The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target

Abstract

The emergence of influenza A viruses resistant to the two existing classes of antiviral drugs highlights the need for additional antiviral drugs, particularly considering the potential threat of a pandemic of H5N1 influenza A viruses. Here, we determine whether influenza A virus replication can be selectively inhibited by blocking the ability of its NS1A protein to inhibit the 3'-end processing of cellular pre-mRNAs, including beta interferon (IFN-beta) pre-mRNA. Pre-mRNA processing is inhibited via the binding of the NS1A protein to the cellular CPSF30 protein, and mutational inactivation of this NS1A binding site causes severe attenuation of the virus. We demonstrate that binding of CPSF30 is mediated by two of its zinc fingers, F2F3, and that the CPSF30/F2F3 binding site on the NS1A protein extends from amino acid 144 to amino acid 186. We generated MDCK cells that constitutively express epitope-tagged F2F3 in the nucleus, although at only approximately one-eighth the level of the NS1A protein produced during virus infection. Influenza A virus replication was inhibited in this cell line, whereas no inhibition was observed with influenza B virus, whose NS1B protein lacks a binding site for CPSF30. Influenza A virus, but not influenza B virus, induced increased production of IFN-beta mRNA in the F2F3-expressing cells. These results, which indicate that F2F3 inhibits influenza A virus replication by blocking the binding of endogenous CPSF30 to the NS1A protein, point to this NS1A binding site as a potential target for the development of antivirals directed against influenza A virus.

FIG. 1.

Identification of the region of CPSF30 that binds to the 186-amino-acid region of the NS1A protein. (A) GST fusions containing the indicated regions of CPSF30 were mixed with ³⁵S-labeled wt NS1A protein, followed by affinity chromatography on glutathione-Sepharose. (B) GST-F2F3 or GST was mixed with ³⁵S-labeled wt or M186 mutant NS1A protein as indicated, followed by affinity chromatography on glutathione-Sepharose (lanes 3 to 5). Lanes 1 and 2 show the input wt and M186 NS1A proteins. RBO, RNA-binding domain.

FIG. 2.

The NS1A protein containing an L-to-A substitution at position 144 did not inhibit the 3′-end processing of β-globin pre-mRNA and did not bind the F2F3 fragment of CPSF30. (A) 3′-end processing assay. 293 cells were cotransfected with a pBC12 plasmid containing a human β-globin gene and either an empty pcDNA3 plasmid (lane 1) or a pcDNA3 plasmid encoding wt NS1A protein (lane 2) or the M144 mutant NS1A protein (lane 3). The M144 sequence, which is diagrammed above the blot, was generated by RT-PCR using appropriate primers. RNA was analyzed by RNase protection using the indicated uniformly labeled RNA probe (270 nucleotides long). The protected RNA fragments were resolved by electrophoresis on a urea-polyacrylamide (5%) gel. The positions of the RNA fragments corresponding to the uncleaved and 3′-end-cleaved pre-mRNAs are indicated. No residual probe containing 270 nucleotides was detected. The amount of the wt and M144 NS1A proteins was determined by a Western blot (WB) using NS1A antibody. (B) GST pull-down assay. GST-F2F3 or GST was mixed with ³⁵S-labeled wt, M186 mutant, or M144 mutant NS1A protein as indicated, followed by affinity chromatography on glutathione-Sepharose.

FIG. 3.

Characterization of the M144 mutant virus. (A) Plaques formed by wt and M144 mutant viruses on MDCK cells. (B) Relative amounts of IFN-β mRNA produced during single-cycle growth by M144 and wt virus. MDCK cells were infected with either M144 or wt virus at an MOI of 5, and at the indicated times after infection the relative amounts of IFN-β mRNA produced were determined by quantitative RT-PCR. The error bars indicate standard deviations for three experiments. WB, Western blot analysis of the viral proteins synthesized in wt and M144 virus-infected cells using either an antibody against virion proteins (top) or NS1A antibody (bottom). HA, hemagglutinin. (C) Localization of the NS1A protein in cells infected by M144 and wt virus at 8 h postinfection was determined by indirect immunofluorescence. The primary antibody was a rabbit polyclonal against the NS1A protein.

