Interactions between influenza A virus nucleoprotein and gene segment UTRs facilitate selective modulation of viral gene expression

The influenza A virus (IAV) genome is divided into eight negative-sense, single-stranded RNA segments. Each segment exhibits a unique level and temporal pattern of expression, however the exact mechanisms underlying the patterns of individual gene segment expression are poorly understood. We previously demonstrated that a single substitution in the viral nucleoprotein (NP:F346S) selectively modulates neuraminidase (NA) gene segment expression while leaving other segments largely unaffected. Given what is currently known about NP function, there is no obvious explanation for how changes in NP can selectively modulate the replication of individual gene segments. We found that the specificity of this effect for the NA segment is virus strain specific and depends on the UTR sequences of the NA segment. While the NP:F346S substitution did not significantly alter the RNA binding or oligomerization activities of NP in vitro, it specifically decreased the ability of NP to promote NA segment vRNA synthesis. In addition to NP residue F346, we identified two other adjacent aromatic residues in NP (Y385 & F479) capable of similarly regulating NA gene segment expression, suggesting a larger role for this domain in gene-segment specific regulation. Our findings reveal a new role for NP in selective regulation of viral gene segment replication and demonstrate how the expression patterns of individual viral gene segments can be modulated during adaptation to new host environments.

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Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA

Journal of Virology ◽

10.1128/jvi.00528-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 15

Author(s):

Carina F. Pereira ◽

Eliot K. C. Read ◽

Helen M. Wise ◽

Maria J. Amorim ◽

Paul Digard

Keyword(s):

Influenza A Virus ◽

Nuclear Export ◽

Influenza A ◽

Matrix Protein ◽

Rna Binding ◽

Viral Gene Expression ◽

Mutant Virus ◽

Viral Gene ◽

Ns1 Protein ◽

Viral Mrnas

ABSTRACT Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery.

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Non-Uniform and Non-Random Binding of Nucleoprotein to Influenza A and B Viral RNA

Viruses ◽

10.3390/v10100522 ◽

2018 ◽

Vol 10 (10) ◽

pp. 522 ◽

Cited By ~ 10

Author(s):

Valerie Le Sage ◽

Adalena Nanni ◽

Amar Bhagwat ◽

Dan Snyder ◽

Vaughn Cooper ◽

...

Keyword(s):

Influenza A ◽

Gene Segment ◽

Influenza Viruses ◽

Viral Rna ◽

Influenza B Virus ◽

Viral Gene ◽

Influenza B ◽

Influenza A Viruses ◽

Binding Profile ◽

Random Manner

The genomes of influenza A and B viruses have eight, single-stranded RNA segments that exist in the form of a viral ribonucleoprotein complex in association with nucleoprotein (NP) and an RNA-dependent RNA polymerase complex. We previously used high-throughput RNA sequencing coupled with crosslinking immunoprecipitation (HITS-CLIP) to examine where NP binds to the viral RNA (vRNA) and demonstrated for two H1N1 strains that NP binds vRNA in a non-uniform, non-random manner. In this study, we expand on those initial observations and describe the NP-vRNA binding profile for a seasonal H3N2 and influenza B virus. We show that, similar to H1N1 strains, NP binds vRNA in a non-uniform and non-random manner. Each viral gene segment has a unique NP binding profile with areas that are enriched for NP association as well as free of NP-binding. Interestingly, NP-vRNA binding profiles have some conservation between influenza A viruses, H1N1 and H3N2, but no correlation was observed between influenza A and B viruses. Our study demonstrates the conserved nature of non-uniform NP binding within influenza viruses. Mapping of the NP-bound vRNA segments provides information on the flexible NP regions that may be involved in facilitating assembly.

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Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development

Scientific Reports ◽

10.1038/srep21662 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 11

Author(s):

Chia-Lin Liu ◽

Hui-Chen Hung ◽

Shou-Chen Lo ◽

Ching-Hui Chiang ◽

I-Jung Chen ◽

...

Keyword(s):

Drug Development ◽

Influenza A Virus ◽

Influenza A ◽

Rna Binding ◽

Antiviral Drug ◽

Conserved Residues ◽

Binding Groove

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Cellular RNA Binding Proteins NS1-BP and hnRNP K Regulate Influenza A Virus RNA Splicing

PLoS Pathogens ◽

10.1371/journal.ppat.1003460 ◽

2013 ◽

Vol 9 (6) ◽

pp. e1003460 ◽

Cited By ~ 46

Author(s):

Pei-Ling Tsai ◽

Ni-Ting Chiou ◽

Sharon Kuss ◽

Adolfo García-Sastre ◽

Kristen W. Lynch ◽

...

Keyword(s):

Influenza A Virus ◽

Rna Splicing ◽

Influenza A ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Hnrnp K ◽

Virus Rna

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A NS1-binding monoclonal antibody interacts with two residues that are highly conserved in seasonal as well as newly emerged influenza A virus

Pathogens and Disease ◽

10.1093/femspd/ftz012 ◽

2019 ◽

Vol 77 (1) ◽

Author(s):

Su Hui Catherine Teo ◽

Jian-Ping Wu ◽

Chee-Keng Mok ◽

Yee-Joo Tan

Keyword(s):

