Structural Basis for Suppression of a Host Antiviral Response by Influenza a Virus

Structural basis for suppression of a host antiviral response by influenza A virus

Kalyan Das*†, Li-Chung Ma*‡, Rong Xiao*‡, Brian Radvansky*‡, James Aramini*‡, Li Zhao*§, Jesper Marklund¶, Rei-Lin Kuo¶, Karen Y. Twu¶, Eddy Arnold*†ʈ, Robert M. Krug¶ʈ, and Gaetano T. Montelione*‡§ʈ

*Center for Advanced Biotechnology and Medicine and †Departments of Chemistry and Chemical Biology and ‡Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854; §Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854; and ¶Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712

Communicated by Aaron J. Shatkin, Center for Advanced Biotechnology and Medicine, Piscataway, NJ, June 11, 2008 (received for review March 24, 2008) Influenza A viruses are responsible for seasonal epidemics and high cally, we report the 1.95-Å resolution X-ray crystal structure of the mortality pandemics. A major function of the viral NS1A protein, a effector domain of the human influenza Ud NS1A protein in complex virulence factor, is the inhibition of the production of IFN-␤ mRNA and with a domain of CPSF30 comprising its second and third zinc (Zn) other antiviral mRNAs. The NS1A protein of the human influenza finger motifs (F2F3). We used the F2F3 domain of CPSF30 because it A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs has been established that this domain binds efficiently to the Ud NS1A by binding the cellular 30-kDa subunit of the cleavage and polyadenyl- protein, and that expression of F2F3 in virus-infected cells leads to the ␤-ation specificity factor (CPSF30), which is required for the 3؅ end pro- inhibition of Ud virus replication and increased production of IFN cessing of all cellular pre-mRNAs. Here we report the 1.95-Å resolution mRNA, presumably by occupying the CPSF30 binding site on the X-ray crystal structure of the complex formed between the second and NS1A protein and hence blocking the binding of endogenous CPSF30 third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of to this site (19, 20). the Ud NS1A protein. The complex is a tetramer, in which each of two This crystal structure reveals an NS1A:F2F3 tetrameric complex F2F3 molecules wraps around two NS1A effector domains that interact with two F2F3 binding pockets. The NS1A amino acids comprising with each other head-to-head. This structure identifies a CPSF30 binding the F2F3 binding pocket are highly conserved among human pocket on NS1A comprised of amino acid residues that are highly influenza A viruses, strongly suggesting that this CPSF30 binding conserved among human influenza A viruses. Single amino acid changes pocket is used by all human influenza A viruses to suppress the within this binding pocket eliminate CPSF30 binding, and a recombinant production of IFN-␤ mRNA. This binding pocket is a potential Ud virus expressing an NS1A protein with such a substitution is atten- target for the development of antivirals directed against influenza uated and does not inhibit IFN-␤ pre-mRNA processing. This binding A virus. The crystal structure also shows that the interaction surface pocket is a potential target for antiviral drug development. The crystal between NS1A and F2F3 extends beyond the primary F2F3 binding structure also reveals that two amino acids outside of this pocket, F103 pocket alone, and that two amino acids in the NS1A protein, Phe and M106, which are highly conserved (>99%) among influenza A at position 103 and Met at position 106, play key roles in stabilizing viruses isolated from humans, participate in key hydrophobic interac- the tetramer. Although F103 and M106 are highly conserved Ͼ tions with F2F3 that stabilize the complex. ( 99%) in the NS1A proteins of human influenza A viruses (21), a few prominent human influenza A viruses encode NS1A proteins antiviral drug discovery ͉ bird flu ͉ vaccine engineering ͉ virology ͉ with different amino acid residues at these positions. The biological X-ray crystallography properties of these few virus variants, however, reinforce the importance of NS1A protein-mediated CPSF30 binding for circulating human influenza A viruses. he NS1 protein of human influenza A viruses (NS1A protein) Tis a small, multifunctional protein that participates in both Results and Discussion protein-RNA and protein–protein interactions. Its N-terminal The Crystal Structure Reveals a Tetrameric NS1A:F2F3 Complex. The RNA-binding domain binds double-stranded RNA (dsRNA) (1–3). Ud NS1A effector domain construct used in our experiments By identifying the replication defect of a recombinant influenza (amino acid residues 85-215) was identified by generation and A/Udorn/72 (Ud) virus that encodes an NS1A protein lacking assessment of the expression levels and solubility of 64 different dsRNA-binding activity, it was established that the primary role of NS1A constructs [supporting information (SI) Table S1]. This Ud ␣ ␤ NS1A dsRNA-binding activity is the inhibition of the IFN- / - NS1A (85-215) effector domain construct comprises 80% of the induced oligo A synthetase/RNase L pathway, and that NS1A effector domain and is well-ordered in solution, as determined by dsRNA-binding activity has no detectable role in inhibiting the its NMR spectra (Fig. S1). The 61-residue F2F3 tandem Zn-finger production of IFN-␤ mRNA or inhibiting the activation of protein construct of CPSF30 comprises ϳ30% of its full-length sequence, kinase R (PKR) (4, 5). The rest of the NS1A protein, which is and is active in vivo in blocking interactions between full-length Ud referred to as the effector domain, has binding sites for several cellular proteins, including: the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular Author contributions: K.D., L.-C.M., R.X., E.A., R.M.K., and G.T.M. designed research; K.D., factor required for the 3Ј end processing of cellular pre-mRNAs, L.-C.M., R.X., B.R., J.A., L.Z., J.M., R.-L.K., and K.Y.T. performed research; R.X., B.R., L.Z., J.M., R.-L.K., and K.Y.T. contributed new reagents/analytic tools; K.D., L.-C.M., R.X., B.R., J.A., thereby inhibiting the production of all cellular mRNAs, including J.M., R.-L.K., K.Y.T., E.A., R.M.K., and G.T.M. analyzed data; and K.D., L.-C.M., J.A., E.A., IFN-␤ mRNA (6–10); p85␤, resulting in the activation of phos- R.M.K., and G.T.M. wrote the paper. phatidylinositol-3-kinase signaling (11–14); and PKR, resulting in The authors declare no conflict of interest. the inhibition of PKR activation (15). Freely available online through the PNAS open access option. Of these multiple protein binding sites on the NS1A protein, only the Data deposition: The atomic coordinates and structure factors have been deposited in the dsRNA-binding site has been structurally characterized (3, 16–18). Protein Data Bank, www.pdb.org (PDB ID code 2RHK). These structural studies have revealed key features of the NS1A ʈTo whom correspondence may be addressed. E-mail: [email protected], dsRNA-binding site that can be targeted for the development of [email protected], or [email protected]. antivirals directed against influenza A virus (18). Here we describe the This article contains supporting information online at www.pnas.org/cgi/content/full/ structure of the interface between CPSF30 and the NS1A protein, a 0805213105/DCSupplemental. molecular interaction that suppresses host antiviral responses. Specifi- © 2008 by The National Academy of Sciences of the USA

13092–13097 ͉ PNAS ͉ September 2, 2008 ͉ vol. 105 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805213105 Downloaded by guest on October 2, 2021 Fig. 1. Crystal structure of F2F3:NS1A (85-215) complex. (A) Gel filtration data demonstrating complex formation between NS1A (85-215) and F2F3. Traces show chromatographic profiles on a Superdex G75 column for NS1A (85-215) alone (red) and the complex of NS1A (85-215) with [S94]-F2F3 (blue). Inset shows calibrated gel filtration data for (A) [S94]-F2F3 (ϳ10 kDa), (B) NS1A (85-215) alone (ϳ27 kDa), and (C) [S94]-F2F3:NS1A complex (ϳ47 kDa). Similar results were obtained by static light scattering analysis of effluent fractions from size exclusion chromatography, as described in SI Materials and Methods: (A) 15 Ϯ 3 kDa, (B) 25 Ϯ 5 kDa, and (C) 48 Ϯ 5 kDa. The molecular mass expected for the tetrameric complex observed in the crystal structure is 49,250 Da, which is in good agreement with these light scattering and gel filtration data. The elution times for isolated NS1A (85-215) (single chain calculated molecular mass 15,943 Da) and [S94]-F2F3 (single chain calculated molecular mass 8,682 Da) domains differ when loaded at different protein concentrations, suggesting that these molecules form weak homodimers under these solution conditions. Calibration standards (A–D) are described in SI Materials and Methods.(B) Two NS1A effector domains (green and red) and two F2F3 domains (blue and yellow) of CPSF30 form the tetramer. Some NS1AЈ amino acid residues that function in complex formation are highlighted in cyan. (C) F3-binding pocket on NS1A (85-215). A hydrophobic pocket on the NS1A surface binds to the F3 Zn finger of F2F3. Both chains of NS1A in the head-to-head dimer interact with each F2F3 molecule. (D) Expanded view of the F3-binding pocket. The NS1A amino acid residues labeled in red interact with the aromatic side chains of residues Y97, F98, and F102 of the F3 Zn finger of F2F3.

NS1A and full-length human CPSF30 (19). The complex between molecule, ϳ1,310 Å2 of ϳ4,300 Å2 of surface area takes part in NS1A (85-215) and F2F3 was formed, purified by gel filtration (Fig. tetramer formation. 1A), and crystallized. The structure of this F2F3:NS1A (85-215) The F2F3 domain of human CPSF30 used in our work (amino complex was then determined using selenomethionine (Se-Met) acid residues 60-120) has a Ser at position 94, compared to the multiwavelength anomalous diffraction (MAD) techniques (22) published sequence of CPSF30 (25), which has a Pro at this position. and refined at 1.95-Å resolution, to Rwork and Rfree of 0.210 and It is not clear if this single nucleotide variant (CCC to TCC) is a 0.234, respectively (Table S2). The chain fold of this Ud NS1A naturally occurring polymorphism or is a result of the cloning domain is similar to that reported for the uncomplexed PR8 NS1A process. In any case, [S94]-F2F3 is biologically active in blocking MICROBIOLOGY effector domain (PDB ID 2GX9) (23). Interestingly, the Zn fingers CPSF30 binding by the NS1A protein in vivo, as it is the same of F2F3 (Cys-X7/X8-Cys-X5/X4-Cys-X3-His) are structurally similar molecule that was used to demonstrate that F2F3 expression in to the C3H Cys-X8-Cys-X5-Cys-X3-His Zn-finger domains of hu- virus-infected cells inhibits virus replication and increases virus- man TIS11d, which binds class II AU-rich elements in the 3Ј induced production of IFN-␤ mRNA (19, 20). Gel filtration data untranslated regions of target mRNAs to regulate mRNA turnover demonstrate that [S94]-F2F3 binds Ud NS1A (85-215), forming a (24). This structural similarity suggests a possible RNA-binding tetrameric complex with a molecular mass of ϳ48 kDa (see Fig. 1A) function for these Zn-finger domains of CPSF30. similar to that obtained using [P94]-F2F3. Attempts to crystallize The structure of the F2F3:NS1A (85-215) complex reveals an the purified [P94]-F2F3:NS1A (85-215) complex provided only tiny unexpected mode of interaction between the NS1A protein and crystals, while crystals of the [S94]-F2F3:NS1A (85-215) complex, CPSF30. The complex is a tetramer, in which two F2F3 molecules with similar morphology, are significantly larger and suitable for wrap around two NS1A effector domains that are interacting with X-ray crystallography. As illustrated in Fig. S3, the S94 residues in each other in a head-to-head orientation (Fig. 1B). The F2F3- both F2F3 molecules of the complex have proline-like backbone binding surface has contributions from both chains of NS1A conformations (␾ ϭϪ72°; ␺ ϭ 172°), essentially identical to the (85-215) in the head-to-head orientation (Fig. S2). The surface area conformation reported for residue P94 (␾ ϭϪ70°; ␺ ϭ 173°) in the of one NS1A (85-215) molecule is ϳ5,600 Å2, of which ϳ1,680 Å2 solution NMR structure of the isolated [P94]-F2F3 molecule (PDB participates in intermolecular interactions, while for each F2F3 accession code 2D9N). In any case, the location of residue 94 in the

Das et al. PNAS ͉ September 2, 2008 ͉ vol. 105 ͉ no. 35 ͉ 13093 Downloaded by guest on October 2, 2021 We next assessed the role of the CPSF30-binding pocket during virus infection by generating a recombinant Ud virus that expresses a NS1A protein with a G184R mutation. This recombinant virus forms plaques only ϳ25% the size of WT plaques (Fig. 2B), and during multiple cycle growth (at low multiplicity of infection) the recombinant replicates 20-fold slower than WT; for example, at 24 h after infection the titers of recombinant and WT are 1.9 ϫ 105 and 3.8 ϫ 106 pfu/ml, respectively. Attenuation of the G184R virus is not because of a reduction in the amount of the NS1A protein synthesized in G184R-infected cells (Fig. 2C). To determine whether this attenuation is because of reduced suppression of pre-mRNA processing, the relative amounts of IFN-␤ pre-mRNA and IFN-␤ mRNA in WT- and G184R-infected cells were determined by quantitative RT-PCR (Fig. 2D). A substantial amount of unprocessed IFN-␤ pre-mRNA was detected in WT virus-infected Fig. 2. Effects of amino acid substitutions in the NS1A protein on its cells, verifying that the Ud virus activates transcription of the IFN-␤ interaction with CPSF30 and on its function in inﬂuenza A virus-infected cells. gene via the activation of interferon regulatory factor (IRF)-3 and (A) GST-pulldown assay. GST-F2F3 or GST were mixed with equal amounts of other transcription factors (6, 9, 26). Approximately 20% as much the