The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells

Abstract

The NS1A proteins of human influenza A viruses bind CPSF30, a cellular factor required for the processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including beta interferon mRNA. Here we show that the NS1A protein of the pathogenic H5N1 influenza A/Hong Kong/483/97 (HK97) virus isolated from humans has an intrinsic defect in CPSF30 binding. It does not bind CPSF30 in vitro and causes high beta interferon mRNA production and reduced virus replication in MDCK cells when expressed in a recombinant virus in which the other viral proteins are encoded by influenza A/Udorn/72. We traced this defect to the identities of amino acids 103 and 106 in the HK97 NS1A protein, which differ from the consensus amino acids, F and M, respectively, found in the NS1A proteins of almost all human influenza A virus strains. X-ray crystallography has shown that F103 and M106, which are not part of the CPSF30 binding pocket of the NS1A protein, stabilize the NS1A-CPSF30 complex. In contrast to the HK97 NS1A protein, the NS1A proteins of H5N1 viruses isolated from humans after 1998 contain F103 and M106 and hence bind CPSF30 in vitro and do not attenuate virus replication. The HK97 NS1A protein is less attenuating when expressed in a virus that also encodes the other internal HK97 proteins and under these conditions binds to CPSF30 to a substantial extent in vivo. Consequently, these internal HK97 proteins largely compensate for the absence of F103 and M106, presumably by stabilizing the NS1A-CPSF30 complex.

FIG. 1.

The HK97 NS1A protein does not bind the F2F3 fragment of CPSF30 and does not inhibit 3′-end processing of cellular pre-mRNAs. (A) GST pulldown assay. GST-F2F3 or GST was mixed with ³⁵S-labeled NS1A protein of the indicated virus. (B) 3′-end processing assay. 293 cells (left panel) or quail embryo fibroblasts (right panel) were cotransfected with a pBC12 plasmid carrying a human β-globin gene and either an empty pcDNA3 plasmid (lane 1) or a pcDNA3 plasmid encoding the indicated NS1A protein. RNA was analyzed by RNase protection using a 270-nucleotide-long RNA probe spanning the 3′ cleavage site of the β-globin pre-mRNA. The protected RNA fragment corresponding to the uncleaved pre-mRNA is 210 nucleotides long, and the RNA fragment corresponding to the 3′-end-cleaved pre-mRNA is 160 nucleotides long. No residual probe containing 270 nucleotides was detected.

FIG. 2.

The Ud virus whose NS gene is replaced by the HK97 NS gene is attenuated. (A) Size of the plaques on MDCK cells of the Ud viruses containing the indicated NS gene. (B) Growth curves of the Ud viruses containing the indicated NS gene after infection of MDCK cells at a MOI of 0.001 PFU/cell.

FIG. 3.

The Ud virus whose NS gene is replaced by the HK97 NS gene does not bind CPSF30 and induces increased levels of IFN-β mRNA during infection. (A) Plaque reduction assays on control and F2F3-expressing MDCK cells using Ud viruses containing the indicated NS gene. The plaque assay for the Ud recombinant containing the HK97 NS gene was incubated for a day longer than the plaque assay shown in Fig. 2A to increase the size of the plaques. (B) The relative amounts of IFN-β mRNA produced during single-cycle growth in MDCK cells of the Ud viruses containing the indicated NS gene.

FIG. 4.

Alignment of the protein sequences of Teal, HK97, VN04, and 1918 viruses (14). The positions of the groups (G1, G2, G3, and G4) of mutations introduced into the HK97 NS1A protein are indicated. G1 consists of R55E, E60A, and H63Q; G2 consists of L103F and I106M; G3 consists of N139D, A143T, and D152E; and G4 consists of T202A.

FIG. 5.

The G2 (+) (I106M and L103F) mutations in the HK97 NS1A protein are required for the acquisition of CPSF30 binding that effectively suppresses IFN-β mRNA synthesis in infected cells and enhances virus replication. (A) GST pulldown assay. GST-F2F3 or GST was mixed with the wt or indicated mutant HK97 ³⁵S-labeled NS1A protein. (B) The relative amounts of IFN-β mRNA produced during single-cycle growth in MDCK cells of the Ud recombinants containing either the wt or G2 (+) mutant HK97 NS gene. (C) Growth curves of the Ud recombinant viruses containing the wt or G2 (+) mutant HK97 NS gene after infection of MDCK cells at low MOI. (D) Plaque reduction assay on control and F2F3-expressing MDCK cells using the Ud recombinant containing the G2 (+) mutant HK97 NS gene.

FIG. 6.

The HK97 NS gene also attenuates virus replication in the context of the other internal HK97 genes due to the identity of the amino acids at 103 and 106 in the NS1A protein. (A) Growth curves of recombinant viruses containing either the wt or G2 (+) mutant HK97 NS gene and all HK97 genes except those encoding HA and NA. (B) The amount of IFN-β mRNA produced during single-cycle growth of the recombinant containing the wt HK97 NS gene relative to that produced by the recombinant containing the G2 (+) mutant HK97 NS gene.

FIG. 7.

The HK97 virus is substantially less attenuated than the Ud/NS-HK97 virus and, unlike the Ud/NS-HK97 virus, is inhibited in F2F3-expressing cells. (A) Growth curves of the HK97 and Ud/NS-HK97 viruses after infection of MDCK cells at low MOI. (B) The amount of IFN-β mRNA produced during single-cycle growth of the HK97 virus relative to that produced by the Ud/NS-HK97 virus. (C) Plaque reduction assays on control and F2F3-expressing MDCK cells of the recombinant viruses expressing the indicated NS and internal (PA, PB1, PB2, NP, M) genes. The plaque assay for the Ud/NS-HK97 virus was incubated for a day longer than the other plaque assays to increase the size of the plaques.

FIG. 8.

The HK97 NS1A protein binds to CPSF30 in cells infected by the HK97 virus but not in cells infected by the Ud/NS-HK97 virus. 293T cells were transfected with the indicated plasmids and infected with the indicated recombinant influenza A viruses. Extracts from cells collected at 12 h after virus infection were immunoprecipitated with anti-FLAG M2 monoclonal antibody, and the immunoprecipitates were analyzed by immunoblotting with either anti-Ud NS1A antibody or anti-FLAG antibody.