Influenza Virus Non-structural Protein Research Articles

Enhancement of systemic and lung-localized CD4<sup>+</sup> T-cell immune responses by truncation of NS1 protein of a seasonal live influenza vaccine strain

Introduction. There is a large variety of licensed influenza vaccines worldwide, but their common limitation is rather narrow specificity and inability to protect against antigenic-drift variants of influenza virus. Therefore, optimization of immunogenic and cross-protective properties of licensed influenza vaccines is an urgent priority of public health agenda. One such approach is to modulate the immunogenic properties of live attenuated influenza vaccine (LAIV) by truncating the open reading frame of influenza virus non-structural protein 1 (NS1). The main objective of this study is to evaluate the immunogenic properties of the H1N1 seasonal LAIV strain by truncation of the NS1 protein to 126 amino acides. Materials and methods. Using reverse genetics technique, two H1N1 LAIV strains with full-length and truncated NS1 protein with three consecutive stop codons added after the 126th amino acid residue were obtained.C57BL/6J mice were immunized intranasally with the vaccine candidates, twice at a three-week interval. Seven days after the second immunization, cells were isolated from spleen and lung tissues and stimulated with whole wild-type H1N1 influenza virus. Levels of systemic and tissue-resident cytokine-producing CD4+ and CD8+ memory T cells were assessed by intracellular cytokine staining assay with flow cytometry. Replication of engineered vaccine strains in in vitro and in vivo systems was also evaluated. Results. Truncation of NS1 protein of the LAIV strain significantly increased the levels of virus-specific CD4+ effector memory T cells in spleens and the levels of CD4+ tissue-resident memory T cells in lungs of mice after two-dose immunization, indicating a higher potential for protection against influenza infection of the LAIV NS126 vaccine strain compared to the classical variant of LAIV. Importantly, the LAIV NS126 strain also had a more pronounced attenuated phenotype in mice than its classical counterpart.

Role of Influenza A virus protein NS1 in regulating host nuclear body ND10 complex formation and its involvement in establishment of viral pathogenesis.

Influenza viral infection is a seasonal infection which causes widespread acute respiratory issues among humans globally. This virus changes its surface receptor composition to escape the recognition process by the host's immune cells. Therefore, the present study focussed to identify some other important viral proteins which have a significant role in establishment of infection and having apparent conserved structural composition. This could facilitate the permanent vaccine development process or help in designing a drug against IAV (influenza A virus) infection which will eliminate the seasonal flu shot vaccination process. The NS1 (Non-structural protein 1) protein of IAV maintains a conserved structural motif. Earlier studies have shown its significant role in infection establishment. However, the mechanism by which viruses escape the host's ND10 antiviral action remains elusive. The present study clearly showed that IAV infection and NS1 transfection in A549 cells degraded the main component of the ND10 anti-viral complex, PML and therefore, inhibited the formation of Daxx-sp100-p53-PML complex (ND10) at the mid phase of infection/transfection. PML degradation activated the stress axis which increased cellular ROS (reactive oxygen species) levels as well as mitochondrial dysfunction. Additionally, IAV/NS1 increased cellular stress and p53 accumulation at the late phase of infection. These collectively activated apoptotic pathway in the host cells. Along with the inactivation of several interferon proteins, IAV was found to decrease p-IKKε. A549 cells transfected with pcDNA3.1-NS1 showed a similar effect in the interferon axis and IKKε. Moreover, NS1 induced the disintegration of the host's ND10 complex through the changes in the SUMOylation pattern of the PML nuclear body. These findings suggest the possible mechanism of how NS1 helps IAV to establish infection in the host cells. However, it demands further detailed study before targeting NS1 to develop permanent vaccines or novel drugs against IAV in future.

An Engineered Influenza a Virus Expressing the Co-Stimulator OX40L as an Oncolytic Agent Against Hepatocellular Carcinoma.

