽

Jad Maamary ◽

John Steel ◽

Randy A. Albrecht ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Highly Pathogenic Avian Influenza ◽

Polymerase Activity ◽

Cellular Proteins ◽

Host Proteins ◽

Highly Pathogenic ◽

Host Interactions ◽

Pathogenic Avian Influenza ◽

Human Proteins

ABSTRACT Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections. IMPORTANCE Highly pathogenic avian influenza A (HPAI) viruses have recently emerged from wild and domestic birds to cause fatal human disease. In human patients, it is thought that adaptation of the viral polymerase, a complex of viral proteins responsible for viral gene expression and RNA genome replication, to interactions with mammalian rather than avian host proteins contributes to disease severity. In this study, we used computational analysis and RNA interference (RNAi) experiments to identify a biological network of human proteins that regulates an H5N1 HPAI virus polymerase, in comparison to a mammalian H1N1 virus. Of 31 proteins tested, 18 (58%) were required for polymerase function in both HPAI and H1N1 viruses. Remarkably, we also found proteins such as DDX17 that governed the HPAI virus polymerase’s adaptation to human cells. These virus-host interactions may thus control pathogenicity of HPAI virus in humans and are promising therapeutic targets for antiviral drugs in severe influenza infections.

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PL Domain at the Carboxyl Terminus of Influenza A Virus NS1 Protein Influences The Nuclear Localization of NS1*

PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS ◽

10.3724/sp.j.1206.2010.00155 ◽

2010 ◽

Vol 37 (9) ◽

pp. 975-982 ◽

Cited By ~ 1

Author(s):

Dan-Gui ZHANG ◽

Wei-Zhong LI ◽

Ge-Fei WANG ◽

Heng ZHANG ◽

Jun ZENG ◽

...

Keyword(s):

Influenza A Virus ◽

Nuclear Localization ◽

Influenza A ◽

Carboxyl Terminus ◽

Ns1 Protein

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Influenza A Virus Virulence Depends on Two Amino Acids in the N-Terminal Domain of Its NS1 Protein To Facilitate Inhibition of the RNA-Dependent Protein Kinase PKR

Journal of Virology ◽

10.1128/jvi.00198-17 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 20

Author(s):

Kristina L. Schierhorn ◽

Fabian Jolmes ◽

Julia Bespalowa ◽

Sandra Saenger ◽

Christin Peteranderl ◽

...

Keyword(s):

Amino Acids ◽

Protein Kinase ◽

Antiviral Activity ◽

Influenza A Virus ◽

Influenza A ◽

Ns1 Protein ◽

Dependent Protein Kinase ◽

N Terminus ◽

Arginine Residues ◽

Dependent Protein

ABSTRACT The RNA-dependent protein kinase (PKR) has broad antiviral activity inducing translational shutdown of viral and cellular genes and is therefore targeted by various viral proteins to facilitate pathogen propagation. The pleiotropic NS1 protein of influenza A virus acts as silencer of PKR activation and ensures high-level viral replication and virulence. However, the exact manner of this inhibition remains controversial. To elucidate the structural requirements within the NS1 protein for PKR inhibition, we generated a set of mutant viruses, identifying highly conserved arginine residues 35 and 46 within the NS1 N terminus as being most critical not only for binding to and blocking activation of PKR but also for efficient virus propagation. Biochemical and Förster resonance energy transfer (FRET)-based interaction studies showed that mutation of R35 or R46 allowed formation of NS1 dimers but eliminated any detectable binding to PKR as well as to double-stranded RNA (dsRNA). Using in vitro and in vivo approaches to phenotypic restoration, we demonstrated the essential role of the NS1 N terminus for blocking PKR. The strong attenuation conferred by NS1 mutation R35A or R46A was substantially alleviated by stable knockdown of PKR in human cells. Intriguingly, both NS1 mutant viruses did not trigger any signs of disease in PKR+/+ mice, but replicated to high titers in lungs of PKR−/− mice and caused lethal infections. These data not only establish the NS1 N terminus as highly critical for neutralization of PKR's antiviral activity but also identify this blockade as an indispensable contribution of NS1 to the viral life cycle. IMPORTANCE Influenza A virus inhibits activation of the RNA-dependent protein kinase (PKR) by means of its nonstructural NS1 protein, but the underlying mode of inhibition is debated. Using mutational analysis, we identified arginine residues 35 and 46 within the N-terminal NS1 domain as highly critical for binding to and functional silencing of PKR. In addition, our data show that this is a main activity of amino acids 35 and 46, as the strong attenuation of corresponding mutant viruses in human cells was rescued to a large extent by lowering of PKR expression levels. Significantly, this corresponded with restoration of viral virulence for NS1 R35A and R46A mutant viruses in PKR−/− mice. Therefore, our data establish a model in which the NS1 N-terminal domain engages in a binding interaction to inhibit activation of PKR and ensure efficient viral propagation and virulence.

