The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

Abstract

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function. A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function. The influenza A virus (IAV) 3The abbreviations used are: IAVinfluenza A virusRBDRNA-binding domainEDeffector domainCARDcaspase activation and recruitment domainHSQCheteronuclear single-quantum coherencem.o.i.multiplicity of infectionLucluciferaseGAGSH-agaroseIACUCInstitutional Animal Care and Use CommitteeMDCKMadin-Darby canine kidneySFserum-freeqPCRquantitative PCRFPfluorescence polarizationEMEMeagle's minimum essential medium6-FAM6-carboxyfluorescein. is a serious public health concern that causes annual epidemics and occasional pandemics, resulting in significant levels of morbidity and mortality each year (1Thompson W.W. Comanor L. Shay D.K. Epidemiology of seasonal influenza: use of surveillance data and statistical models to estimate the burden of disease.J. Infect. Dis. 2006; 194 (17163394): S82-S9110.1086/507558Crossref PubMed Scopus (211) Google Scholar, 2Molinari N.A. Ortega-Sanchez I.R. Messonnier M.L. Thompson W.W. Wortley P.M. Weintraub E. Bridges C.B. The annual impact of seasonal influenza in the US: measuring disease burden and costs.Vaccine. 2007; 25 (17544181): 5086-509610.1016/j.vaccine.2007.03.046Crossref PubMed Scopus (1284) Google Scholar). The ability of IAV to adapt to various hosts and undergo genetic reassortment ensures constant generation of unique strains with unknown degrees of pathogenicity, transmissibility, and pandemic potential. Currently, our knowledge of the precise combination of factors that drives the emergence of strains with pandemic potential is incomplete. However, several proteins expressed by IAV have been identified as key determinants of virulence during infection. One such protein encoded by IAV is nonstructural protein 1 (NS1). influenza A virus RNA-binding domain effector domain caspase activation and recruitment domain heteronuclear single-quantum coherence multiplicity of infection luciferase GSH-agarose Institutional Animal Care and Use Committee Madin-Darby canine kidney serum-free quantitative PCR fluorescence polarization eagle's minimum essential medium 6-carboxyfluorescein. NS1 is highly multifunctional and ranges from 215–237 amino acids in length, depending on the strain from which it is derived. During infection, NS1 plays a critical role in antagonizing the cellular innate immune response (3Ayllon J. García-Sastre A. The NS1 protein: a multitasking virulence factor.Curr. Top Microbiol. Immunol. 2015; 386 (25007846): 73-107PubMed Google Scholar, 4Hale B.G. Randall R.E. Ortín J. Jackson D. The multifunctional NS1 protein of influenza A viruses.J. Gen. 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Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I.Cell Host Microbe. 2009; 5 (19454348): 439-44910.1016/j.chom.2009.04.006Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 26Rajsbaum R. Albrecht R.A. Wang M.K. Maharaj N.P. Versteeg G.A. Nistal- Villán E. García-Sastre A. Gack M.U. Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.PLoS Pathog. 2012; 8 (23209422): e100305910.1371/journal.ppat.1003059Crossref PubMed Scopus (234) Google Scholar). Although it is has been shown that this interaction is a critical mechanism through which NS1 antagonizes RIG-I 2CARD ubiquitination (6Gack M.U. Albrecht R.A. Urano T. Inn K.S. Huang I.C. Carnero E. Farzan M. Inoue S. Jung J.U. García-Sastre A. Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I.Cell Host Microbe. 2009; 5 (19454348): 439-44910.1016/j.chom.2009.04.006Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 26Rajsbaum R. Albrecht R.A. Wang M.K. Maharaj N.P. Versteeg G.A. Nistal- Villán E. García-Sastre A. Gack M.U. Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein.PLoS Pathog. 