Oncolytic virus (OV) therapy has emerged as a promising novel form of immunotherapy. Moreover, an increasing number of studies have shown that the therapeutic efficacy of OV can be further improved by arming OVs with immune-stimulating molecules. In this study, we used reverse genetics to produce a novel influenza A virus, termed IAV-OX40L, which contained the immune-stimulating molecule OX40L gene in the influenza virus nonstructural (NS1) protein gene. The oncolytic effect of IAV-OX40L was explored on hepatocellular carcinoma (HCC)HCC cells in vitro and in vivo. Hemagglutination titers of the IAV-OX40L virus were stably 27-28 in specific-pathogen-free chicken embryos. The morphology and size distribution of IAV-OX40L are similar to those of the wild-type influenza. Expression of OX40L protein was confirmed by Western blot and immunofluorescence. MTS assays showed that the cytotoxicity of IAV-OX40L was higher in HCC cells (HepG2 and Huh7) than in normal liver cells (MIHA) in a time- and dose-dependent manner in vitro. We found that intratumoral injection of IAV-OX40L reduced tumor growth and increased the survival rate of mice compared with PR8-treated controls in vivo. In addition, the pathological results showed that IAV-OX40L selectively destroyed tumor tissues without harming liver and lung tissues. CD4+ and CD8+ T cells of the IAV-OX40L group were significantly increased in the splenic lymphocytes of mice. Further validation confirmed that IAV-OX40L enhanced the immune response mainly by activating Th1-dominant immune cells, releasing interferon-γ and interleukin-2. Taken together, our findings demonstrate the novel chimeric influenza OV could provide a potential therapeutic strategy for combating HCC and improve the effectiveness of virotherapy for cancer therapy.

Cellular PSMB4 Protein Suppresses Influenza A Virus Replication through Targeting NS1 Protein.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) possesses multiple functions, such as the inhibition of the host antiviral immune responses, to facilitate viral infection. To search for cellular proteins interacting with the IAV NS1 protein, the yeast two-hybrid system was adopted. Proteasome family member PSMB4 (proteasome subunit beta type 4) was found to interact with the NS1 protein in this screening experiment. The binding domains of these two proteins were also determined using this system. The physical interactions between the NS1 and cellular PSMB4 proteins were further confirmed by co-immunoprecipitation assay and confocal microscopy in mammalian cells. Neither transiently nor stably expressed NS1 protein affected the PSMB4 expression in cells. In contrast, PSMB4 reduced the NS1 protein expression level, especially in the presence of MG132. As expected, the functions of the NS1 protein, such as inhibition of interferon activity and enhancement of transient gene expression, were suppressed by PSMB4. PSMB4 knockdown enhances IAV replication, while its overexpression attenuates IAV replication. Thus, the results of this study suggest that the cellular PSMB4 protein interacts with and possibly facilitates the degradation of the NS1 protein, which in turn suppresses IAV replication.

Influenza virus NS1 interacts with 14-3-3ε to antagonize the production of RIG-I-mediated type I interferons

For influenza A viruses (IAVs), non-structural protein 1 (NS1) protein was recognized to be the key factor to enhance virulence by antagonizing host innate anti-viral responses. However, for the pathways allowing NS1 to regulate the type I interferon (IFN) response, the identification of the substrates was still incomplete. Here a recombinant IAV encoding a NS1 containing an affinity tag (NS1-Strep) was generated to capture the NS1-interactome in the lungs of infected mice. Several scaffold proteins of the 14-3-3 family were distinguished as the most potent candidates. Based on the conserved motif RxxTxxT of NS1, the interaction between NS1 and 14-3-3ε was enabled, which competed for the binding of RIG-I to 14-3-3ε and prevented RIG-I translocation to the adaptor MAVS, consequently inhibiting IFN-β expression. A recombinant mutant IAV deficient in 14-3-3ε binding elicited a markable innate immune responses and showed impaired growth kinetics.

Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity

BackgroundInfluenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infection. The influenza A virus (IAV) non-structural protein 1 (NS1) is a key viral factor shown to counteract type I IFN antiviral response mainly through targeting RIG-I signaling. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. Yet, it remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, then promoting viral propagation in the host.MethodsLuciferase reporter assays were conducted to determine the effect of NS1 and its mutants on the RIG-I and TLR3 pathways to the activation of the IFN-β and NF-κB promoters. Coimmunoprecipitation and confocal microscopic analyses were used to the interaction and colocalization between NS1 and TRAF3. Ubiquitination assays were performed to study the effect of NS1 and its mutants on TRAF3 ubiquitination. A recombinant mutant virus carrying NS1 E152A/E153A mutations was generated by reverse genetics for biochemical, ex vivo, and in vivo analyses to explore the importance of NS1 E152/E153 residues in targeting the RNA sensing-TRAF3-type I IFN axis and IAV pathogenicity.ResultsHere we report that NS1 subverts the RIG-I, TLR3, and TLR7 pathways to type I IFN production through targeting TRAF3 E3 ubiquitin ligase. NS1 harbors a conserved FTEE motif (a.a. 150-153), in which the E152/E153 residues are critical for binding TRAF3 to block TRAF3 ubiquitination and type I IFN production by these RNA sensing pathways. A recombinant mutant virus carrying NS1 E152A/E153A mutations induces higher type I IFN production ex vivo and in vivo, and exhibits the attenuated phenotype in infected mice, indicating the importance of E152/E153 residues in IAV pathogenicity.ConclusionsTogether our work uncovers a novel mechanism of IAV NS1-mediated immune evasion to promote viral infection through targeting the RNA sensing-TRAF3-type I IFN axis.

