Multiple anti-interferon actions of the influenza A virus NS1 protein - PubMed (original) (raw)

Multiple anti-interferon actions of the influenza A virus NS1 protein

Georg Kochs et al. J Virol. 2007 Jul.

Abstract

The replication and pathogenicity of influenza A virus (FLUAV) are controlled in part by the alpha/beta interferon (IFN-alpha/beta) system. This virus-host interplay is dependent on the production of IFN-alpha/beta and on the capacity of the viral nonstructural protein NS1 to counteract the IFN system. Two different mechanisms have been described for NS1, namely, blocking the activation of IFN regulatory factor 3 (IRF3) and blocking posttranscriptional processing of cellular mRNAs. Here we directly compare the abilities of NS1 gene products from three different human FLUAV (H1N1) strains to counteract the antiviral host response. We found that A/PR/8/34 NS1 has a strong capacity to inhibit IRF3 and activation of the IFN-beta promoter but is unable to suppress expression of other cellular genes. In contrast, the NS1 proteins of A/Tx/36/91 and of A/BM/1/18, the virus that caused the Spanish influenza pandemic, caused suppression of additional cellular gene expression. Thus, these NS1 proteins prevented the establishment of an IFN-induced antiviral state, allowing virus replication even in the presence of IFN. Interestingly, the block in gene expression was dependent on a newly described NS1 domain that is important for interaction with the cleavage and polyadenylation specificity factor (CPSF) component of the cellular pre-mRNA processing machinery but is not functional in A/PR/8/34 NS1. We identified the Phe-103 and Met-106 residues in NS1 as being critical for CPSF binding, together with the previously described C-terminal binding domain. Our results demonstrate the capacity of FLUAV NS1 to suppress the antiviral host defense at multiple levels and the existence of strain-specific differences that may modulate virus pathogenicity.

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Figures

FIG. 1.

FIG. 1.

Control of IFN-β induction and IRF3 activation by NS1. (A) Induction of IFN-β by recombinant A/WSN/33 carrying the NS gene segment of A/PR/8/34 or A/Tx/36/91 or an inactive NS1 mutant. A549 cells were infected with 0.5 PFU/cell of the recombinant viruses or were left uninfected (ctrl). At 10 h postinfection, total RNA was isolated, and transcripts for IFN-β, viral NP, and β-actin were detected by RT-PCR. (B) IRF3 dimerization assay. 293 cells were infected with the recombinant viruses (1 PFU/cell) or were left uninfected (ctrl). After 12 h, the cells were superinfected with 1 PFU/cell of SeV for an additional 6 h. The cells were then lysed and analyzed for the presence of IRF3 monomers and dimers by nondenaturing gel electrophoresis followed by Western blotting using an IRF3-specific antibody (a). In parallel, the lysates were analyzed by denaturing gel electrophoresis and Western blotting for the accumulation of viral NP (b), NS1 (c), and β-actin (d) as an internal standard. The lanes were cut out from the same gel. The numbers in panel c indicate the relative intensities of the NS1 protein bands normalized to the expression of NP and actin, with PR NS1 activity defined as 100. (C) NS1 association with RIG-I. 293T cells were transfected with a Flag-RIG-I expression construct or empty vector and were infected with the recombinant viruses (2 PFU/cell) 48 h later. At 8 h postinfection, lysates of the cells were subjected to immunoprecipitation (co-IP) using an anti-Flag antibody. The precipitated proteins were analyzed by Western blotting using polyclonal antibodies directed against NS1 and the Flag peptide. Infection of the cells was monitored by the accumulation of viral NP and NS1 in the cell lysates (input). β-Actin was used as a loading control.

FIG. 2.

FIG. 2.

NS1 of A/Tx/36/91 mediates a general inhibition of gene expression. (A) Schematic representation of functional domains in the NS1 protein, showing the RNA-binding domain (positions 1 to 73) (40), the eIF4G-binding domain (positions 81 to 113) (1), the CPSF interaction domain (around position 184) (51), and the PABII-binding domain (positions 223 to 230) (12). Numbers indicate amino acid positions. 293T (B, E, F, and I) and Vero (C, D, G, and H) cells were cotransfected with reporter plasmids carrying the FF-Luc gene under the control of the IFN-β promoter (B and F) or the Mx1 promoter (C and G) and REN-Luc under the control of the constitutive SV40 promoter (D and H), together with expression plasmids encoding HA-tagged versions of NS1, an NS1 chimera of A/PR/8/34 and A/Tx/36/91, or C-terminally truncated Tx NS1 constructs or with an empty plasmid (ctrl). At 16 h posttransfection, the cells were infected with SeV (B and F) or treated with 200 U/ml of IFN-α2a (C and G) for 16 h and then analyzed for reporter gene expression. (E and I) Lysates of 293T cells were analyzed for NS1 protein expression by Western blotting using an HA-specific antiserum. The numbers indicate the relative intensities of the NS1 protein bands, with PR NS1 activity defined as 100. The data from one representative experiment are shown.