2012; 8 (23209422): e100305910.1371/journal.ppat.1003059Crossref PubMed Scopus (234) Google Scholar), it has not been shown whether additive antagonistic effects could result from additional direct interactions between NS1 and RIG-I. Recently, we reported that the NS1RBD from 1918H1N1 IAV (A/Brevig Mission/1/1918) interacts directly with the second CARD of RIG-I and that a naturally occurring mutation in the NS1RBD (R21Q) abrogated this direct interaction (8Jureka A.S. Kleinpeter A.B. Cornilescu G. Cornilescu C.C. Petit C.M. Structural basis for a novel interaction between the NS1 protein derived from the 1918 influenza virus and RIG-I.Structure. 2015; 23 (26365801): 2001-201010.1016/j.str.2015.08.007Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Because of the critical role the 2CARDs play in RIG-I activation, we hypothesized that this direct interaction would enhance the ability of NS1 to inhibit ubiquitination of the RIG-I 2CARDs. Furthermore, this inhibition would be independent of other previously studied NS1 interactions involving TRIM25 (6Gack M.U. Albrecht R.A. Urano T. Inn K.S. Huang I.C. Carnero E. Farzan M. Inoue S. Jung J.U. García-Sastre A. Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I.Cell Host Microbe. 2009; 5 (19454348): 439-44910.1016/j.chom.2009.04.006Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 7Guo Z. Chen L.M. Zeng H. Gomez J.A. Plowden J. Fujita T. Katz J.M. Donis R.O. Sambhara S. NS1 protein of influenza A virus inhibits the function of intracytoplasmic pathogen sensor, RIG-I.Am. J. Respir. Cell Mol. Biol. 2007; 36 (17053203): 263-26910.1165/rcmb.2006-0283RCCrossref PubMed Scopus (243) Google Scholar, 8Jureka A.S. Kleinpeter A.B. Cornilescu G. Cornilescu C.C. Petit C.M. Structural basis for a novel interaction between the NS1 protein derived from the 1918 influenza virus and RIG-I.Structure. 2015; 23 (26365801): 2001-201010.1016/j.str.2015.08.007Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 9Mibayashi M. Martínez-Sobrido L. Loo Y.M. Cárdenas W.B. Gale Jr., M. García-Sastre A. Inhibition of retinoic acid-inducible gene I-mediated induction of beta interferon by the NS1 protein of influenza A virus.J. Virol. 2007; 81 (17079289): 514-52410.1128/JVI.01265-06Crossref PubMed Scopus (493) Google Scholar, 10Opitz B. Rejaibi A. Dauber B. Eckhard J. Vinzing M. Schmeck B. Hippenstiel S. Suttorp N. Wolff T. IFNβ induction by influenza A virus is mediated by RIG-I, which is regulated by the viral NS1 protein.Cell Microbiol. 2007; 9 (17140406): 930-93810.1111/j.1462-5822.2006.00841.xCrossref PubMed Scopus (228) Google Scholar, 11Pichlmair A. Schulz O. Tan C.P. Näslund T.I. Liljeström P. Weber F. Reis e Sousa C. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates.Science. 2006; 314 (17038589): 997-100110.1126/science.1132998Crossref PubMed Scopus (1767) Google Scholar) and dsRNA structures (3Ayllon J. García-Sastre A. The NS1 protein: a multitasking virulence factor.Curr. Top Microbiol. Immunol. 2015; 386 (25007846): 73-107PubMed Google Scholar) also known to inhibit activation of RIG-I. In this study, we demonstrate that the naturally occurring R21Q mutation in NS1 (NS1R21Q) significantly impacts NS1's ability to control RIG-I signaling. Specifically, we used reverse genetics to generate WT A/Puerto Rico/8/1934 IAV (rPR8WT) and a mutant IAV encoding the R21Q mutation in NS1 (rPR8R21Q) to test the effects of the mutation on aspects of the viral life cycle. We observed that rPR8R21Q induced significantly more IRF3 phosphorylation and IFN-β expression compared with rPR8WT. These data indicate that rPR8R21Q is a significantly more potent RIG-I activator compared with rPR8WT. Further analysis revealed that NS1R21Q is significantly less efficient at inhibiting TRIM25-dependent ubiquitination of the RIG-I 2CARDs, a critical step in activation of RIG-I signaling. Additionally, we determined that the R21Q mutation markedly diminished the ability of NS1 to interact with the RIG-I 2CARDs. Finally, we confirmed that the R21Q mutation did not have an effect on any of the other NS1 functions known to inhibit the RIG-I pathway. Taken together, this is the first study to identify a natural polymorphism