WT or indicated mutant 35S-labeled full-length NS1A protein of Ud, which IFN-␤ pre-mRNA accumulated in G184R-infected cells, whereas were prepared as described in SI Materials and Methods. The labeled proteins ␤ eluted with glutathione from GST-F2F3 or GST were resolved by SDS- the amount of mature IFN- mRNA was approximately five times polyacrylamide gels, which were analyzed by exposure to X-ray ﬁlm. (B) more than that in WT-infected cells. Consequently, the processing Plaque sizes of the WT and G184R mutant Ud viruses in Madin-Darby canine of IFN-␤ pre-mRNA, which is largely blocked in WT virus-infected kidney (MDCK) cells. (C) The G184R mutation in the Ud NS1A protein does not cells, occurs much more efficiently in G184R virus-infected cells. affect the amount of the NS1A protein synthesized in MDCK cells infected with This functional analysis demonstrates in vivo the biological signif- 5 pfu/cell. Immunoblots of cell extracts collected at 6 h after infection were icance of the tetrameric [S94]-F2F3:NS1A (85-215) complex struc- carried out with either anti-NS1A or antitubulin antibody. (D) Quantitative ␤ ␤ ture, and particularly the importance of the CPSF30 binding pocket. RT-PCR measuring amounts of IFN- pre-mRNA (Left) and IFN- mRNA (Right) It also provides definitive evidence for the essential role of NS1A- in WT and G184R Ud-infected cells. Pre-mRNA results were normalized to WT, ␤ and mRNA results were normalized to G184R data. The results show the CPSF30 binding in the inhibition of IFN- mRNA production average and standard deviation for the relative levels of G184R pre-mRNA and during infection with influenza A/Udorn/72 virus. WT mRNA from three different virus infections. Of the eight amino acid residues identified in the CPSF30- binding pocket of the Ud NS1A protein by this crystal structure, six are almost completely (Ͼ98%) conserved among influenza A 3D structure of the complex is distant from, and not in contact with, viruses isolated from humans (21), strongly suggesting that this the NS1A effector domain (see Fig. S2). These results demonstrate CPSF30 binding site is used by all human influenza A viruses to that the S94 substitution does not disrupt the structure of F2F3, and suppress the production of IFN-␤ mRNA. These residues are also that [S94]-F2F3 and [P94]-F2F3 can form complexes with NS1A conserved in H5N1 viruses isolated from humans and in the (85-215) with similar structures. Most importantly, as described pandemic 1918 virus (A/Brevig Mission/1/18). The exceptions are below, the [S94]-F2F3:NS1A (85-215) crystal structure accurately I119 and V180, at the edge of the pocket shown in Fig. 1D, which predicts effects of single-site mutations on specific functions of the in some sequences are replaced by similar hydrophobic Met and Ile NS1A protein in virus-infected cells, verifying the biological validity residues, respectively, preserving the hydrophobicity of the pocket. of this crystal structure. The Role of F103 and M106 of the NS1A Protein in Stabilizing the The F2F3 Binding Pocket on the NS1A Protein. The crystal structure Tetrameric Complex. The interaction surface between NS1A and identifies the F2F3 binding pocket on the surface of NS1A (Fig. 1 F2F3 in the tetrameric complex extends beyond the primary F2F3 C and D). This largely hydrophobic pocket, primarily defined by binding pocket shown in Fig. 1D. Two NS1A amino acids outside amino acid residues K110, I117, I119, Q121, V180, G183, G184, and the binding pocket, F103 and M106, are also critically involved in W187, interacts with aromatic side chains of residues Y97, F98, and formation of the tetrameric complex (Fig. 3 A and B, and Fig. 4). F102 of the F3 Zn finger of the corresponding F2F3 molecule. As illustrated in Fig. 4, the side chain of residue M106 is positioned To validate the biological relevance of the binding pocket, at the tetrameric epicenter and interacts with the side chain of site-specific Ud NS1A protein variants were designed and evaluated M106Ј of the NS1AЈ (molecule II) and with residues in both the for their effect on CPSF30-binding. Because the viral NS2/NEP F2F3 and F2F3Ј domains. The aromatic side chain F103 of NS1A protein and NS1A are coded for by the same region of the viral (molecule I) interacts extensively with hydrophobic residues L72Ј, genome, but in a different translation frame (21), substitutions were Y88Ј, and P111Ј of F2F3Ј (molecule II). Residues F103 and M106 selected such that the amino acid sequence