Influenza A virus NS1 induces degradation of sphingosine 1-phosphate lyase to obstruct the host innate immune response

The type I interferon (IFN)-mediated innate immune response is one of the central obstacles influenza A virus (IAV) must overcome in order to successfully replicate within the host. We have previously shown that sphingosine 1-phosphate (S1P) lyase (SPL) enhances IKKϵ-mediated type I IFN responses. Here, we demonstrate that the nonstructural protein 1 (NS1) of IAV counteracts the SPL-mediated antiviral response by inducing degradation of SPL. SPL was ubiquitinated and downregulated upon IAV infection or NS1 expression, whereas NS1-deficient IAV failed to elicit SPL ubiquitination or downregulation. Transiently overexpressed SPL increased phosphorylation of IKKϵ, resulting in enhanced expression of type I IFNs. However, this induction was markedly inhibited by IAV NS1. Collectively, this study reveals a novel strategy employed by IAV to subvert the type I IFN response, providing new insights into the interplay between IAV and host innate immunity.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Altering Intracellular Localization of the RNA Interference Factors by Influenza A Virus Non-structural Protein 1.

Influenza A virus (IAV) causes seasonal infections and periodic pandemics in humans. The non-structural protein 1 (NS1) of IAV is the main viral antagonist of the innate immune responses that play a key role in influenza pathogenesis. However, the mechanism to disrupt the host cell homeostasis by IAV NS1 remains poorly understood. Here, we show that expression of NS1 from the WSN strain, but not PR8 strain, of IAV, markedly induced nuclear import of the host RNA interference (RNAi) factors such as Argonaute-2 and microRNA 16. We found that the single residue substitution of aspartic acid with histidine at position 101 (D101H) of IAV-PR8 NS1 was sufficient to induce the nuclear import process and to enhance the virulence of IAV-PR8 in mice. However, we observed no significant differences between the wild-type and mutant IAV-PR8 in virus titers or induction of the interferon response in lung tissues, indicating a novel role of NS1 in the virulence determination of IAV in a mammalian host. Moreover, our bioinformatic analysis of 69,057 NS1 sequences from all IAV subtypes deposited in the NCBI database revealed that the NS1-H101 gene of IAV-WSN was widespread among H1N1 viruses isolated in 1933 but disappeared completely after 1940. Thus, IAV NS1 (H101) is a mutation selected against during evolution of IAV, suggesting that mutation H101 confers an important biological phenotype.

Examining effects of NS1 specific antibodies on sublethal influenza infection and secondary bacterial pneumonia in mice

Influenza is a highly contagious respiratory disease widespread throughout the world that causes disease in humans, birds and many mammalian species. Annually, around 20% of the global human gets sick with influenza so that more than 500,000 people die its various complications. Secondary bacterial pneumonia poses the peak threat during influenza infection, being most frequently caused by S. pneumoniae. Multiple studies in humans confirm the negative impact of influenza virus infection on subsequent outcome of bacterial pneumonia and provides insight into increased morbidity and mortality due to complicated influenza infection. In particular, the last 2009 influenza pandemic caused by H1N1 virus revealed that 25-56% cases of severe disease forms were associated with secondary pneumonia, among which 14-46% of them were fatal. Based on the aforementioned, it is of high priority to investigate a role of influenza virus proteins in developing of pathogen synergism in viral-bacterial pneumonia, particularly influenza virus non-structural protein NS1. The study objective was to examine effects of NS1-specific antibodies on course of influenza infection and secondary bacterial pneumonia in mice. For this, we used an experimental model of sublethal influenza infection followed by secondary Streptococcus pneumoniae bacterial pneumonia. Influenza A/Puerto Rico/8/34 (H1N1) virus and S. pneumoniae No. 3405 strain were used to simulate influenza infection. Rabbit serum containing antibodies against recombinant NS1 protein from A/Puerto Rico/8/34 virus and native rabbit serum (contain no specific antibodies) were used for vaccination. The study was carried out with female BALB/c mice, weighing 20-22 g. Protective activity of animal serum was assessed by using the three criteria: infection-related mortality, life expectancy and body weight change. The data obtained showed that passive transfer of antibodies specific to influenza virus NS1 protein did not lowered viral replication in sublethal murine model of influenza infection. Subsequent secondary bacterial pneumonia induced by S. pneumoniae revealed no protective effect of anti-NS1 protein antibodies assessed by measuring survival rate, lung viral and bacterial titers in treated vs. control mice.