in the NS1RBD that mitigates its ability to control RIG-I 2CARD ubiquitination by TRIM25. This study demonstrates that strain-specific polymorphisms in NS1 not only have a significant impact on its ability to efficiently antagonize RIG-I activation but that the effects of these polymorphisms may be conditional to species-specific activation of RIG-I. Previously, we demonstrated that an R21Q mutation in the NS1RBD abrogated its ability to interact with the second CARD domain of RIG-I. Residue 21 is distal to other residues in the NS1RBD (Arg35, Arg38, and Lys41) that are known to have functions in RNA binding and NS1 cellular localization (Fig. 1) (28Cheng A. Wong S.M. Yuan Y.A. Structural basis for dsRNA recognition by NS1 protein of influenza A virus.Cell Res. 2009; 19 (18813227): 187-19510.1038/cr.2008.288Crossref PubMed Scopus (100) Google Scholar, 29Melén K. Kinnunen L. Fagerlund R. Ikonen N. Twu K.Y. Krug R.M. Julkunen I. Nuclear and nucleolar targeting of influenza A virus NS1 protein: striking differences between different virus subtypes.J. Virol. 2007; 81 (17376915): 5995-600610.1128/JVI.01714-06Crossref PubMed Scopus (155) Google Scholar, 30Greenspan D. Palese P. Krystal M. Two nuclear location signals in the influenza virus NS1 nonstructural protein.J. Virol. 1988; 62 (2969057): 3020-3026Crossref PubMed Google Scholar). Disrupting these residues (Arg35, Arg38, and Lys41) by mutation is well-known to cause significant defects in viral fitness, including decreased replication and inability to control the cellular antiviral response. What is not known, however, is the effect of the R21Q mutation on the viral life cycle, as mutations in this region of the NS1RBD have yet to be studied. Given that residue 21 is in an unstudied region of the NS1RBD, we first wanted to ensure that there were no gross structural changes in the NS1RBD associated with the R21Q mutation. To assess any potential structural changes caused by the mutation, we used NMR to analyze the 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of both the WT NS1RBD (RBDWT) and the R21Q mutation (RBDR21Q). Both HSQC spectra showed a single set of dispersed peaks, indicative of a well-folded protein with a single, major conformation (Fig. 2A). Because the NS1RBD is a symmetric homodimer, only 72 amide resonances were expected and ultimately observed (not including the side-chain amides of glutamine and asparagine) for both the RBDWT and RBDR21Q. We then obtained resonance assignments for RBDR21Q using three-dimensional NMR experiments such as HNCACB and CBCA(CO)NH for further analysis. High-quality triple-resonance spectra allowed backbone resonance assignments to be obtained for 70 of the 72 (97%) possible assigned residues of the RBDR21Q. Analysis of the 13Cα chemical shifts indicated that each monomer of the RBDR21Q mutant is composed of α1–turn–α2–turn–α3 (Fig. 2B) (31Wishart D.S. Sykes B.D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data.J. Biomol. NMR. 1994; 4 (8019132): 171-180Crossref PubMed Scopus (1908) Google Scholar). This six-helix dimeric structure of the RBDR21Q homodimer is consistent with both 13Cα chemical shift analysis of the RBDWT (Fig. 2B) and our previously solved solution structure of the 1918H1N1 NS1RBD. Because the NS1 R21Q mutation had not been characterized previously, it was critical to determine whether the mutation impacted basic aspects of the viral life cycle, such as IAV replication and NS1 cellular localization. To accomplish this, we used a previously described IAV reverse genetics system (32Quinlivan M. Zamarin D. García-Sastre A. Cullinane A. Chambers T. Palese P. Attenuation of equine influenza viruses through truncations of the NS1 protein.J. Virol. 2005; 79 (15956587): 8431-843910.1128/JVI.79.13.8431-8439.2005Crossref PubMed Scopus (200) Google Scholar) to generate and rescue recombinant A/Puerto Rico/8/1934 viruses encoding WT NS1 (rPR8WT) and R21Q NS1 mutant (rPR8R21Q). Upon infecting adenocarcinomic human alveolar basal epithelial (A549) cells at m.o.i. 0.01, we observed no significant differences in viral replication between rPR8WT and rPR8R21Q (Fig. 3A). Similarly, we observed no significant change in the cellular localization of NS1 in A549 cells infected with rPR8WT or rPR8R21Q 12 h post-infection (m.o.i. 2) (Fig. 3B). Given that R21Q is a naturally occurring mutation, it is not surprising that replication and intracellular localization are not affected; numerous viruses capable of productive infection in humans also possess a Gln at position 21. However, the observation of similar replication kinetics does not adjudicate the question of whether the two viruses will result in differential activation of the RIG-I signaling pathway upon infection, nor is it prognostic of potential differences in virulence between the two viruses, as influenza replication levels do not necessarily correlate with virulence (33Askovich P.S. Sanders C.J. Rosenberger C.M. Diercks A.H. Dash P. Navarro G. Vogel P. Doherty P.C. Thomas P.G. Aderem A. Differential host response, rather than early viral replication efficiency, correlates with pathogenicity caused by influenza viruses.PLoS ONE. 2013; 8 (24073225): e7486310.1371/journal.pone.0074863Crossref PubMed Scopus (22) Google Scholar, 34Fan S. Hatta M. Kim J.H. Halfmann P. Imai M. Macken C.A. Le M.Q. Nguyen T. Neumann G. Kawaoka Y. Novel residues in avian influenza virus PB2 protein affect virulence in mammalian hosts.Nat. Commun. 2014; 5 (25289523): 502110.1038/ncomms6021Crossref PubMed Scopus (50) Google Scholar). Activation of the RIG-I pathway results in phosphorylation of the antiviral transcription factor IRF3, which then translocates to the nucleus and induces transcription of type I interferons (e.g. IFN-β). IAV NS1 has been shown previously to inhibit IRF3-mediated signaling through its antagonism of the RIG-I pathway (9Mibayashi M. Martínez-Sobrido L. Loo Y.M. Cárdenas W.B. Gale Jr., M. García-Sastre A. Inhibition of retinoic acid-inducible gene I-mediated induction of beta interferon by the NS1 protein of influenza A virus.J. Virol. 2007; 81 (17079289): 514-52410.1128/JVI.01265-06Crossref PubMed Scopus (493) Google Scholar, 35Talon J. Horvath C.M. Polley R. Basler C.F. Muster T. Palese P. García-Sastre A. Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein.J. Virol. 2000; 74 (10933707): 7989-799610.1128/JVI.74.17.7989-7996.2000Crossref PubMed Scopus (502) Google Scholar). Based on our previous data demonstrating that NS1R21Q is unable to interact with the second CARD of RIG-I, we hypothesized that rPR8R21Q would be less efficient at antagonizing RIG-I signaling. To test this hypothesis, we infected A549 cells with rPR8WT and rPR8R21Q (m.o.i. 2) to determine the respective levels of IRF3 phosphorylation 6, 12, and 24 h post-infection. We observed that infection with rPR8R21Q resulted in significantly increased IRF3 phosphorylation (Fig. 4, A and B) 12 and 24 h post-infection relative to rPR8WT levels. Furthermore, these observed differences are not due to disparities in NS1 expression levels between the two viruses, as NS1 expression was determined to be equal across all time points measured (Fig. 4A, quantification not shown). These data suggest that a Gln at position 21 in NS1 results in significantly less efficient antagonism of the RIG-I signaling pathway without altering NS1 expression. Given that phosphorylated IRF3 acts as a transcription factor for up-regulation of type I IFNs, a corresponding increase in IFN-β expression upon infection with rPR8R21Q compared with rPR8WT was expected. We observed significantly increased IFN-β mRNA expression at all time points (Fig. 4C) and significantly increased IFN-β protein expression 12 and 24 h post-infection (Fig. 4D) using identical experimental conditions as those described previously. Increased IRF3 phosphorylation and IFN-β mRNA and protein expression suggest that rPR8R21Q is a significantly more potent activator of the RIG-I pathway during infection. Although increased IRF3 phosphorylation and IFN-β