H5N1 NS1 Research Articles

A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection.

The non-structural protein, NS1, is a virulence factor encoded by influenza A viruses (IAVs). In this report, we provide evidence that the conserved residue, tyrosine (Y) 84, in a conserved putative SH2-binding domain in A/Duck/Hubei/2004/L-1 [H5N1] NS1 is critical for limiting an interferon (IFN) response to infection. A phenylalanine (F) substitution of this Y84 residue abolishes NS1-mediated downregulation of IFN-inducible STAT phosphorylation, and surface IFNAR1 expression. Recombinant IAV (rIAV) [H1N1] expressing A/Grey Heron/Hong Kong/837/2004 [H5N1] NS1-Y84F (rWSN-GH-NS1-Y84F) replicates to lower titers in human lung epithelial cells and is more susceptible to the antiviral effects of IFN-β treatment compared with rIAV expressing the intact H5N1 NS1 (rWSN-GH-NS1-wt). Cells infected with rWSN-GH-NS1-Y84F express higher levels of IFN stimulated genes (ISGs) associated with an antiviral response compared with cells infected with rWSN-GH-NS1-wt. In mice, intranasal infection with rWSN-GH-NS1-Y84F resulted in a delay in onset of weight loss, reduced lung pathology, lower lung viral titers and higher ISG expression, compared with mice infected with rWSN-GH-NS1-wt. IFN-β treatment of mice infected with rWSN-GH-NS1-Y84F reduced lung viral titers and increased lung ISG expression, but did not alter viral titers and ISG expression in mice infected with rWSN-GH-NS1-wt. Viewed altogether, these data suggest that the virulence associated with this conserved Y84 residue in NS1 is, in part, due to its role in regulating the host IFN response.

The Influenza A Virus Protein NS1 Displays Structural Polymorphism

NS1 of influenza A virus is a potent antagonist of host antiviral interferon responses. This multifunctional protein with two distinctive domains, an RNA-binding domain (RBD) and an effector domain (ED) separated by a linker region (LR), is implicated in replication, pathogenesis, and host range. Although the structures of individual domains of NS1 from different strains of influenza viruses have been reported, the only structure of full-length NS1 available to date is from an H5N1 strain (A/Vietnam/1203/2004). By carrying out crystallographic analyses of full-length H6N6-NS1 (A/blue-winged teal/MN/993/1980) and an LR deletion mutant, combined with mutational analysis, we show here that these full-length NS1 structures provide an exquisite structural sampling of various conformational states of NS1 that based on the orientation of the ED with respect to RBD can be summarized as "open," "semi-open," and "closed" conformations. Our studies show that preference for these states is clearly dictated by determinants such as linker length, residue composition at position 71, and a mechanical hinge, providing a structural basis for strain-dependent functional variations in NS1. Because of the flexibility inherent in the LR, any particular NS1 could sample the conformational space around these states to engage ED in different quaternary interactions so that it may participate in specific protein-protein or protein-RNA interactions to allow for the known multifunctionality of NS1. We propose that such conformational plasticity provides a mechanism for autoregulating NS1 functions, depending on its temporal distribution, posttranslational modifications, and nuclear or cellular localization, during the course of virus infection. NS1 of influenza A virus is a multifunctional protein associated with numerous strain-specific regulatory functions during viral infection, including conferring resistance to antiviral interferon induction, replication, pathogenesis, virulence, and host range. NS1 has two domains, an RNA-binding domain and an effector domain separated by a linker. To date, the only full-length NS1 structure available is that from an H5N1 strain (A/Vietnam/1203/2004). Here, we determined crystal structures of the wild type and a linker region mutant of the H6N6 NS1 (A/blue-winged teal/MN/993/1980), which together with the previously determined H5N1 NS1 structure show that NS1 exhibits significant strain-dependent structural polymorphism due to variations in linker length, residue composition at position 71, and a mechanical hinge. Such a structural polymorphism may be the basis for strain-specific functions associated with NS1.

The non-structural (NS) gene segment of H9N2 influenza virus isolated from backyard poultry in Pakistan reveals strong genetic and functional similarities to the NS gene of highly pathogenic H5N1

Apart from natural reassortment, co-circulation of different avian influenza virus strains in poultry populations can lead to generation of novel variants and reassortant viruses. In this report, we studied the genetics and functions of a reassorted non-structural gene (NS) of H9N2 influenza virus collected from back yard poultry (BYP) flock. Phylogenetic reconstruction based on hemagglutinin and neuraminidase genes indicates that an isolate from BYP belongs to H9N2. However, the NS gene-segment of this isolate cluster into genotype Z, clade 2.2 of the highly pathogenic H5N1. The NS gene plays essential roles in the host-adaptation, cell-tropism, and virulence of influenza viruses. However, such interpretations have not been investigated in naturally recombinant H9N2 viruses. Therefore, we compared the NS1 protein of H9N2 (H9N2/NS1) and highly pathogenic H5N1 (H5N1/NS1) in parallel for their abilities to regulate different signaling pathways, and investigated the molecular mechanisms of IFN-β production in human, avian, and mink lung cells. We found that H9N2/NS1 and H5N1/NS1 are comparably similar in inhibiting TNF-α induced nuclear factor κB and double stranded RNA induced activator protein 1 and interferon regulatory factor 3 transcription factors. Thus, the production of IFN-β was inhibited equally by both NS1s as demonstrated by IFN stimulatory response element and IFN-β promoter activation. Moreover, both NS1s predominantly localized in the nucleus when transfected to human A549 cells. This study therefore suggests the possible increased virulence of natural reassortant viruses for their efficient invasion of host immune responses, and proposes that these should not be overlooked for their epizootic and zoonotic potential.

Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30

BackgroundThe genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species.MethodsNS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed.ResultsEach of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.ConclusionsThe F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

Highly Pathogenic H5N1 Influenza A Virus Strains Provoke Heterogeneous IFN-α/β Responses That Distinctively Affect Viral Propagation in Human Cells

The fatal transmissions of highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-α/β) are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-α/β gene activation by different avian and human H5N1 isolates, if the IFN-α/β response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-α/β system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-β, whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-α/β activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-α/β induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-α/β activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to overcome the human IFN-α/β barrier involve mutations in multiple H5N1 genes.

Methionine-101 from one strain of H5N1 NS1 protein determines its IFN-antagonizing ability and subcellular distribution pattern

Influenza A virus NS1 protein has developed two main IFN-antagonizing mechanisms by inhibiting retinoic-acid-inducible gene I (RIG-I) signal transduction, or by suppressing cellular pre-mRNA processing through binding to cleavage and polyadenylation specific factor 30 (CPSF30). However, the precise effects of NS1 on suppressing type I IFN induction have not been well characterized. Here we report that compared with PR/8/34 NS1, which is localized partially in the cytoplasm and has strong IFN-antagonizing ability via specifically inhibiting IFN-β promoter activity, H5N1 NS1 has strikingly different characteristics. It mainly accumulates in the nucleus of transfected cells and exerts rather weak IFN-counteracting ability through suppression of the overall gene expression. The M101I mutation of H5N1 NS1, namely H5-M101I, fully reversed its functions. H5-M101I gained the ability to specifically inhibit IFN-β promoter activity, translocate to the cytoplasm, and release CPSF30. The previously reported NES (nuclear export signal) (residues 138-147) was unable to lead H5N1 NS1 to translocate. This suggests that other residues may serve as a potent NES. Findings indicated that together with leucine-100, methionine-101 enhanced the regional NES. In addition, methionine-101 was the key residue for the NS1-CPSF30 interaction. This study reveals the importance of methionine-101 in the influenza A virus life cycle and may provide valuable information for antiviral strategies.

The Virulence of 1997 H5N1 Influenza Viruses in the Mouse Model Is Increased by Correcting a Defect in Their NS1 Proteins

The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3' end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-β) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.

Modification of Nonstructural Protein 1 of Influenza A Virus by SUMO1

Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.

Inhibition of host innate immune responses and pathogenicity of recombinant Newcastle disease viruses expressing NS1 genes of influenza A viruses

The NS1 protein has been associated with the virulence of influenza A viruses. To evaluate the role of the NS1 protein in pathogenicity of pandemic H5N1 avian influenza and H1N1 2009 influenza viruses, recombinant Newcastle disease viruses (rNDVs) expressing NS1 proteins were generated. Expression of the NS1 proteins resulted in inhibition of host innate immune responses (beta interferon and protein kinase R production). In addition, the NS1 proteins were localized predominantly in the nucleus of virus-infected cells. Consequently, expression of the NS1 protein contributed to an increase in pathogenicity of rNDV in chickens. In particular, mutational analysis of H5N1 NS1 protein indicated that both the RNA-binding and effector domains affect virus pathogenicity synergistically. Our study also demonstrated that expression of H1N1/09 NS1 resulted in enhanced replication of rNDV in human cells, indicating that function of the NS1 proteins can be host-species-specific.

H5N1 NS1 change the cell cytoskeleton and interferes with host cell motility through the GTPase

H5N1 NS1 change the cell cytoskeleton and interferes with host cell motility through the GTPase

A new panel of NS1 antibodies for easy detection and titration of influenza A virus

The non-structural protein NS1 of the influenza A virus is a good target for the development of diagnostic assays. In this study, three NS1 monoclonal antibodies (mAbs) were generated by using recombinant NS1 protein of H5N1 virus and found to bind both the native and denatured forms of NS1. Two of the mAbs, 6A4 and 2H6, bind NS1 of three different strains of influenza A virus, namely H1N1, H3N2, and H5N1. Epitope mapping revealed that residues 42-53 of H5N1 NS1 are essential for the interaction with both mAbs. Between the three strains, there is only one amino acid difference in this domain, which is consistent with the observed cross-reactivities. On the other hand, mAb 1G1 binds to residues 206-215 of H5N1 NS1 and does not bind NS1 of H1N1 or H3N2. Furthermore, all three mAbs detected NS1 proteins expressed in virus infected MDCK cells and indirect immunofluorescence staining with mAbs 6A4 and 2H6 provided an alternative method for viral titer determination. Quantifying the numbers of fluorescent foci units yielded viral titers for three different isolates of H5N1 virus that are highly comparable to that obtained by observing cytopathic effect induced by virus infection. Importantly, this alternative method yields results at 1 day post-infection while the conventional method using cytopathic effect yields results at 3 days post-infection. The results showed that this new panel of NS1 antibodies can detect NS1 protein expressed during viral infection and can be used for fast and easy titration of influenza A virus. J. Med. Virol. 82:467-475, 2010. (c) 2010 Wiley-Liss, Inc.