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Development of an HTS Assay for the Search of Anti-influenza Agents Targeting the Interaction of Viral RNA with the NS1 Protein

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108318754 ◽

2008 ◽

Vol 13 (7) ◽

pp. 581-590 ◽

Cited By ~ 23

Author(s):

Marta Maroto ◽

Yolanda Fernandez ◽

Juan Ortin ◽

Fernando Pelaez ◽

M. Angerles Cabello

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Cytopathic Effect ◽

Nonstructural Protein ◽

Specific Interaction ◽

Chemical Compounds ◽

Viral Rna ◽

Ns1 Protein ◽

Rna Molecules ◽

Novel Target

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. ( Journal of Biomolecular Screening 2008:581-590)

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Nuclear Translocation of Crk Adaptor Proteins by the Influenza A Virus NS1 Protein

Viruses ◽

10.3390/v8040101 ◽

2016 ◽

Vol 8 (4) ◽

pp. 101 ◽

Cited By ~ 4

Author(s):

Leena Ylösmäki ◽

Riku Fagerlund ◽

Inka Kuisma ◽

Ilkka Julkunen ◽

Kalle Saksela

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Nuclear Translocation ◽

Adaptor Proteins ◽

Ns1 Protein

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Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins

Journal of Virology ◽

10.1128/jvi.02097-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 4

Author(s):

Hannah L. Turkington ◽

Mindaugas Juozapaitis ◽

Nikos Tsolakos ◽

Eugenia Corrales-Aguilar ◽

Martin Schwemmle ◽

...

Keyword(s):

Influenza A Virus ◽

Virulence Factor ◽

Influenza A ◽

Nonstructural Protein ◽

Functional Divergence ◽

Protein A ◽

Regulatory Subunit ◽

Ns1 Protein ◽

Influenza A Viruses ◽

Hydrophobic Patch

ABSTRACT Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.

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Role of the chaperone protein 14-3-3ε in the regulation of influenza A virus-activated beta interferon

Journal of Virology ◽

10.1128/jvi.00231-21 ◽

2021 ◽

Author(s):

Ee-Hong Tam ◽

Yen-Chin Liu ◽

Chian-Huey Woung ◽

Helene Minyi Liu ◽

Guan-Hong Wu ◽

...

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Influenza A ◽

Critical Role ◽

Type I ◽

Type I Ifn ◽

Ns1 Protein ◽

Chaperone Protein ◽

Influenza A Virus Infection

The NS1 protein of the influenza A virus plays a critical role in regulating several biological processes in cells, including the type I interferon (IFN) response. We previously profiled the cellular factors that interact with the NS1 protein of influenza A virus and found that the NS1 protein interacts with proteins involved in RNA splicing/processing, cell cycle regulation, and protein targeting processes, including 14-3-3ε. Since 14-3-3ε plays an important role in RIG-I translocation to MAVS to activate type I IFN expression, the interaction of the NS1 and 14-3-3ε proteins may prevent the RIG-I-mediated IFN response. In this study, we confirmed that the 14-3-3ε protein interacts with the N-terminal domain of the NS1 protein and that the NS1 protein inhibits RIG-I-mediated IFN-β promoter activation in 14-3-3ε-overexpressing cells. In addition, our results showed that knocking down 14-3-3ε can reduce IFN-β expression elicited by influenza A virus and enhance viral replication. Furthermore, we found that threonine in the 49 th amino acid position of the NS1 protein plays a role in the interaction with 14-3-3ε. Influenza A virus expressing C-terminus-truncated NS1 with T49A mutation dramatically increases IFN-β mRNA in infected cells and causes slower replication than that of virus without the T-to-A mutation. Collectively, this study demonstrates that 14-3-3ε is involved in influenza A virus-initiated IFN-β expression and that the interaction of the NS1 protein and 14-3-3ε may be one of the mechanisms for inhibiting type I IFN activation during influenza A virus infection. IMPORTANCE Influenza A virus is an important human pathogen causing severe respiratory disease. The virus has evolved several strategies to dysregulate the innate immune response and facilitate its replication. We demonstrate that the NS1 protein of influenza A virus interacts with the cellular chaperone protein 14-3-3ε, which plays a critical role in RIG-I translocation that induces type I IFN expression, and that NS1 protein prevents RIG-I translocation to mitochondrial membrane. The interaction site for 14-3-3ε is the RNA-binding domain (RBD) of the NS1 protein. Therefore, this research elucidates a novel mechanism by which the NS1 RBD mediates IFN-β suppression to facilitate influenza A viral replication. Additionally, the findings reveal the antiviral role of 14-3-3ε during influenza A virus infection.

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