of NS2/NEP would not are required for the tight binding of F2F3 in vitro. Thus, for example, be affected when a NS1A-mutant recombinant influenza A virus no observable binding between F2F3 and an Ud NS1A protein with was generated (described below); for example, only Arg substitu- simultaneous F103L and M106I substitutions occurs in vitro GST tions are possible at the positions G184 or W187 of NS1A without pull-down experiments (Fig. 3C). affecting the NS2/NEP sequence. Either of these two Arg substi- In contrast, such F103L and M106I substitutions in the NS1A tutions, or substitution of Ala for Q121, eliminated detectable protein reduce, but do not completely eliminate, its binding to binding of the full-length Ud NS1A protein to F2F3 in GST- CPSF30 in infected cells because other viral proteins bind to and pulldown experiments (Fig. 2A), confirming our hypotheses based stabilize the NS1A:CPSF30 complex (20). This phenomenon was on the crystal structure that these three amino acids are required observed with the NS1A protein of the 1997 pathogenic H5N1 for the formation of the F2F3:NS1A complex. To ascertain if the influenza A/Hong Kong/483/97 (HK97) virus, which contains L G184R substitution alters the structure of the NS1A protein, (instead of F) at 103 and I (instead of M) at 106. The HK97 NS1A [G184R]-NS1A (85-215) was cloned, purified, and characterized. protein does not bind to F2F3 in vitro (20), like the mutant Ud This amino acid substitution has little or no effect on the overall fold NS1A protein that contains L103 and I106 (see Fig. 3C), but does of NS1A (85-215), as indicated by amide circular dichroism and two bind CPSF30 to a significant extent in vivo when it is expressed in dimensional (1H-15N)-HSQC NMR spectra (Fig. S4). a virus that also encodes the other internal HK97 proteins (20). The

13094 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805213105 Das et al. Downloaded by guest on October 2, 2021 Fig. 4. Molecular graphic showing the locations of NS1A residues F103 and M106 with respect to the F3-binding pocket. The tetrameric interface extends beyond the hydrophobic pocket of the NS1A effector domain (orange) which binds one F2F3 molecule (blue). Residues M106 and F103 (green surfaces) of the same NS1A effector domain interact with both the F2F3 (blue) and F2F3Ј (yellow) molecules. The M106 sidechain of this NS1A effector domain also interacts with the M106Ј side chain of the second NS1AЈ molecule (red) at the tetrameric epicenter.

binding of CPSF30 to the HK97 NS1A protein requires the HK97 polymerase complex (PB1, PB2, PA, and NP), but not the HK97 M protein, and that the viral polymerase complex is actually part of the CPSF30:NS1A complex in infected cells, even when the NS1A protein has the optimum F103 and M106 amino acids (R.-L.K. and R.M.K., unpublished data). Despite encoding a NS1A protein with less than optimum CPSF30 binding, the HK97 virus was pathogenic in birds, humans, mice, and ferrets (27, 28). Consequently, pathogenicity does not require a fully functional NS1A protein, and other viral proteins are sufficient to confer a pathogenic phenotype, consistent with a large body of literature indicating that pathogenicity/virulence is polygenic; that is, it cannot be ascribed to a single specific viral gene, but rather requires a combination of several, but not necessarily all, viral genes (29–31). Nonetheless, attenuated CPSF30 binding by the HK97 virus is suboptimal for virulence: changing L103 to F and I106 to M results in not only a 20-fold enhancement in virus replication in tissue culture (20), but also an even larger 250-fold, enhancement of virulence in mice (L.-M. Chen, R.T. Davis, R.-L.K., M. Malur, R.M.K., R.O. Donis, unpublished data). Thus, Fig. 3. Structural role of F103 and M106 in formation of the tetrameric enhanced CPSF30 binding because of these two amino acid changes MICROBIOLOGY complex. (A) Molecular graphics showing how two F2F3 molecules (repre- leads to enhanced influenza virulence in mice, demonstrating the sented as the electrostatic potential surface) wrap around two NS1A (85-215) importance of the intermolecular interactions involving the highly molecules. The head-to-head interaction of NS1A molecules forms a docking conserved F103 and M106 amino acids of the NS1A protein in the surface for F2F3 binding. The side chain of residue M106 is critically positioned virulence of influenza A viruses. at the tetrameric epicenter and interacts with the other three molecules. (B) Unlike the NS1A protein of the HK97 virus, all of the NS1A Expanded regions showing the structural environments of amino acid residues F103 and M106 of NS1A. The aromatic side chain of NS1A F103 interacts proteins of H5N1 viruses isolated from humans since 2003 contain primarily with the hydrophobic amino acid residues L72Ј, Y88Ј and P111Ј that the optimum F103 and M106 amino acids (20, 21). The origin of the are present on the surface of F2F3Ј.