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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

ABSTRACT: 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

PROVIDER: PRJNA209214 | ENA |

REPOSITORIES: ENA

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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.

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.

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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

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