Influenza C virus NS1 protein counteracts RIG-I-mediated IFN signalling

The nonstructural proteins 1 (NS1) from influenza A and B viruses are known as the main viral factors antagonising the cellular interferon (IFN) response, inter alia by inhibiting the retinoic acid-inducible gene I (RIG-I) signalling. The cytosolic pattern-recognition receptor RIG-I senses double-stranded RNA and 5'-triphosphate RNA produced during RNA virus infections. Binding to these ligands activates RIG-I and in turn the IFN signalling. We now report that the influenza C virus NS1 protein also inhibits the RIG-I-mediated IFN signalling. Employing luciferase-reporter assays, we show that expression of NS1-C proteins of virus strains C/JJ/50 and C/JHB/1/66 considerably reduced the IFN-β promoter activity. Mapping of the regions from NS1-C of both strains involved in IFN-β promoter inhibition showed that the N-terminal 49 amino acids are dispensable, while the C-terminus is required for proper modulation of the IFN response. When a mutant RIG-I, which is constitutively active without ligand binding, was employed, NS1-C still inhibited the downstream signalling, indicating that IFN inhibitory properties of NS1-C are not necessarily linked to an RNA binding mechanism

Non-structural protein 1 of avian influenza A viruses differentially inhibit NF-κB promoter activation

<p>Abstract</p> <p>Background</p> <p>Influenza virus infection activates NF-κB and is a general prerequisite for a productive influenza virus infection. On the other hand, non-structural protein 1 (NS1) suppresses this viral activated NF-κB, presumably to prevent expression of NF-κB mediated anti-viral response. NS1 proteins of influenza A viruses are divided into two groups, known as allele A and allele B. The possible functional relevance of this NS1 division to viral pathogenicity is lacking.</p> <p>Findings</p> <p>The ability of NS1 protein from two avian influenza subtypes, H6N8 and H4N6, to inhibit NF-κB promoter activation was assessed. Further, efforts were made to characterize the genetic basis of this inhibition. We found that allele A NS1 proteins of H6N8 and H4N6 are significantly better in preventing dsRNA induced NF-κB promoter activation compared to allele B of corresponding subtypes, in a species independent manner. Furthermore, the ability to suppress NF-κB promoter activation was mapped to the effector domain while the RNA binding domain alone was unable to suppress this activation. Chimeric NS1 proteins containing either RNA binding domain of allele A and effector domain of allele B or vice versa, were equally potent in preventing NF-κB promoter activation compared to their wt. NS1 protein of allele A and B from both subtypes expressed efficiently as detected by Western blotting and predominantly localized in the nucleus in both A549 and MiLu cells as shown by <it>in situ </it>PLA.</p> <p>Conclusions</p> <p>Here, we present another aspect of NS1 protein in inhibiting dsRNA induced NF-κB activation in an allele dependent manner. This suggests a possible correlation with the virus's pathogenic potential.</p

A transient homotypic interaction model for the influenza A virus NS1 protein effector domain

Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins

Differences in the ability to suppress interferon β production between allele A and allele B NS1 proteins from H10 influenza A viruses

BACKGROUND: In our previous study concerning the genetic relationship among H10 avian influenza viruses with different pathogenicity in mink (Mustela vison), we found that these differences were related to amino acid variations in the NS1 protein. In this study, we extend our previous work to further investigate the effect of the NS1 from different gene pools on type I IFN promoter activity, the production of IFN-β, as well as the expression of the IFN-β mRNA in response to poly I:C. RESULTS: Using a model system, we first demonstrated that NS1 from A/mink/Sweden/84 (H10N4) (allele A) could suppress an interferon-stimulated response element (ISRE) reporter system to about 85%. The other NS1 (allele B), from A/chicken/Germany/N/49 (H10N7), was also able to suppress the reporter system, but only to about 20%. The differences in the abilities of the two NS1s from different alleles to suppress the ISRE reporter system were clearly reflected by the protein and mRNA expressions of IFN-β as shown by ELISA and RT-PCR assays. CONCLUSIONS: These studies reveal that different non-structural protein 1 (NS1) of influenza viruses, one from allele A and another from allele B, show different abilities to suppress the type I interferon β expression. It has been hypothesised that some of the differences in the different abilities of the alleles to suppress ISRE were because of the interactions and inhibitions at later stages from the IFN receptor, such as the JAK/STAT pathway. This might reflect the additional effects of the immune evasion potential of different NS1s

Influenza Virus Non-Structural Protein 1 (NS1) Disrupts Interferon Signaling

Type I interferons (IFNs) function as the first line of defense against viral infections by modulating cell growth, establishing an antiviral state and influencing the activation of various immune cells. Viruses such as influenza have developed mechanisms to evade this defense mechanism and during infection with influenza A viruses, the non-structural protein 1 (NS1) encoded by the virus genome suppresses induction of IFNs-α/β. Here we show that expression of avian H5N1 NS1 in HeLa cells leads to a block in IFN signaling. H5N1 NS1 reduces IFN-inducible tyrosine phosphorylation of STAT1, STAT2 and STAT3 and inhibits the nuclear translocation of phospho-STAT2 and the formation of IFN-inducible STAT1:1-, STAT1:3- and STAT3:3- DNA complexes. Inhibition of IFN-inducible STAT signaling by NS1 in HeLa cells is, in part, a consequence of NS1-mediated inhibition of expression of the IFN receptor subunit, IFNAR1. In support of this NS1-mediated inhibition, we observed a reduction in expression of ifnar1 in ex vivo human non-tumor lung tissues infected with H5N1 and H1N1 viruses. Moreover, H1N1 and H5N1 virus infection of human monocyte-derived macrophages led to inhibition of both ifnar1 and ifnar2 expression. In addition, NS1 expression induces up-regulation of the JAK/STAT inhibitors, SOCS1 and SOCS3. By contrast, treatment of ex vivo human lung tissues with IFN-α results in the up-regulation of a number of IFN-stimulated genes and inhibits both H5N1 and H1N1 virus replication. The data suggest that NS1 can directly interfere with IFN signaling to enhance viral replication, but that treatment with IFN can nevertheless override these inhibitory effects to block H5N1 and H1N1 virus infections

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

Background TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. However, there is scarce knowledge about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Results Here, we reveal that the RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25’s endogenous RNA targets and protein binding partners. We demonstrate that TRIM25 controls the levels of Zinc Finger Antiviral Protein (ZAP). Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase activity towards itself (autoubiquitination) and its physiologically relevant target ZAP. Conclusions Our results suggest that many other proteins with the PRY/SPRY domain could have yet uncharacterized RNA-binding potential. Together, our data reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor in their enzymatic activity