FIG. 3.

FIG. 3.

Identification of NS1 domains responsible for inhibition of gene expression and CPSF binding. (A) Vero cells were cotransfected with reporter plasmids carrying the FF-Luc gene under the control of the Mx1 promoter and the REN-Luc gene under the control of the SV40 promoter, together with expression plasmids encoding HA-tagged versions of NS1 of A/PR/8/34 and A/Tx/36/91 or with empty plasmid (ctrl). At 16 h posttransfection, the cells were treated with 200 U/ml of IFN-α2a for 16 h and then analyzed for reporter gene expression. (B) Lysates of transfected cells were analyzed for NS1 protein expression by Western blotting using an HA-specific antiserum. The numbers indicate the relative intensities of the NS1 protein bands, with PR NS1 activity defined as 100. (C) Analysis of subcellular accumulation of NS1 proteins. MDCK cells were transfected with the NS1 expression plasmids for 48 h. The cells were then fixed and analyzed by immunofluorescence, using a polyclonal antibody directed against the HA tag. (D) Analysis of CPSF binding. The HA-tagged NS1 constructs were expressed by in vitro translation in the presence of [35S]methionine (input). Flag-tagged CPSF was expressed in transfected 293T cells. The cell lysate was mixed with the 35S-labeled NS1 proteins and subjected to coimmunoprecipitation (IP) using a monoclonal anti-Flag antibody coupled to protein A-Sepharose. The beads were washed three times, and the precipitated proteins were analyzed for the presence of 35S-labeled NS1 by autoradiography and for Flag-CPSF by Western blot analysis using a Flag-specific rabbit antiserum. The numbers indicate the relative intensities of the protein bands, with the strongest signal defined as 100.

FIG. 4.

FIG. 4.

NS1 of A/Tx/36/91, but not that of A/PR/8/34, blocks IFN action. A549 cells were infected with recombinant viruses (1 PFU/cell) encoding NS gene segments of A/PR/8/34 and A/Tx/36/91 or were left uninfected (ctrl). The cells were then treated with 400 U/ml of IFN-α2a for 20 h or were left untreated (ctrl-IFN). Cell lysates were analyzed for the induction of MxA (a) and the accumulation of viral NP (b), NS1 (c), and β-tubulin (d) by Western blot analysis. The numbers indicate the relative intensities of the protein bands, with the strongest signal defined as 100.

FIG. 5.

FIG. 5.

NS1 of A/BM/1/18 inhibits RNA polymerase II-driven gene expression. 293T (A, C, and D), Vero (B and E), and BSR-T7 (F) cells were cotransfected with a reporter plasmid carrying the Luc gene under the control of the IFN-β promoter (A), the Mx1 promoter (B), the IRF1 promoter (C), an NF-κB-driven promoter (D), the constitutive SV40 promoter (E), or the T7 promoter (F) together with expression plasmids encoding NS1 proteins or with empty plasmid (ctrl). At 16 h posttransfection, the cells were infected with SeV (A) or were treated with 200 U/ml of IFN-α2a (B) or 100 U/ml of IFN-γ (C) for 16 h and then analyzed for reporter gene expression. Two independent experiments yielded similar results. The data from one experiment are shown.

FIG. 6.

FIG. 6.

NS1 proteins of A/Tx/36/91 and A/BM/1/18 suppress the antiviral effect of IFN-α. A549 cells were transfected with empty vector (a and b), with an expression plasmid encoding the V protein of SV5 (c), or with NS1 expression plasmids (d to f). After 16 h, the cells were treated with 150 U/ml of IFN-α2a (b to f) for 12 h or were left untreated (a) and then infected with 1 PFU/cell of NDV-GFP. At 24 h postinfection, GFP expression was analyzed by fluorescence microscopy. (g) The numbers of GFP-positive cells for a given area were determined for three independent experiments.

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