(C) GST-pulldown assay. GST-F2F3 or GST selective pressure favoring replacement of F for L at 103 and of M was mixed with the WT or 103/106 mutant [F103L, M106I]-NS1A protein, and for I at 106 in human H5N1 NS1A proteins is not known. Surpris- analyzed as described in the legend of Fig. 2A. ingly, in 1999 to 2002, a period during which no H5N1 viruses were isolated from humans, the vast majority of the H5N1 viruses isolated from avian species encoded NS1A proteins with F and M F103L and M106I mutations weaken, but do not prevent, complex at these positions (21). It is likely that these avian H5N1 viruses formation in vivo because cognate HK97 internal proteins interact were the source of the H5N1 viruses that were transmitted to with, and hence substantially stabilize, the CPSF30: HK97 NS1A humans in 2003. It is not known what caused this change in the avian complex in infected cells (20). Recent experiments show that such H5N1 NS1A protein, in light of the fact that the identities of the

Das et al. PNAS ͉ September 2, 2008 ͉ vol. 105 ͉ no. 35 ͉ 13095 Downloaded by guest on October 2, 2021 amino acids at positions 103 and 106 in the NS1A proteins of other types of avian viruses (e.g., H9N2 and H6N1) showed considerable variability from 1997 to 2006 (21). F103 and M106 are highly conserved in seasonal (H1N1, H3N2, H2N2) human influenza A viruses. Of the 2,284 seasonal influenza A viruses isolated from humans since 1933, 2,276 (99.6%) contain F103 and M106 (21). Only four seasonal viruses have encoded a NS1A protein with Leu instead of the consensus Phe at position 103 (21) and these viruses, like the H5N1 HK97 virus (20), presumably Fig. 5. PR8 virus infection, unlike infection by Ud virus, does not activate bind CPSF30 suboptimally in infected cells. Only five seasonal IRF-3. HEL299 cells were either mock-infected (M, lane 1), infected with 5 viruses have encoded an NS1A protein with a hydrophilic amino pfu/cell of Ud virus (Ud, lane 2), infected with 5 pfu/cell of PR8 virus (PR8, lane acid (S) at position 103, namely, three viruses isolated in 1934 to 3), or infected with 5 pfu/cell of a recombinant Ud virus in which the Ud NS 1936 (including influenza A/PR/8/34), one virus isolated in 1954 gene was replaced by the PR8 NS gene (Ud/NS-PR8, lane 4). At 7 h after (A/Leningrad/54), and one virus isolated in 1976 (A/New Jersey/76) infection, cell extracts were prepared, subjected to electrophoresis on a 7.5% (21). The absence of such viruses since 1976 shows that influenza native gel, and IRF-3 monomers and dimers were detected by Western immu- A viruses encoding NS1A proteins with S103 are selected against noblotting using rabbit anti-IRF-3 antibody (36). An immunoblot with anti- during replication in humans. NS1A antibody conﬁrmed that equivalent amounts of the NS1A protein were Our F2F3:Ud NS1A structure predicts that a hydrophilic residue synthesized in Ud, PR8, and Ud/NS-PR8 virus-infected cells (lanes 2–4). Further details are provided in the SI Materials and Methods. at position 103 in the NS1A protein should attenuate CPSF30 binding (see Figs. 3B and 4), and indeed the A/PR/8/34 (PR8) NS1A protein that contains S103 does not bind CPSF30 in vitro (32). In because IRF-3 is not activated in PR8 virus-infected cells, and the addition, the PR8 NS1A protein does not inhibit cellular gene resulting activation of IFN-␤ gene expression does not occur, expression in infected cells (32), indicating that it does not bind CPSF30 binding by the PR8 NS1A protein is not required in PR8 CPSF30 in infected cells (which we have confirmed). The 2.1 Å infected cells. resolution crystal structure of the PR8 NS1A effector domain is a To determine whether this lack of IRF-3 activation is caused by ␤ dimer, stabilized by intermolecular -sheet interactions (23). As the PR8 NS1A protein itself, we generated a recombinant Ud virus illustrated in Fig. S5, the oligomer orientations in the PR8 NS1A that expresses the PR8 instead of the Ud NS1A protein. In cells structure are completely different from