Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-␤ mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3 end processing of all cellular pre-mRNAs. Here we report the 1.95-Å resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-␤ pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.antiviral drug discovery ͉ bird flu ͉ vaccine engineering ͉ virology ͉ X-ray crystallography

The influenza A virus NS1 protein (NS1A protein) binds and inhibits the function of the 30-kDa subunit of CPSF, a cellular factor that is required for the 3'-end processing of cellular pre-mRNAs. Here we generate a recombinant influenza A/Udorn/72 virus that encodes an NS1A protein containing a mutated binding site for the 30-kDa subunit of CPSF. This mutant virus is substantially attenuated, indicating that this binding site in the NS1A protein is required for efficient virus replication. Using this mutant virus, we show that NS1A binding to CPSF mediates the viral posttranscriptional countermeasure against the initial cellular antiviral response--the interferon-alpha/beta (IFN-alpha/beta)-independent activation of the transcription of cellular antiviral genes, which requires the interferon regulatory factor-3 (IRF-3) transcription factor that is activated by virus infection. Whereas the posttranscriptional processing of these cellular antiviral pre-mRNAs is inhibited in cells infected by wild-type influenza A virus, functional antiviral mRNAs are produced in cells infected by the mutant virus. These results establish that the binding of 30-kDa CPSF to the NS1A protein is largely responsible for the posttranscriptional inhibition of the processing of these cellular antiviral pre-mRNAs. Mutation of this binding site in the NS1A protein also affects a second cellular antiviral response: in cells infected by the mutant virus, IFN-beta mRNA is produced earlier and in larger amounts.

Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology.

Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin ␣ isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin ␣/␤ pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine-or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.The influenza A virus genome consisting of eight separate RNA segments encodes 11 viral structural and nonstructural proteins. In addition to the viral hemagglutinin, nonstructural protein 1 (NS1A) is one of the major viral virulence factors. The evolution of NS1A genes appears to be species specific, and the evolution of the present human NS1A genes began in 1918 when H1N1 type viruses emerged and became pandemic (20).The NS1A protein is a multifunctional protein that participates in both protein-RNA (7, 16, 28, 57) and protein-protein (23, 25, 38) interactions. The NS1A protein contains an Nterminal double-stranded RNA (dsRNA)-binding domain and a C-terminal effector domain (45). The three-dimensional structures of the dsRNA-binding and effector domains of NS1A have been determined (3,6,27). The NS1A protein exists as a dimer, and the structure of its RNA-binding domain differs markedly from all other known RNA-binding proteins. The effector domain binds two cellular proteins that are essential for the 3Ј end processing of cellular pre-mRNAs (5, 26, 38). As a result, the processing of cellular pre-mRNAs, including beta interferon (IFN-␤) pre-mRNA and the pre-mRNAs of other antiviral proteins, is inhibited, thereby suppressing the amount of mature IFN-␤ mRNA that is produced in infected cells (38,39,49,55). The role of the dsRNA-binding activity is controversial and may be virus strain specific. The role of the dsRNA-binding activity of the NS1A protein of the human H3N2 influenza A/Udorn/72 virus was determined using a recombinant virus expressing a NS1 protein lacking dsRNAbinding activity. Analysis of the defect in virus replication demonstrated that the primary ...

. 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.