Role of diverse NS1 influenza segments in the infection of human bronchial epithelial cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE184017 | GEO | 2021/11/22
REPOSITORIES: GEO
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Project description:Role of diverse NS1 influenza segments in the infection of human bronchial epithelial cells
| PRJNA762860 | ENA
Project description:The influenza virus is opportunistic by nature and is capable of subverting cellular signalling pathways for the benefit of viral replication. The function of non-structural protein 1 (NS1) as a core component for the influenza virus to interfere with host antiviral activity has been well cited in literature. However, the mechanism as to how NS1 is capble of subverting the host's innate immune system is unknown. NS1 has a unique capability to migrate between both the cytoplasm and nucleus of infected cells. To assess if NS1 interference operates a stratagy pertaining to a mechanism involved with nuclear-located signalling processes we designed two mutant virsus; WSNdelNS1 which encodes a deletion to prevent complete NS1 expression and WSN3M which encodes a NS1-variant that is defective in NS1 nuclear-translocation, thus preventing NS1 from entering the nuclus of infected cells.
2020-08-12 | GSE156060 | GEO
Project description:Gene transcription effects of mutations in the infuenza virus A/Hong Kong/1/1968(H3N2) nonstructural 1 NS1 gene in infected human A549 (lung epithilium) cells 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 (manuscript submitted to Virology Journal). Human cells were infected with influenza viruses mutants with specific gain of function mutations in the NS1 gene in order to assess the affects of each mutation on host gene expression. Human (A549) and mouse (M1) cells were infected at a multiplicity of infection of 2 (infectious viruses/cell) and incubated for 8 hr before collection of total RNA and microarray anlaysis using the Affymetrix platforms. Samples were compared in triplicate to mock PBS infected (uninfected) cells to detecte dysregulated genes for A/HK/1/1968(H3N2) wt, and the following NS1 gene mutants: F103L, M106I, M106V, and F103L + M106I. Background: The 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/1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. Methods: NS1 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 proprieties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, effect on host gene transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. Results: Each 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-M-NM-2 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 partially or totally restored by the F103L or M106I mutations respectively. Conclusions: The 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 viruses to switch hosts and cause severe disease in mammals. Triplicate biological replicates of mock PBS treated (uninfected) cells to detecte dysregulated genes for A/HK/1/1968(H3N2) wt, and the following NS1 gene mutants: F103L, M106I, M106V, and F103L + M106I. Cells were infected at a multiplicty of infection of 2 and cells were incubated for 8 hr at 37 C for 8 hrs before RNA extraction and analysis of 3 biological replicates relative to mock PBS infected cells.
2013-06-22 | E-GEOD-48200 | biostudies-arrayexpress
Project description:Gene transcription effects of mutations in the infuenza virus A/Hong Kong/1/1968(H3N2) nonstructural 1 NS1 gene in infected human A549 (lung epithilium) cells 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 (manuscript submitted to Virology Journal). Human cells were infected with influenza viruses mutants with specific gain of function mutations in the NS1 gene in order to assess the affects of each mutation on host gene expression. Human (A549) and mouse (M1) cells were infected at a multiplicity of infection of 2 (infectious viruses/cell) and incubated for 8 hr before collection of total RNA and microarray anlaysis using the Affymetrix platforms. Samples were compared in triplicate to mock PBS infected (uninfected) cells to detecte dysregulated genes for A/HK/1/1968(H3N2) wt, and the following NS1 gene mutants: F103L, M106I, M106V, and F103L + M106I. Background: The 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/1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. Methods: NS1 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 proprieties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, effect on host gene transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. Results: Each 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 partially or totally restored by the F103L or M106I mutations respectively. Conclusions: The 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 viruses to switch hosts and cause severe disease in mammals.
2013-06-22 | GSE48200 | GEO
Project description:We defined the major transcriptional responses in primary human bronchial epithelial cells (HBECs) after either infection with influenza or treatment with relevant ligands. We used four different strategies, each highlighting distinct aspects of the response. (1) cells were infected with the wild-type PR8 influenza virus that can mount a complete replicative cycle. (2) cells were transfected with viral RNA (âvRNAâ) isolated from influenza particles. This does not result in the production of viral proteins or particles and identifies the effect of RNA-sensing pathways (e.g., RIG-I.). (3) Cells were treated with interferon beta (IFNb), to distinguish the portion of the response which is mediated through Type I IFNs. (4) Cells were infected with a PR8 virus lacking the NS1 gene (âDNS1â). The NS1 protein normally inhibits vRNA- or IFNb-induced pathways, and its deletion can reveal an expanded response to infection. HBECs were stimulated with a 15 minute pulse of 1000U/ml IFNÃ (PBL, Piscataway, NJ), 100ng/ml vRNA (purified directly from PR8 virus) with LTX transfection reagent (Invitrogen; Carlsbad, California), wild type H1N1 influenza (A/PR/8/34) or ?NS1 virus (PR8 with a deleted NS1 gene, gift from Dr. Garcia-Sastre). Viruses were used at a multiplicity of infection (moi) of 5. Control samples were incubated with media or LTX under the same conditions. Cells were washed, supplemented with warm media and harvested at 11 timepoints (0, .25, .5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). HBECs were seeded in 6 well plates at a concentration of 250,000/well 18 hours prior to stimultaion. Cells were stimulated with a 15 minute pulse of IFNb, vRNA, infected with PR8 influenza or NS1 deleted influenza, or mock treated
2010-04-13 | E-GEOD-19392 | biostudies-arrayexpress
Project description:Influenza A virus (IAV), one of the most prevalent respiratory diseases, causes pandemics around the world. The multifunctional non-structural protein 1 (NS1) of IAV is a viral antagonist that suppresses host antiviral response. However, the mechanism by which NS1 modulates the RNA interference (RNAi) pathway remains unclear. Here, we identified interactions between NS1 proteins of Influenza A/PR8/34 (H1N1; IAV-PR8) and Influenza A/WSN/1/33 (H1N1; IAV-WSN) and Dicer’s cofactor TAR-RNA binding protein (TRBP). We found that the N-terminal RNA binding domain (RBD) of NS1 and the first two domains of TRBP protein mediated this interaction. Furthermore, two amino acid residues (Arg at position 38 and Lys at position 41) in NS1 were essential for the interaction. We generated TRBP knockout cells and found that NS1 instead of NS1 mutants (two-point mutations within NS1, R38A/K41A) inhibited the process of microRNA (miRNA) maturation by binding with TRBP. PR8-infected cells showed masking of short hairpin RNA (shRNA)-mediated RNAi, which was not observed after mutant virus-containing NS1 mutation (R38A/K41A, termed PR8/3841) infection. Moreover, abundant viral small interfering RNAs (vsiRNAs) were detected in vitro and in vivo upon PR8/3841 infection. We identify, for the first time, the interaction between NS1 and TRBP that affects host RNAi machinery.
2022-04-15 | GSE189776 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE39199: Gene Expression Response to Interferon Treatment at 12h and 24 in A549 cells GSE39200: The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways Refer to individual Series
2012-07-09 | E-GEOD-39202 | biostudies-arrayexpress
Project description:Influenza non-structural protein 1 (NS1) has been well cited to ellicit an inhibitory effect of immune gene expression thus resulting in the curtailment of efficient antiviral immunity. However the mechanisms and gene networks subjected to this immuno-suppression are unclear. We designed an RNA-Seq study to explore this viral trait and characterize the induced gene expression in response to wildtype (native) Influenza A infection. We have also developed an Influenza mutant virus that is deficient in its ability to expression NS1 (WSNdelNS1), thus our study incorporates a virus that will enable induced transcriptomic comparison between wildtype and mutant viral infections.
2020-08-13 | GSE156152 | GEO
Project description:We defined the major transcriptional responses in primary human bronchial epithelial cells (HBECs) after either infection with influenza or treatment with relevant ligands. We used four different strategies, each highlighting distinct aspects of the response. (1) cells were infected with the wild-type PR8 influenza virus that can mount a complete replicative cycle. (2) cells were transfected with viral RNA (‘vRNA’) isolated from influenza particles. This does not result in the production of viral proteins or particles and identifies the effect of RNA-sensing pathways (e.g., RIG-I.). (3) Cells were treated with interferon beta (IFNb), to distinguish the portion of the response which is mediated through Type I IFNs. (4) Cells were infected with a PR8 virus lacking the NS1 gene (‘DNS1’). The NS1 protein normally inhibits vRNA- or IFNb-induced pathways, and its deletion can reveal an expanded response to infection.
2010-03-30 | GSE19392 | GEO
Project description:The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models.
2012-07-10 | GSE39200 | GEO