Influenza virus NS1- C/EBPβ gene regulatory complex inhibits RIG-I transcription

Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPβ) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPβ phosphorylation and its recruitment to the RIG-I promoter as a C/EBPβ/NS1 complex. C/EBPβ overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPβ. Further, C/EBPβ phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses.

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

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

Structural basis for influenza virus NS1 protein block of mRNA nuclear export.

Influenza viruses antagonize key immune defense mechanisms via the virulence factor NS1 protein. A major NS1 strategy is to block nuclear export of host mRNAs including those encoding immune factors1–3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor NXF1•NXT1, which is the principal receptor to mediate docking and translocation of mRNAs through the nuclear pore complex by interacting with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1•NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1•NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1•NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. Together, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.

Sphingosine 1-phosphate lyase promotes the type I interferon-mediated innate immune response to influenza but is subjected to degradation by influenza A virus NS1

Abstract Influenza viruses cause seasonal and pandemic influenza, remaining a threat to human health. The type I interferon (IFN)-mediated innate immune response is one of the central obstacles influenza A virus (IAV) must overcome to successfully replicate within the host. Here, we demonstrate that sphingosine 1-phosphate (S1P) lyase (SPL) enhances the type I IFN response, but this antiviral innate immunity is counteracted by IAV infection. Although SPL is an enzyme that metabolizes S1P, SPL was found to interact with IKKɛ and promote IKKɛ-mediated type I IFN responses. Thus, when SPL was knocked out of host cells, IAV replication increased and the elements of IKKɛ-induced type I IFN response decreased. However, IAV infection destabilized the SPL-mediated type I IFN response by inducing the degradation of SPL. Importantly, nonstructural protein 1 (NS1) of IAV triggered the depletion of SPL. SPL was ubiquitinated upon IAV infection or NS1 expression, whereas NS1-deficient IAV failed to elicit ubiquitination or downregulation of SPL. Transiently overexpressed SPL increased the levels of auto-phosphorylation of IKKɛ, resulting in enhanced expression of type I IFN and IFN-stimulated genes. However, this induction was markedly inhibited by IAV NS1. Collectively, this study reveals a pro-IFN function of SPL as well as a novel strategy employed by IAV to subvert the type I IFN response, providing new insights into the interplay between IAV and host innate immunity.

Influenza virus NS1 protein upregulates epithelial sodium channel expression and function by inhibiting ubiquitin ligase Nedd4‐2