the F2F3-assisted head-to- infected by this recombinant virus, IRF-3 is activated (Fig. 5, lane head dimer of Ud NS1A effector domains observed in the 4), demonstrating that the PR8 NS1A protein does not suppress F2F3:NS1A (85-215) complex structure. The Ud NS1A dimer IRF-3 activation and that other mechanisms are responsible for the formation is mediated by extensive hydrophobic interactions with lack of IRF-3 activation in PR8 virus-infected cells. This recombi- two F2F3 molecules, whereas the PR8 NS1A dimer interface ␤ nant virus is attenuated: during multiple cycle growth the recom- primarily involves hydrogen bonds between two small -strands binant replicates 40-fold more slowly than WT; that is, at 24 h after (␤1). The Ud NS1A effector domain also forms weak oligomers at infection the titers of the recombinant and Ud are 2 ϫ 105 and 8 ϫ higher concentrations and in the absence of F2F3 (as described in 106 pfu/ml, respectively. Despite encoding an NS1A protein that is the legend to Fig. 1). However, these interactions are weak and defective in CPSF30 binding (32), as well as defective in another could readily dissociate to form other oligomeric states with ap- function (34), the PR8 virus is pathogenic in mice (31), again propriate binding partners, such as the F2F3-stabilized head-to- attesting to the polygenic nature of pathogenicity (29–31). head dimer seen in the Ud NS1A:F2F3 complex. Why is CPSF30 binding by the PR8 NS1A protein not required Conclusions during virus infection? Previous studies have reported that PR8 virus infection does not activate transcription factor IRF-3 (32, 33). The X-ray crystal structure of the Ud NS1A (85-215):F2F3 complex In one set of experiments, IRF-3 activation was assayed by deter- described here provides unique insights into the binding interface mining whether the large amount of the NS1A protein that accu- between NS1A and CPSF30 and its linked suppression of a crucial mulated after 12 h of influenza A virus infection inhibited the IRF-3 host antiviral response. These insights are not anticipated by the dimerization induced by a subsequent 6 h superinfection by Sendai available structures of the F2F3 fragment alone (PDB ID 2D9N) or virus (32). Based on this assay, it was claimed that the NS1A protein of the PR8 NS1A effector domain that does not bind F2F3 or of PR8 virus, as well as the NS1A proteins of other influenza A CPSF30 (23, 32). The key structural features include two symmetric viruses, inhibits IRF-3 activation. However, it was not established F2F3 binding pockets that are formed at a protein–protein interface that the effect of such accumulated NS1A protein on subsequent in the tetrameric structure of the Ud NS1A:F2F3 complex. The two Sendai virus-induced IRF-3 activation accurately mirrors the ac- Ud NS1A effector domains in this complex interact with each other tions of the NS1A protein on IRF-3 activation induced by influenza in a head-to-head orientation, which is unexpected and different ␤ A virus itself at earlier times of infection. To address this issue, we from the extended -sheet dimer interface observed in the PR8 directly assayed IRF-3 activation in influenza A virus-infected cells NS1A effector domain (23). As illustrated in Fig. S6, this head-to- by measuring the formation of the homodimer of IRF-3 that results head dimer structure is also compatible with the known dimeric from the phosphorylation-dependent activation of IRF-3 (6, 29). structure of the N-terminal RNA-binding domain (16, 17). The Ud Afterinfectionofcellsfor7hwiththeUdvirus,ϳ50% of the NS1A:F2F3 structure also explains the roles of NS1A residues F103 endogenous IRF-3 migrates in the position of the activated dimer and M106 in stabilizing the functional complex and their strong (Fig. 5, lane 2), confirming our previous study (6). This activated evolutionary conservation. dimeric IRF-3 functions to activate high level transcription of the Based on these insights, we can conclude that CPSF30 binding by IFN-␤ gene, as documented here by the accumulation of a sub- the NS1A protein is the primary, if not the only, mechanism by stantial amount of unprocessed IFN-␤ pre-mRNA in infected cells which circulating human influenza A viruses suppress the produc- (see Fig. 2B). In fact, we have found that IRF-3 is activated by tion of IFN-␤ mRNA in infected cells. The X-ray crystal structure influenza A viruses expressing NS1A proteins encoded by many presented here reveals the atomic details underlying this binding other influenza A virus strains [e.g., two H5N1 viruses (HK97; process. Significantly, six of the amino acids comprising the F2F3/ A/Vietnam/1203/04) and the 1918 virus] (data not shown). In CPSF30-binding pocket are almost completely (Ͼ98%) conserved contrast, the PR8 virus does not activate IRF-3 (see Fig. 5, lane 3), among human influenza A viruses, including H5N1 viruses and the confirming previous studies by others (32, 33). Consequently, 1918 virus (21). In addition, the two NS1A residues, F103 and

13096 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0805213105 Das et al. Downloaded by guest on October 2, 2021 M106, that stabilize the F2F3:NS1A tetrameric structure are also Materials and Methods Ͼ almost completely conserved ( 99%) among human influenza A NS1A effector domain NS1A (85-215) and the F2F3 (60-120) fragments of CPSF30, viruses (21). Interestingly, the properties of two prominent influ- were cloned into modified pET21c and pET14c (Novagen) vectors (35). The enza A viruses, H5N1 HK97 and PR8, that encode NS1A proteins constructs were verified by DNA sequence analysis and expressed in E. coli with different amino acids at positions 103 and 106, reinforce the BL21(DE3) cells containing the rare tRNA expression plasmid pMGK. The over- importance of the CPSF30 binding site on the NS1A protein. The expressed proteins were purified as described in SI Materials and Methods. The HK97 NS1A protein contains different hydrophobic amino acids at crystals of the purified [S94]-F2F3:NS1A complex were obtained by hanging drop positions 103 and 106, and its binding to CPSF30 is stabilized in vapor diffusion against the well solution containing 0.1 M sodium acetate pH 5.5, infected cells via the interaction of the viral polymerase with the 0.5 M KNO3, and 10% sucrose at 20°C. The structure was solved by Se-Met MAD NS1A:CPSF30 complex (20), demonstrating that a supplementary technique (22) and refined at 1.95 Å resolution to final Rwork and Rfree of 0.210 and viral mechanism is used in infected cells to ensure that the 0.234, respectively. Details of the X-ray crystallography methods are presented in NS1A:CPSF30 complex is formed. The PR8 NS1A protein has a the SI Materials and Methods. Recombinant Ud viruses were generated from hydrophilic (S) amino acid at position 103 that essentially eliminates cloned DNA as described in the SI Materials and Methods. IFN-␤ pre-mRNA and CPSF30 binding affinity, but PR8 virus uses a unique strategy to IFN-␤ mRNA in virus-infected cells were measured by real-time quantitative suppress the production of IFN-␤ mRNA, namely suppressing RT-PCR as described in the SI Materials and Methods. IRF-3 activation by an undetermined mechanism. This PR8 strategy has been selected against during replication in humans, in Note Added in Proof. While this paper was in press, another X-ray crystal competition with influenza A viruses that both activate IRF-3 and structure of an apo-NS1A effector domain was published (37), in which the require CPSF30 binding by the NS1A protein to suppress the NS1A dimer interface differs from that previously reported (as depicted in Fig. production of mature IFN-␤ mRNA. This selection further dem- S5b). The dimer interface in this new structure involves residues which con- onstrates the crucial importance of NS1A protein-mediated tribute to the F3-binding pocket in our structure (e.g., Trp-187). CPSF30 binding for circulating human influenza A viruses. Con- sidering that F2F3 expression in cells inhibits the replication of ACKNOWLEDGMENTS. We thank T. Acton, A. Ertekin, Y.J. Huang, A. Shatkin, influenza A viruses with no apparent effect on the cells (19), the and C. Zhao for helpful discussions, and G. DeTitta from Hauptman- intermolecular interfaces characterized here at atomic resolution, Woodward Medical Research Institute for the High-Throughput Crystalliza- tion Facility. This work was supported by institutional funds provided by particularly the interface between the CPSF30 F3 finger and Rutgers University and by National Institutes of Health Grant AI11772 (to specific conserved amino acid residues of NS1A (see Fig. 1), are R.M.K.). Protein sample production was supported in part by the Northeast candidate target sites for the development of small-molecule anti- Structural Genomics Consortium of the National Institutes of Health Protein viral drugs. Structure Initiative, Grant U54-074958 (to G.T.M.).

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