Journal: Journal of Virology

Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

doi: 10.1128/JVI.01039-16

Figure Lengend Snippet: Effect of IAV H3N2 NS1 protein mutations on innate immune responses induced by SeV infection. Human 293T cells were transiently transfected using the calcium phosphate method with the indicated pCAGGS NS1-expressing plasmids, together with plasmids expressing Fluc under the control of an IFN-β (A) or ISRE promoter (B). At 24 hpt, cells were mock infected (M) or infected with SeV (Cantell strain) (+SeV) to induce activation of the promoters, and 16 hpi cell lysates were prepared for reporter gene expression. (A and B) Reporter Firefly expression was measured by luminescence. Data represented show the means and standard deviations of the results determined for triplicate wells. Experiments were repeated 3 times in triplicate wells with similar results. P values determined using Student's t test are indicated. (C) At 16 h after SeV infection, TCS were collected and, after UV inactivation, were used to treat fresh A549 cells. Alternatively, A549 cells were treated with 2,500 U/ml of universal IFN-α as a control (picture on the right). After 24 h of incubation, cells were infected (MOI, 0.001) with the IFN-sensitive VSV-GFP. At 16 hpi, VSV-GFP-infected cells were observed under a fluorescence microscope. (D) At 16 h after SeV infection, cellular extracts were obtained and the expression of ISG15 and actin were evaluated by Western blotting, using antibodies specific to ISG15 and to actin (as a loading control). Western blots were quantified by densitometry using the software ImageJ (v1.46), and the amounts of ISG15 protein were normalized to the amounts of actin protein (numbers below the ISG15 blot; ND, not detected). Molecular mass markers (in kilodaltons) are indicated on the right. The squares represent the NS1 variants found in the subjects , showing the numbers of patients for each NS1 variant below the squares.

Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

Techniques: Infection, Transfection, Expressing, Activation Assay, Incubation, Fluorescence, Microscopy, Western Blot, Software, Variant Assay

Journal: Journal of Virology

Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

doi: 10.1128/JVI.01039-16

Figure Lengend Snippet: Mutation I64T in IAV 1918 H1N1 NS1 affects its ability to inhibit general gene expression and innate immune responses. (A, B, and C) Human 293T cells were transiently cotransfected with pCAGGS plasmids expressing the different NS1 variants indicated and the WT 1918-NS1 (E26, I64, and R224), along with pCAGGS plasmids expressing the reporter proteins GFP and Gluc. (A) At 30 hpt, GFP expression was visualized using a fluorescence microscope. (B) Gluc expression was analyzed at 30 hpt using luminescence. Error bars represent the standard deviations for triplicates. P values using Student's t test are indicated. (C) In addition, NS1 and actin expression levels were analyzed by Western blotting from cell extracts using antibodies specific to the HA tag (to detect the NS1 protein) and to actin as a loading control. Western blots were quantified by densitometry using the software ImageJ (v1.46), and the amounts of NS1 protein were normalized to the amounts of actin protein. Protein expression in cells transfected with the PR8-NS1-expressing plasmid was considered to be 100% for comparison with the level of expression by the other NS1 variants (numbers below the NS1 blot). Molecular mass markers (in kilodaltons) are indicated on the right. Three different experiments were performed, with similar results. (D and E) Human A549 cells were transfected with pCAGGS plasmids expressing the 1918-NS1-I64, 1918-NS-T64, or PR8-NS1 protein using DNA-IN. At 24 hpt, cells were either transfected with 100 ng of poly(I·C) (D) or treated with 250 U/ml of universal IFN-α (E) to induce an antiviral cellular state. At 16 h posttreatment, cells were infected (MOI, 0.001) with VSV-GFP, and viral titers in the TCS were determined at 21 hpi. Bars represent the standard deviations from triplicates. Experiments were repeated 3 times in triplicate wells with similar results. P values using Student's t test are indicated.

Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

Techniques: Mutagenesis, Expressing, Fluorescence, Microscopy, Western Blot, Software, Transfection, Plasmid Preparation, Infection

Journal: Journal of Virology

Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

doi: 10.1128/JVI.01039-16

Figure Lengend Snippet: Recombinant virus growth kinetics in vitro . (A) Scheme of the pDZ plasmid encoding the NS split segment used to rescue the viruses r65 and r85 (top lane) as well as the NS segment encoding the NS1 and NEP proteins (second lane) being processed by the porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site (third lane). Viral 3′ and 5′ noncoding regions are indicated with black boxes at the end of the viral segment. Viral products from the NS (NS1 and NEP) segment are indicated with white boxes. The region before the splicing donor in the viral segments is indicated with light gray boxes. The sequence of the PTV-1 2A autoproteolytic cleavage site (2A) is indicated with dark gray boxes. The sequences encoding the polymerase (Pol) I and II promoters, the Pol I promoter terminator (T), and the poly(A) tail (pA) are indicated. The position of restriction sites EcoRI and BsmBI, used to clone the NS1 sequences from patients 65 and 85, is shown. (B) Canine MDCK (left) and human A549 (right) cells were infected in duplicate with recombinant PR8 viruses expressing either 65 and 85 H3N2 NS1 proteins or the PR8 NS1 protein (wt-PR8-NSs) at an MOI of 0.001. Virus titers in infected cell supernatants were determined at different times postinfection by immunofocus assay. (C) MDCK cells were infected (MOI, 0.001) with viruses r65 and r85, and just after the infection, cells were left untreated or were treated with 2,000 U of IFN-α/well. Supernatants were collected at 24 and 48 hpi, and virus titers were determined by immunofocus assay. (Left) The percentage of growth in MDCK cells treated with IFN-α (+) was normalized to the growth in the nontreated cells (−) at 24 and 48 hpi (considered 100% growth). (Right) Virus titers (in FFU/ml) in nontreated (−) and IFN-treated (+) cells are shown. The experiments were repeated 3 times in duplicate wells. The dotted line in panel B indicates the limit of detection.

Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

Techniques: Recombinant, In Vitro, Plasmid Preparation, Sequencing, Infection, Expressing

Journal: Journal of Virology

Article Title: NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

doi: 10.1128/JVI.01039-16

Figure Lengend Snippet: Induction of IFN responses on PBMCs from infected subjects. PBMCs from subject 85 (infected with the virus encoding T64-NS1; in gray) and from subjects 64, 68, 87, 21, 23, and 44 (infected with viruses encoding I64-NS1; in black) were either treated with 2,000 U/ml of IFN-α (A) or infected (MOI, 1) with virus r85 (B). The levels of mRNA expression of cellular IFIT2 (A), IFN-λ1 (B), or gene M mRNA in the infected PBMCs (C) were analyzed by qRT-PCR. Bars represent standard deviations of the means from triplicates. P values determined using Student's t test are indicated. The experiments were repeated twice independently, using technical triplicates, with similar results. r.u., relative units.

Article Snippet: In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora).

Techniques: Infection, Expressing, Quantitative RT-PCR