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

Figures

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  FIGURE 1. NS1 protein of WSN induces subcellular redistribution of RNAi factors in infected cells. Western and Northern blotting detection of RNA interference protein components and control RNAs in nuclear, cytoplasmic, and total extracts isolated from 293T cells 12 h after inoculation with buffer (mock), WSN, WSNΔNS1, Sendai, and PR8 viruses. Fractionation controls include β-actin, Mito-tRNA (cytoplasmic RNA), and U6 snRNA (nuclear RNA). Each experiment was repeated at least three times independently.

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  FIGURE 2. WSN NS1 protein presents in a complex with AGO2 in the 293T cells. Immunofluorescent analysis of 293T cells ectopically expressing Flag-AGO2 with WSN infection. (A) Flag-AGO2 and antibody-stained viral NS1 are co-localized in the infected cells. (B) Flag-AGO2 and antibody-stained viral NP are not co-localized in the infected cells. (C) Fluorescence intensity of nuclear DAPI and AGO2 in virus-infected or uninfected cells in (A). FLAG-tagged epitope representing AGO2 labeling with AlexaFluor488 (Green). Viral NS1 (A) and NP (B) labeling with AlexaFluor633 (Red). Nuclei counterstaining with DAPI (Blue). Immunoprecipitation of WSN NS1 with AGO2 protein. (D) 293T cells were co-transfected with a plasmid encoding WSN NS1 plus an empty plasmid or a plasmid encoding Flag-tagged AGO2 or Flag-tagged T7. (E) 293T cells were transfected with a plasmid encoding Flag-tagged AGO2, Flag-tagged T7, or empty vector before infection by IAV-WSN. Immunoprecipitations were performed with anti-FLAG antibody. Flag-tagged proteins and NS1 were detected with specific antibodies.

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  FIGURE 3. WSN NS1 induces nuclear importing of miRNAs in infected cells. Northern blotting detection of miR-16 or miR-21 in cytoplasmic and nuclear fractions from 293T cells transfected with a miRNA expression plasmid and infected by buffer (−), WSN or WSNΔNS1 viruses. Fractionation controls include Mito-tRNA (cytoplasmic RNA) and U6 snRNA (nuclear RNA). Each experiment was repeated at least three times independently.

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  FIGURE 4. Induction of AGO2 subcellular redistribution requires histidine at position 101 (H101) of NS1. (A) Western and northern blotting detection of RNAi protein components and miR-16 in nuclear and cytoplasmic extracts isolated from 293T cells 12 h after inoculation with buffer (Mock), PR8 7 + 1, PR8 D101H, PR8 A155T, or PR8 D189N viruses. Fractionation controls include β-actin, GAPDH (cytoplasmic protein), Sp1 (nuclear protein), Mito-tRNA (cytoplasmic RNA), and U6 snRNA (nuclear RNA). (B) Amino acid sequence alignment of the NS1 proteins of influenza A virus WSN, PR8, and TX 91; three non-conservative amino acids are labeled at positions 101, 155, and 189. (C) Representative plaque assay images of the WT and recombinant mutant viruses on MDCK cells. (D) Multiple-cycle growth curves of the WT and recombinant mutant viruses on MDCK cells. Cells were infected by WT and recombinant mutant viruses of the same titer. Every 12 h post infection (h.p.i.), the virus titers were determined by plaque assay and quantified as plaque-forming units (p.f.u.) per ml. Mean titer values at each time point were plotted. Each experiment was repeated at least three times independently.

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  FIGURE 5. H101 mutation of NS1 contributes to viral pathogenesis in mice. (A) The survival of BALB/c mice was monitored. Weight loss of BALB/c mice was measured daily after intranasal inoculation with Mock, PR8 WT, or PR8 D101H virus (**p < 0.01; log rank). Mock infected (n = 5); PR8 WT infected (n = 10); PR8 D101H infected (n = 10). (B) Representative lung H&E slice images of BALB/c mice after intranasal inoculation with Mock (left panel), PR8 WT (middle panel), or PR8 D101H virus (right panel). Lungs were harvested 3 days after infection. Sections through the main bronchiole of the left lobe were stained with hematoxylin and eosin. Black arrows indicate hemorrhage, red arrow indicates inflammation, and blue arrows indicate necrosis. Scale bar: 100 μm.

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  FIGURE 6. No significant difference in the accumulation of the two viruses in lung tissue. (A) Influenza viral titers were determined by qPCR in the lungs of BALB/c mice infected with PR8 WT and PR8 D101H viruses. Lungs were harvested 3 days after infection. Virus titers were quantified by relative HA expression. Differences between groups were analyzed by an unpaired t-test (ns, not significant). (B) Representative lung IHC slice images of BALB/c mice intranasally inoculated with Mock (left panel), PR8 WT virus (middle panel), or PR8 D101H virus (right panel). Lungs were harvested 3 days after infection. Viral infection was confirmed by immunohistochemistry with influenza A virus M1 polyclonal antibody (upper panel) and NS1 monoclonal antibody (lower panel). Sections through the main bronchiole of the left lobe were stained with antibody. Scale bar: 50 μm.

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  FIGURE 7. No significant difference in the expression level of innate immune-related genes between the two viruses in vivo. Expression of type I IFNs (A), RIG-I (B), and Mavs (C) in the lungs of BALB/c mice following influenza infection were determined by qPCR. Lungs were harvested 3 days after infection. Differences between groups were analyzed by unpaired t-test (ns, not significant), N.D., not detectable.

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  FIGURE 8. NS1 (H101)-encoding IAV isolates are rare. The pattern of the 101st amino acids and underlying codons of 69,057 influenza NS1 proteins. The dominant codon GAC encoding aspartic acid (56%) is regarded as the ancient codon, whereas GAU (aspartic acid, 6%), AAC (asparagine, 30%), GAA (glutamic acid, 7%), and CAC (histidine, 0.032%) are thought to be derived from GAC with minimal nucleotide mutations.