The NS1A protein of influenza A virus: its crucial role in the inhibition of 3' end processing of cellular pre-mRNAs
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Influenza A viruses, one of the four influenza virus genera, are responsible for the human pandemics that have caused high mortality rates, and the highly pathogenic virus transmitted directly from chickens to humans in 1997 is an influenza A virus. It has been shown previously that influenza A viruses, like many mammalian viruses induce the interferon-α/β (IFN-α/β)-independent activation of interferon regulatory factor-3 (IRF-3) and transcription of some IFN-α/β–stimulated response element (ISRE)-controlled cellular genes. This cellular defense against virus infection takes place prior to the synthesis of IFN-α/β. In addition, influenza A viruses are unique in that their nonstructural protein (NS1A protein) inhibits the posttranscriptional processing of these cellular antiviral pre-mRNAs. The NS1A protein contains two functional domains: an RNA-binding/dimerization domain at the amino terminus and an effector domain in the carboxyl half. The effector domain physically associates with two essential components of the machinery of the 3′ end processing of cellular pre-mRNAs: the 30kDa subunit of the cleavage and polyadenylation specificity factor (CPSF) and poly(A)-binding protein (PABII). The consequent inhibition of CPSF and PABII function inhibits the 3′ end processing of cellular pre-mRNAs in virus-infected cells, and as a result, cellular premRNAs are not cleaved and nuclear export of mature mRNAs is inhibited. This action of the NS1A protein also inhibits the production of IFN-β mRNA. Binding of CPSF30 to NS1A is mediated by two of its zinc finger domains, F2 and F3, and the CPSF30/F2F3 binding site on the NS1A protein extending from amino acid 144 to 186. We generated MDCK cells that constitutively express epitope-tagged F2F3, and showed that influenza A virus replication was selectively inhibited in this cell line. Influenza A virus induced increased production of IFN-β 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. Moreover, we found the NS1A protein encoded by HK/483/97, the initial H5N1 virus that was transmitted from chicken to human in 1997, does not inhibit posttranscriptional processing of cellular pre-mRNAs. As a consequence, a chimeric Udorn/H5N1 virus that contains only the NS gene from A/HK/483/97 virus induced the production of a high level of IFN-β mRNA and was highly attenuated. In contrast, the NS1A protein encoded by the pathogenic H5N1 virus isolated in 2004 inhibited pre-mRNA processing, resulting in decreased production of IFN-β mRNA. These results demonstrate that the NS1A protein in H5N1 viruses acquired a function between 1997 and 2004 that enhances virus replication in mammalian cells.