Prediction of micro RNAs against H5N1 and H1N1 NS1 Protein: a Window to Sequence Specific Therapeutic Development

Motivation: Conventional Influenza drugs might result in failure of action due to highly variable nature of influenza antigenic proteins. in silico prediction of sequence specific therapeutics is less time consuming and cost effective. Most of the miRNA target prediction platforms differ in their specificity and sensitivity in prediction. Here we propose a bioinformatics approach to predict micro RNAs against non-structural protein 1 of avian influenza H5N1 virus and against segment 8 swine influenza H1N1 virus. Results: hsa-miR-138, hsa-miR-525-5p and hsa-miR-124 were sorted out as potential sequence specific therapeutic agents against NS1 protein of H5N1 virus. Similar studies on different H1N1 segment 8 genomes resulted in the prediction of miRNAs which can be used as anti-influenza agents. The predicted micro RNAs also possess roles in regulatory processes of host cell, stress related pathways including MAPK pathway, mTOR pathway and are found to be involved in pathways related to cancer.

X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus

Recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns1,2. Several studies have underscored the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains3,4. NS1, which consists of two domains, a dsRNA binding domain (RBD)5,6 and the effector domain (ED)7 separated through a linker, is an antagonist of antiviral type-I interferon (IFN) response in the host8,9. Here we report the x-ray structure of the full length NS1 from a H5N1 (A/Vietnam/1203/2004) strain that was associated with 60% of human deaths in an outbreak in Vietnam1,2. Compared to the individually determined structures of RBD and ED from non-H5N1 strains, the RBD within H5N1 NS1 exhibits modest structural changes, while the H5N1 ED shows significant alteration particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions observed in the cryo-EM images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1 in which residues implicated in dsRNA binding face a 20 Å wide central tunnel provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.

Avian Influenza Virus A/HK/483/97(H5N1) NS1 Protein Induces Apoptosis in Human Airway Epithelial Cells

Avian H5N1 influenza virus causes a remarkably severe disease in humans, with an overall case fatality rate of greater than 50%. Human influenza A viruses induce apoptosis in infected cells, which can lead to organ dysfunction. To verify the role of H5N1-encoded NS1 in inducing apoptosis, the NS1 gene was cloned and expressed in human airway epithelial cells (NCI-H292 cells). The apoptotic events posttransfection were examined by a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling assay, flow cytometric measurement of propidium iodide, annexin V staining, and Western blot analyses with antibodies specific for proapoptotic and antiapoptotic proteins. We demonstrated that the expression of H5N1 NS1 protein in NCI-H292 cells was sufficient to induce apoptotic cell death. Western blot analyses also showed that there was prominent cleavage of poly(ADP-ribose) polymerase and activation of caspase-3, caspase-7, and caspase-8 during the NS1-induced apoptosis. The results of caspase inhibitor assays further confirmed the involvement of caspase-dependent pathways in the NS1-induced apoptosis. Interestingly, the ability of H5N1 NS1 protein to induce apoptosis was much enhanced in cells pretreated with Fas ligand (the time posttransfection required to reach >30% apoptosis was reduced from 24 to 6 h). Furthermore, 24 h posttransfection, an increase in Fas ligand mRNA expression of about 5.6-fold was detected in cells transfected with H5N1 NS1. In conclusion, we demonstrated that the NS1 protein encoded by avian influenza A virus H5N1 induced apoptosis in human lung epithelial cells, mainly via the caspase-dependent pathway, which encourages further investigation into the potential for the NS1 protein to be a novel therapeutic target.

Homology modeling and examination of the effect of the D92E mutation on the H5N1 nonstructural protein NS1 effector domain

Virulent H5N1 strains of influenza virus often harbor a D92E point mutation in the nonstructural protein NS1. This crucial mutation has been correlated with increased virulence and/or cytokine resistance, but the structural implications of such a change are still unclear. Furthermore, NS1 protein could also be a potential target for the development of novel antiviral agents against H5N1 strains. Therefore, a reasonable 3D model of H5N1 NS1 is important for the understanding of the molecular basis of increased virulence and the design of novel antiviral agents. Based on the crystal structure of a non-H5N1 NS1 protein, a model of H5N1 NS1 was developed by homology modeling, molecular mechanics and molecular dynamics simulations. It was found that the D92E mutation could result in weakened interactions of the carboxylate side chain with other phosphorylated residues, thereby activating phosphorylation of NS1.