The CPSF30 Binding Site on the NS1A Protein of Influenza A Virus Is a Potential Antiviral Target

Abstract

. 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-␤) 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-␤ 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.

Large-scale analysis of influenza A virus sequences reveals potential drug target sites of non-structural proteins

“…The high variability of these residues clearly demonstrates that this interaction is not universal for all influenza A viruses, but is restricted to avian influenza viruses, as reported previously (Heikkinen et al, 2008). The NS1 protein has been implicated in the inhibition of mRNA maturation via interaction with PABPII (Chen et al, 1999) and CPSF30 (Twu et al, 2006). There is no significant conservation among the PABPII interacting residues reported (218-232).…”

“…The highest-ranking binding site (site 1 reported by Q-SiteFinder) is surrounded by the highly conserved residues Trp102, Met104, Asp120, Arg148, Gly158 and Glu159, some of which are in the region that interacts with translation initiation factor eIF4F (see above). This site could form a target for small molecule inhibitors of protein-protein interactions (Twu et al, 2006). Binding sites 2 and 6 are located between highly conserved residues on the RBD, such as Arg19, Arg35 and Arg46.…”

The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2′-5′ oligo (A) synthetase/RNase L pathway

“…In contrast, it has been definitively established that the NS1A protein effectively inhibits the production of IFN-␤ mRNA by another mechanism: it binds CPSF30, thereby inhibiting the production of mature cellular mRNAs, including IFN-␤ mRNA (6,7,13). Recombinant viruses that express an NS1A protein containing a mutated binding site for CPSF30 are highly attenuated and induce the production of a substantially increased amount of IFN-␤ mRNA (7,27). In addition, we have shown that this NS1A-binding site is a potential target for the development of antivirals, because blocking the access of CPSF30 for this NS1A site results in increased production of IFN-␤ mRNA and the inhibition of virus replication (27).…”

“…Recombinant viruses that express an NS1A protein containing a mutated binding site for CPSF30 are highly attenuated and induce the production of a substantially increased amount of IFN-␤ mRNA (7,27). In addition, we have shown that this NS1A-binding site is a potential target for the development of antivirals, because blocking the access of CPSF30 for this NS1A site results in increased production of IFN-␤ mRNA and the inhibition of virus replication (27). Because inhibition of IFN-␤ mRNA production occurs in the nucleus of influenza A virus-infected cells, a failure of the NS1A protein to enter the nucleus would be expected to result in substantially increased production of IFN-␤ mRNA.…”

“…As shown previously, WT Udorn virus activates IFN regulatory factor 3 (IRF-3), but the production of mature IFN-␤ mRNA is low because the NS1A protein binds and inhibits the function of two cellular proteins that are essential for the 3Ј end processing of cellular pre-mRNAs, the 30-kDa subunit of CPSF (CPSF30) and poly(A)-binding protein II (7,26). Mutational inactivation of the NS1A site for binding CPSF30 was shown to result in a large increase in IFN-␤ mRNA production and substantial (1,000-fold) attenuation of the virus (CPSF mutant virus) (7,27). This increase in IFN-␤ mRNA production serves as a benchmark for the levels of IFN-␤ mRNA that result in substantial virus attenuation.…”