Monoclonal Antibody ◽

Influenza A Virus ◽

Influenza A ◽

Rna Binding ◽

Intracellular Localization ◽

Cross Reactivity ◽

Structural Protein ◽

Multifunctional Protein ◽

Good Target ◽

Conformational Plasticity

Abstract The non-structural protein 1 (NS1) of influenza A virus (IAV) is a multifunctional protein that antagonizes host antiviral responses, modulating virus pathogenesis. As such, it serves as a good target for research and diagnostic assay development. In this study, we have generated a novel monoclonal antibody (mAb) 19H9 and epitope mapping revealed that two residues, P85 and Y89, of NS1 are essential for interacting with this mAb. Furthermore, residues P85 and Y89 are found to be highly conserved across different IAV subtypes, namely seasonal H1N1 and H3N2, as well as the highly pathogenic H5N1 and H5N6 avian strains. Indeed, mAb 19H9 exhibits broad cross-reactivity with IAV strains of different subtypes. The binding of mAb 19H9 to residue Y89 was further confirmed by the abrogation of interaction between NS1 and p85β. Additionally, mAb 19H9 also detected NS1 proteins expressed in IAV-infected cells, showing NS1 intracellular localization in the cytoplasm and nucleolus. To our knowledge, mAb 19H9 is the first murine mAb to bind at the juxtaposition between the N-terminal RNA-binding domain and C-terminal effector domain of NS1. It could serve as a useful research tool for studying the conformational plasticity and dynamic changes in NS1.

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Characterization of influenza A virus with nine segments: Effect gene segment on virus property

Research in Veterinary Science ◽

10.1016/j.rvsc.2011.12.020 ◽

2012 ◽

Vol 93 (2) ◽

pp. 1076-1080

Author(s):

Rui Wu ◽

Xuke Zhang ◽

Huabin Shao ◽

Qingping Luo ◽

Jun Yang ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Gene Segment ◽

Effect Gene

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The accumulation of influenza A virus segment 7 spliced mRNAs is regulated by the NS1 protein

Journal of General Virology ◽

10.1099/vir.0.035485-0 ◽

2012 ◽

Vol 93 (1) ◽

pp. 113-118 ◽

Cited By ~ 35

Author(s):

Nicole C. Robb ◽

Ervin Fodor

Keyword(s):

Influenza Virus ◽

Viral Infection ◽

Influenza A Virus ◽

Influenza A ◽

Rna Binding ◽

Mrna Splicing ◽

Ns1 Protein ◽

Binding Ability ◽

Alternatively Spliced ◽

Transcription And Replication

The influenza A virus M1 mRNA is alternatively spliced to produce M2 mRNA, mRNA3, and in some cases, M4 mRNA. Splicing of influenza mRNAs is carried out by the cellular splicing machinery and is thought to be regulated, as both spliced and unspliced mRNAs encode proteins. In this study, we used radioactively labelled primers to investigate the accumulation of spliced and unspliced M segment mRNAs in viral infection and ribonucleoprotein (RNP) reconstitution assays in which only the minimal components required for transcription and replication to occur were expressed. We found that co-expression of the viral NS1 protein in an RNP reconstitution assay altered the accumulation of spliced mRNAs compared with when it was absent, and that this activity was dependent on the RNA-binding ability of NS1. These findings suggest that the NS1 protein plays a role in the regulation of splicing of influenza virus M1 mRNA.

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Interaction of Host Nucleolin with Influenza A Virus Nucleoprotein in the Early Phase of Infection Limits the Late Viral Gene Expression

PLoS ONE ◽

10.1371/journal.pone.0164146 ◽

2016 ◽

Vol 11 (10) ◽

pp. e0164146 ◽

Cited By ~ 10

Author(s):

Deepshikha Kumar ◽

Shobha Broor ◽

Maitreyi S. Rajala

Keyword(s):

Gene Expression ◽

Influenza A Virus ◽

Early Phase ◽

Influenza A ◽

Viral Gene Expression ◽

Viral Gene

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Functional Replacement of the Carboxy-Terminal Two-Thirds of the Influenza A Virus NS1 Protein with Short Heterologous Dimerization Domains

Journal of Virology ◽

10.1128/jvi.76.24.12951-12962.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12951-12962 ◽

Cited By ~ 80

Author(s):

Xiuyan Wang ◽

Christopher F. Basler ◽

Bryan R. G. Williams ◽

Robert H. Silverman ◽

Peter Palese ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Influenza A Virus ◽

Influenza A ◽

Rna Binding ◽

Influenza Viruses ◽

Ns1 Protein ◽

Wild Type ◽

Dimerization Domain ◽

Carboxy Terminal

ABSTRACT The NS1 protein of influenza A/WSN/33 virus is a 230-amino-acid-long protein which functions as an interferon alpha/beta (IFN-α/β) antagonist by preventing the synthesis of IFN during viral infection. In tissue culture, the IFN inhibitory function of the NS1 protein has been mapped to the RNA binding domain, the first 73 amino acids. Nevertheless, influenza viruses expressing carboxy-terminally truncated NS1 proteins are attenuated in mice. Dimerization of the NS1 protein has previously been shown to be essential for its RNA binding activity. We have explored the ability of heterologous dimerization domains to functionally substitute in vivo for the carboxy-terminal domains of the NS1 protein. Recombinant influenza viruses were generated that expressed truncated NS1 proteins of 126 amino acids, fused to 28 or 24 amino acids derived from the dimerization domains of either the Saccharomyces cerevisiae PUT3 or the Drosophila melanogaster Ncd (DmNcd) proteins. These viruses regained virulence and lethality in mice. Moreover, a recombinant influenza virus expressing only the first 73 amino acids of the NS1 protein was able to replicate in mice lacking three IFN-regulated antiviral enzymes, PKR, RNaseL, and Mx, but not in wild-type (Mx-deficient) mice, suggesting that the attenuation was mainly due to an inability to inhibit the IFN system. Remarkably, a virus with an NS1 truncated at amino acid 73 but fused to the dimerization domain of DmNcd replicated and was also highly pathogenic in wild-type mice. These results suggest that the main biological function of the carboxy-terminal region of the NS1 protein of influenza A virus is the enhancement of its IFN antagonist properties by stabilizing the NS1 dimeric structure.

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