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

ABSTRACTThe nucleoprotein (NP) binds the viral RNA genome and associates with the polymerase in a ribonucleoprotein complex (RNP) required for transcription and replication of influenza A virus. NP has no cellular counterpart, and the NP sequence is highly conserved, which led to considering NP a hot target in the search for antivirals. We report here that monomeric nucleoprotein can be inhibited by a small molecule binding in its RNA binding groove, resulting in a novel antiviral against influenza A virus. We identified naproxen, an anti-inflammatory drug that targeted the nucleoprotein to inhibit NP-RNA association required for NP function, by virtual screening. Further docking and molecular dynamics (MD) simulations identified in the RNA groove two NP-naproxen complexes of similar levels of interaction energy. The predicted naproxen binding sites were tested using the Y148A, R152A, R355A, and R361A proteins carrying single-point mutations. Surface plasmon resonance, fluorescence, and otherin vitroexperiments supported the notion that naproxen binds at a site identified by MD simulations and showed that naproxen competed with RNA binding to wild-type (WT) NP and protected active monomers of the nucleoprotein against proteolytic cleavage. Naproxen protected Madin-Darby canine kidney (MDCK) cells against viral challenges with the H1N1 and H3N2 viral strains and was much more effective than other cyclooxygenase inhibitors in decreasing viral titers of MDCK cells. In a mouse model of intranasal infection, naproxen treatment decreased the viral titers in mice lungs. In conclusion, naproxen is a promising lead compound for novel antivirals against influenza A virus that targets the nucleoprotein in its RNA 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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Chios mastic gum inhibits influenza A virus replication and viral pathogenicity

Journal of General Virology ◽

10.1099/jgv.0.001550 ◽

2021 ◽

Author(s):

Dong-In Kim ◽

Yong-Bin Cho ◽

Younghyun Lim ◽

So-Hee Hong ◽

Bumsuk Hahm ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Bacterial Infections ◽

Early Stage ◽

Antiviral Drug ◽

Virus Infectivity ◽

Viral Attachment ◽

Cellular Processes ◽

Chios Mastic Gum

Chios mastic gum (CMG), a resin of the mastic tree (Pistacia lentiscus var. chia), has been used to treat multiple disorders caused by gastrointestinal malfunctions and bacterial infections for more than 2500 years. However, little is known about CMG’s antiviral activity. CMG is known to influence multiple cellular processes such as cell proliferation, differentiation and apoptosis. As virus replication is largely dependent on the host cellular metabolism, it is conceivable that CMG regulates virus infectivity. Therefore, in this study, we evaluated CMG’s potential as an antiviral drug to treat influenza A virus (IAV) infection. CMG treatment dramatically reduced the cytopathogenic effect and production of RNAs, proteins and infectious particles of IAV. Interestingly, CMG interfered with the early stage of the virus life cycle after viral attachment. Importantly, the administration of CMG greatly ameliorated morbidity and mortality in IAV-infected mice. The results suggest that CMG displays a potent anti-IAV activity by blocking the early stage of viral replication. Thus, mastic gum could be exploited as a novel therapeutic agent against IAV infection.

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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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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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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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Structural Definition of Duck Major Histocompatibility Complex Class I Molecules That Might Explain Efficient Cytotoxic T Lymphocyte Immunity to Influenza A Virus

Journal of Virology ◽

10.1128/jvi.02511-16 ◽

2017 ◽

Vol 91 (14) ◽

Cited By ~ 8

Author(s):

Yanan Wu ◽

Junya Wang ◽

Shuhua Fan ◽

Rong Chen ◽

Yanjie Liu ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Cytotoxic T Lymphocyte ◽

Peptide Binding ◽

Class I ◽

Binding Motif ◽

Mhc I ◽

Peptide Binding Groove ◽

Peptide Binding Motif ◽

Binding Groove

ABSTRACT A single dominantly expressed allele of major histocompatibility complex class I (MHC I) may be responsible for the duck's high tolerance to highly pathogenic influenza A virus (HP-IAV) compared to the chicken's lower tolerance. In this study, the crystal structures of duck MHC I (Anpl-UAA*01) and duck β2-microglobulin (β2m) with two peptides from the H5N1 strains were determined. Two remarkable features were found to distinguish the Anpl-UAA*01 complex from other known MHC I structures. A disulfide bond formed by Cys95 and Cys112 and connecting the β5 and β6 sheets at the bottom of peptide binding groove (PBG) in Anpl-UAA*01 complex, which can enhance IAV peptide binding, was identified. Moreover, the interface area between duck MHC I and β2m was found to be larger than in other species. In addition, the two IAV peptides that display distinctive conformations in the PBG, B, and F pockets act as the primary anchor sites. Thirty-one IAV peptides were used to verify the peptide binding motif of Anpl-UAA*01, and the results confirmed that the peptide binding motif is similar to that of HLA-A*0201. Based on this motif, approximately 600 peptides from the IAV strains were partially verified as the candidate epitope peptides for Anpl-UAA*01, which is a far greater number than those for chicken BF2*2101 and BF2*0401 molecules. Extensive IAV peptide binding should allow for ducks with this Anpl-UAA*01 haplotype to resist IAV infection. IMPORTANCE Ducks are natural reservoirs of influenza A virus (IAV) and are more resistant to the IAV than chickens. Both ducks and chickens express only one dominant MHC I locus providing resistance to the virus. To investigate how MHC I provides IAV resistance, crystal structures of the dominantly expressed duck MHC class I (pAnpl-UAA*01) with two IAV peptides were determined. A disulfide bond was identified in the peptide binding groove that can facilitate Anpl-UAA*01 binding to IAV peptides. Anpl-UAA*01 has a much wider recognition spectrum of IAV epitope peptides than do chickens. The IAV peptides bound by Anpl-UAA*01 display distinctive conformations that can help induce an extensive cytotoxic T lymphocyte (CTL) response. In addition, the interface area between the duck MHC I and β2m is larger than in other species. These results indicate that HP-IAV resistance in ducks is due to extensive CTL responses induced by MHC I.

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