FIG. 4.

Transient expression of the F2F3 protein fragment did not inhibit the 3′-end processing of cellular pre-mRNAs. 293 cells were cotransfected with a pBC12 plasmid containing a human β-globin gene and either an empty pcDNA3 plasmid (lane 1) or a pcDNA3 plasmid encoding wt NS1A protein (lane 2) or the F2F3 fragment (lane 3). The sequence of the F2F3 protein fragment is diagrammed above. Cells were collected 40 h posttransfection, and RNA was analyzed by RNase protection as described in the legend to Fig. 2A. The positions of the RNA fragments corresponding to the uncleaved and 3′-end-cleaved pre-mRNAs are indicated. No residual probe containing 270 nucleotides was detected.

FIG. 5.

Characterization of F2F3-expressing MDCK cells. (A) Localization of the F2F3 fragment was determined by indirect immunofluorescence using anti-myc antibody (Ab). (B) Determination of the relative amounts of the F2F3 protein fragment and the NS1A protein in influenza A virus-infected F2F3-expressing MDCK cells. An aliquot of the infected cells was analyzed by immunoblotting using either anti-myc (left) or anti-NS1A (right) antibody. To estimate the amount of the F2F3 protein fragment, increasing amounts of GST-NLS-13xmyc were applied to the anti-myc immunoblot (left). The GST-NLS-13xmyc was generated using the pAJ1026 plasmid, which contains 13 myc epitopes (12). Based on this immunoblot, as well as another immunoblot containing 10 to 30 ng of GST-NLS-13xmyc, we estimated that the aliquot from the virus-infected cells contained 10 ng of the F2F3 protein fragment. To estimate the amount of the NS1A protein, increasing amounts of GST-NS1A protein were applied to the anti-NS1A immunoblot (right). Based on this immunoblot, we estimated that the aliquot from the virus-infected cells contained approximately 80 ng. Because the molecular weights of the F2F3 protein fragment and the NS1A protein were approximately the same, the molar ratio of the F2F3 fragment/NS1A protein was approximately 1/8.

FIG. 6.

Plaque reduction assays and virus yields after low-MOI infections in control and F2F3-expressing MDCK cells. The viruses used in these assays were influenza A/WSN/33, influenza A/Udorn/72, and influenza B/Lee/40 viruses.

FIG. 7.

Production of IFN-β mRNA and infectious virus during single-cycle growth in control and F2F3-expressing cells. (A) Relative amounts of IFN-β mRNA produced in F2F3-expressing and control cells after high-MOI infection (5 PFU/cell) with either influenza A/Udorn/72 or influenza B/Lee/40 virus. The error bars indicate standard deviations for three experiments. (B) Viral-protein synthesis in F2F3-expressing and control cells after high-MOI infection with either influenza A/Udorn/72 virus (left) or influenza B/Lee/40 virus (right). At the indicated times after infection, the cells were washed twice with methionine-free DMEM, 5 μl of a mixture of [³⁵S]methionine and [³⁵S]cysteine (Promix; Amersham) was added in a final volume of 1 ml of serum-free DMEM, followed by incubation for 30 min. After incubation, the cells were washed twice with phosphate-buffered saline and lysed in 200 μl of Laemmli sample buffer. An aliquot was loaded onto SDS-polyacrylamide gels (12 to 15%) for analysis by autoradiography. (C) Replication of influenza A/Udorn/72 and influenza B/Lee/40 after high-MOI infection of F2F3-expressing and control cells.

FIG. 8.

Proposed mechanism for the selective inhibition of influenza A virus replication by the F2F3 fragment of CPSF30.