Influenza is a contagious respiratory virus that infects epithelial cells throughout the respiratory tract. The viral infection is facilitated by the attachment of the viral hemagglutinin to the sialic acid residues on the host cell membrane. Once the virus is inside the host cell, the acidic endosomal environment facilitates the release of the viral ribonucleoproteins, which are, subsequently, transported to the host cell nucleus for replication. The mechanism by which replicating influenza viruses affect the function of ion channels in the lung epithelia is not well understood. We have studied the effects of the matrix protein 2 (M2) on the Epithelial Sodium Channel (ENaC) and the Cystic Fibrosis Transmembrane Conductance regulator1,2. However, the effects of other viral proteins is still unknown. In this study we explored the mechanisms by which the Non‐Structural protein 1 (NS1) regulates the expression and the function of ENaC heterologously expressed in Xenopus Laevis oocytes. in‐vivo, NS1 acts by, mainly, inhibiting the host immune response. Herein, we found NS1 to act by a dual mechanism when heterologously expressed with ENaC in Xenopus Laevis oocytes. 1) We observe an inhibition of ENaC expression and function (−5.64±2.7 μA to −1.14±0.71 μA; p<0.001) when both cRNAs, ENaC's and NS1's, were consecutively injected the same day, NS1 acted by inhibiting the replication and proteins synthesis, a known effect of NS1 on host proteins and RNAs. 2) NS1 acted by enhancing ENaC expression and function when its cRNA was injected 24 hours after ENaC cRNA was injected, a situation simulating the infection of an epithelail cell by a flu virus. NS1 increased the expression of ENaC proteins at the surface of the oocytes, at least, three times the control values, measured by western blot. The increased ENaC proteins at the membrane of the oocyte resulted in a 3 fold increase of recorded amiloride sensitive sodium currents (from −6.62±0.67 μA to −17.15±0.62 μA; p<0.001) measured by the double voltage clamp technique. In conclusion, NS1 acted by increasing the number of active ENaC at the surface of oocytes. NS1 may have acted by inhibiting Nedd4‐2, the ubiquitin ligase, responsible th regulation of ENaC expression at the cell surface. Nedd4‐2 acts by ubiquitinilating and marking ENaC proteins for removal by endocytosis, internalization, and degradation in the proteasome or lysosome.Support or Funding Information5U01ES026458 and 1U01ES02769701This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-β gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.

MicroRNA expression profiles from HEK293 cells expressing H5N1 avian influenza virus non-structural protein 1.

H5N1 avian influenza poses a serious threat to the poultry industry and human health. Non-structural protein 1 (NS1) plays an important role in the replication and pathogenesis of avian influenza virus (AIV). However, the function of the NS1 gene is still unclear. In this study, illumina genome analyzer iix screening was used to identify the differentially expressed microRNAs (miRNAs) in HEK293 cells expressing H5N1 AIV NS1. There were 13 differentially expressed miRNAs (hsa-miR-17-5p, hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-31-5p, hsa-miR-20a-5p, hsa-miR-222-3p, hsa-miR-24-3p, hsa-miR-3613-3p, hsa-miR-3178, hsa-miR-4505, hsa-miR-345-3p, hsa-miR-3648, and hsa-miR-455-3p) (P < 0.01). The qRT-PCR validation results demonstrated that hsa-miR-221-3p, hsa-miR-22-3p, hsa-miR-20a-5p, and hsa-miR-3613-3p were upregulated, while hsa-miR-3178 and hsa-miR-4505 were down-regulated. The softwares targetscan and miranda were further used to predict their target genes, and the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that 20 GO terms and 20 KEGG pathways were significantly enriched. Our findings are the first to report expression profiling of miRNA and their functions in H5N1 AIV NS1-expressing HEK293 cells, and pave the way to further elucidating the accurate interaction mechanism between NS1 and virus replication, thus providing brand new insight into the prophylaxis and treatment of H5N1 AIV.

Global Interactomics Connect Nuclear Mitotic Apparatus Protein NUMA1 to Influenza Virus Maturation.

Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells.

Structural analyses reveal the mechanism of inhibition of influenza virus NS1 by two antiviral compounds

The influenza virus is a significant public health concern causing 250,000-500,000 deaths worldwide each year. Its ability to change quickly results in the potential for rapid generation of pandemic strains for which most individuals would have no antibody protection. This pandemic potential highlights the need for the continuous development of new drugs against influenza virus. As an essential component and well established virulence determinant, NS1 (nonstructural protein 1) of influenza virus is a highly prioritized target for the development of anti-influenza compounds. Here, we used NMR to determine that the NS1 effector domain (NS1ED) derived from the A/Brevig Mission/1/1918 (H1N1) strain of influenza (1918H1N1) binds to two previously described anti-influenza compounds A9 (JJ3297) and A22. We then used X-ray crystallography to determine the three-dimensional structure of the 1918H1N1 NS1ED Furthermore, we mapped the A9/A22-binding site onto our 1918H1N1 NS1ED structure and determined that A9 and A22 interact with the NS1ED in the hydrophobic pocket known to facilitate binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), suggesting that the two compounds likely attenuate influenza replication by inhibiting the NS1ED-CPSF30 interaction. Finally, our structure revealed that NS1ED could dimerize via an interface that we termed the α3-α3 dimer. Taken together, the findings presented here provide strong evidence for the mechanism of action of two anti-influenza compounds that target NS1 and contribute significant structural insights into NS1 that we hope will promote and inform the development and optimization of influenza therapies based on A9/A22.

A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity

Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals.IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines.