Structural Basis of the Influenza A Virus RNA Polymerase PB2 RNA-binding Domain Containing the Pathogenicity-determinant Lysine 627 Residue

Abstract

Because the influenza A virus has an RNA genome, its RNA-dependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNA primers/promoters to the polymerases is an initiation step in viral transcription. In our current study, we reveal the 2.7 Å tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2, and this residue is associated with the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique “φ”-shaped structure consisting of a helix and an encircled loop within the PB2 domain. By electrostatic analysis, we identified a highly basic groove along with this φ loop and found that lysine 627 is located in the φ loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. This is the first report to show a relationship between RNA binding activity and the pathogenicity-determinant lysine 627. Using the Matras program for protein three-dimensional structural comparisons, we further found that the helix bundles in the PB2 domain are similar to that of activator 1, the 40-kDa subunit of DNA replication clamp loader (replication factor C), which is also an RNA-binding protein. This suggests a functional and structural relationship between the RNA-binding mechanisms underlying both influenza A viral transcription and cellular DNA replication. Our present results thus provide important new information for developing novel drugs that target the primer/promoter RNA binding of viral RNA polymerases. Because the influenza A virus has an RNA genome, its RNA-dependent RNA polymerase, comprising the PA, PB1, and PB2 subunits, is essential for viral transcription and replication. The binding of RNA primers/promoters to the polymerases is an initiation step in viral transcription. In our current study, we reveal the 2.7 Å tertiary structure of the C-terminal RNA-binding domain of PB2 by x-ray crystallography. This domain incorporates lysine 627 of PB2, and this residue is associated with the high pathogenicity and host range restriction of influenza A virus. We found from our current analyses that this lysine is located in a unique “φ”-shaped structure consisting of a helix and an encircled loop within the PB2 domain. By electrostatic analysis, we identified a highly basic groove along with this φ loop and found that lysine 627 is located in the φ loop. A PB2 domain mutant in which glutamic acid is substituted at position 627 shows significantly lower RNA binding activity. This is the first report to show a relationship between RNA binding activity and the pathogenicity-determinant lysine 627. Using the Matras program for protein three-dimensional structural comparisons, we further found that the helix bundles in the PB2 domain are similar to that of activator 1, the 40-kDa subunit of DNA replication clamp loader (replication factor C), which is also an RNA-binding protein. This suggests a functional and structural relationship between the RNA-binding mechanisms underlying both influenza A viral transcription and cellular DNA replication. Our present results thus provide important new information for developing novel drugs that target the primer/promoter RNA binding of viral RNA polymerases. In 1918, a pandemic expansion of influenza A virus resulted in ten million deaths worldwide (1Taubenberger J.K. Reid A.H. Lourens R.M. Wang R. Jin G. Fanning T.G. Nature. 2005; 437: 889-893Crossref PubMed Scopus (848) Google Scholar), and strategies to prevent any future expansions of this virus are therefore an important endeavor (2Morse S.S. Nat. Med. 2007; 13: 681-684Crossref PubMed Scopus (10) Google Scholar, 3Horimoto T. Kawaoka Y. Nat. Rev. Microbiol. 2005; 3: 591-600Crossref PubMed Scopus (591) Google Scholar). Although inhibitors of neuraminidase and the M2 ion channel are widely used as anti-influenza A virus drugs (4De Clercq E. Nat. Rev. Drug Discov. 2006; 5: 1015-1025Crossref PubMed Scopus (596) Google Scholar, 5Hayden F.G. Atmar R.L. Schilling M. Johnson C. Poretz D. Paar D. Huson L. Ward P. Mills R.G. N. Engl. J. Med. 1999; 341: 1336-1343Crossref PubMed Scopus (471) Google Scholar), some adverse effects of these agents and also the emergence of drug-resistant viruses have been reported (6Reece P.A. J. Med. Virol. 2007; 79: 1577-1586Crossref PubMed Scopus (96) Google Scholar, 7Izumi Y. Tokuda K. O'dell K.A. Zorumski C.F. Narahashi T. Neurosci. Lett. 2007; 426: 54-58Crossref PubMed Scopus (80) Google Scholar). Because the transcription and replication of influenza A virus requires the activity of its RNA-dependent RNA polymerase, this enzyme represents a very promising target for anti-viral drug development. Influenza A virus digests host mRNA via the PB1 subunit of its RNA polymerase (8Shi L. Summers D.F. Peng Q. Galarz J.M. Virology. 1995; 208: 38-47Crossref PubMed Scopus (69) Google Scholar). A 5′-RNA fragment of 10–15 nucleotides is used as a primer for transcription initiation (8Shi L. Summers D.F. Peng Q. Galarz J.M. Virology. 1995; 208: 38-47Crossref PubMed Scopus (69) Google Scholar) through primer and promoter RNA binding activities within the C-terminal region of its RNA polymerase PB2 domain (9Honda A. Ishihama A. Biol. Chem. 1997; 378: 483-488PubMed Google Scholar, 10Crescenzo-Chaigne B. van der Werf S. Naffakh N. Virology. 2002; 303: 240-252Crossref PubMed Scopus (43) Google Scholar, 11Jung T.E. Brownlee G.G. J. Gen. Virol. 2006; 87: 679-688Crossref PubMed Scopus (42) Google Scholar). The PA subunit has protease activity and binds to PB (2Morse S.S. Nat. Med. 2007; 13: 681-684Crossref PubMed Scopus (10) Google Scholar). The determination of the tertiary structure of a protein is a valuable tool in the development of a targeting drug, and low resolution structures have previously been determined for the PA, PB1, and PB2 subunits of influenza A virus RNA polymerase by electron microscopy (12Torreira E. Schoehn G. Fernández Y. Jorba N. Ruigrok R.W. Cusack S. Ortín J. Llorca O. Nucleic Acids Res. 2007; 35: 3774-3783Crossref PubMed Scopus (69) Google Scholar). In addition, the structure of PB2 nuclear localization signal domain (amino acid (aa) 3The abbreviations used are: aa, amino acids; SeMet, seleno-methionine; MAD, multiple anomalous dispersion; vRNA, viral RNA; CM, carboxymethyl. residues 687–759), the PB2 cap (7-methyl guanosine triphosphate)-binding domain (aa 318–483), and the C-terminal domain of PA with the N-terminal domain of PB1 have also been reported (13Tarendeau F. Boudet J. Guilligay D. Mas P.J. Bougault C.M. Boulo S. Baudin F. Ruigrok R.W. Daigle N. Ellenberg J. Cusack S. Simorre J.P. Hart D.J. Nat. Struct. Mol. Biol. 2007; 14: 229-233Crossref PubMed Scopus (265) Google Scholar, 14Guilligay D. Tarendeau F. Resa-Infante P. Coloma R. Crepin T. Sehr P. Lewis J. Ruigrok R.W. Ortin J. Hart D.J. Cusack S. Nat. Struct. Mol. Biol. 2008; 15: 500-506Crossref PubMed Scopus (415) Google Scholar, 15Obayashi E. Yoshida H. Kawai F. Shibayama N. Kawaguchi A. Nagata K. Tame J.R. Park S.Y. Nature. 2008; 454: 1127-1131Crossref PubMed Scopus (227) Google Scholar, 16He X. Zhou J. Bartlam M. Zhang R. Ma J. Lou Z. Li X. Li J. Joachimiak A. Zeng Z. Ge R. Rao Z. Liu Y. Nature. 2008; 454: 1123-1126Crossref PubMed Scopus (254) Google Scholar, 17Collins P.J. Haire L.F. Lin Y.P. Liu J. Russell R.J. Walker P.A. Skehel J.J. Martin S.R. Hay A.J. Gamblin S.J. Nature. 2008; 453: 1258-1261Crossref PubMed Scopus (452) Google Scholar). The C-terminal domain of PB2 includes the Lys627 residue, which plays a role in the high pathogenicity and host range restriction of the influenza virus. The structure of PB2 has not been previously elucidated, and the structure-function relationships between this domain and Lys627 represent a significant advance in our understanding of the pathogenicity of influenza A. Expression and Purification of the Influenza A Virus RNA Polymerase PB2 3/3 RNA-binding Domain-A cDNA fragment of the influenza A virus RNA polymerase PB2 3/3 domain (aa 535–759) was amplified by PCR from pBMSA-PB2 (RIKEN) using the following primers: PB2 Met 3/3, 5′-GCCGTTCATATGATGTGGGAGATTAATGGT-3′, and PB2 stop BamHI, 5′-GCCGTTGGATCCTTAATTGATGGCCATCCGAAT-3′. The amplified DNA fragment was then subcloned into the pET28a(+) plasmid (Novagen, Madison, WI) at the NdeI and BamHI restriction sites. This construct was then introduced into BL21-CodonPlus (Stratagene, La Jolla, CA) Escherichia coli. The induction of His6-tagged recombinant protein expression was achieved by the addition of isopropyl β-d-thiogalactopyranoside in TBG-M9 medium, and this was followed by purification using Ni2+-agarose. For further purification, the His-tagged proteins were cleaved by thrombin and purified using a HiTrap™ carboxymethyl (CM) FF column (GE Healthcare, Buckinghamshire, UK) with the ÄKTAprime™ plus system (GE Healthcare). For phase determination, we expressed the recombinant proteins using seleno-methionine (SeMet)-containing M9 medium (18Doublié S. Methods Enzymol. 1997; 276: 523-530Crossref PubMed Scopus (801) Google Scholar) in E. coli B836 (DE3) cells (Novagen) to enable multiple wavelength anomalous dispersion (MAD) analysis. Site-directed mutagenesis for lysine 627 was performed by PCR. We purified PB2 3/3 Glu627 and PB2 3/3 for reference using a HiTrap™ SP XL column (GE Healthcare). RNA Binding Analysis by Surface Plasmon Resonance (Biacore) Assay-A Biacore 3000 instrument (Biacore AB, Uppsala, Sweden) was used to perform surface plasmon resonance assays in accordance with the manufacturer’s instructions. The buffers used in these analyses were as follows: HBS-EP running buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 3 mm EDTA, 0.005% surfactant P20) and sensor chip streptavidin to which 100 μl of 20 μm biotinylated RNA oligomers were immobilized. A 5′-vRNA promoter (5′-AGUAGAAACAAGGCC-3′), capped primer (5′-m7GmAAAUACUCAAG-3′), or non-capped primer (5′-AmAAUACUCAAG-3′) were synthesized by Hokkaido System Science Co. Ltd., (Hokkaido, Japan), and successful capping of the primers was confirmed by mass spectrometry (Voyager 2073 system). These molecules were then tested in the PB2 3/3 domain protein binding assays. Various concentrations (0.015, 0.044, 0.13, 0.40, 1.2, and 3.6 μm) of the purified influenza A virus RNA polymerase PB2 3/3 proteins were then applied to the RNA-immobilized sensor chip in HBS-EP buffer at a 20 μl/min flow rate. A blank control was used as a reference, and these values were subtracted from the raw data. Estimation of the Kd (Dissociation Constant) Values of the PB2 3/3 Protein for Different RNA Substrates-For affinity analyses, the association-dissociation curves for various concentrations of PB2 3/3 or PB2 3/3 Glu627 protein were plotted using the BIAevaluation steady state affinity program (GE Healthcare) from which equilibrium dissociation constant (Kd) values (mol/liter) were calculated. Crystallization-Purified samples were concentrated to 4–10 mg/ml by ultrafiltration. Optimal conditions for crystallization were determined initially using Crystal Screen 1™ and Crystal Screen 2™ (Hampton Research, Aliso Viejo, CA), which involved the hanging drop vapor diffusion method at 4 °C. Native crystals were obtained using a reservoir solution containing 0.1 m sodium cacodylate hydrochloric acid, pH 6.2, and 2 m sodium formate. Crystals of selenomethionine-labeled PB2 3/3 protein were obtained using a reservoir solution containing 0.1 m sodium cacodylate hydrochloric acid, pH 6.0, and 3 m sodium formate. X-ray Diffraction Analysis-Diffraction data sets for Native and SeMet crystals were collected at 100 K, using a mixture of paratone-N (Hampton Research) and paraffin oil as a cryoprotectant, on a beamline PF-AR NW12 instrument at the Photon Factory (Tsukuba, Japan). The image data were processed using HKL2000 (19Otowinovski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38771) Google Scholar) and SCALA (20Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19877) Google Scholar). The phase was obtained by MAD with three wavelength SeMet data sets using SOLVE and RESOLVE (21Terwilliger T.C. Methods Enzymol. 2003; 374: 22-37Crossref PubMed Scopus (435) Google Scholar). The initial models were constructed using the XtalView program (22McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2023) Google Scholar). Iterative refinement and model building were subsequently performed using the REFMAC5 module of the CCP4 suite (22McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2023) Google Scholar). There were no residues found in the disallowed region of the Ramachandran plot as determined by PROCHECK (23Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). To perform x-ray crystallographic analysis of the RNA polymerase PB2 of influenza A virus (H1N1, A/PR/8/34), it was necessary to obtain soluble quantities of the enzyme. The PB2 subunit was therefore divided into three portions. We expressed PB2 3/3 (aa 535–759) in E. coli as a His6-tagged recombinant protein that was subsequently purified from soluble fractions using Ni2+-agarose. We further purified the PB2 3/3 domain protein by cation-exchange chromatography to near homogeneity (98%). We then subjected the recombinant PB2 3/3 protein to crystallization as described under “Experimental Procedures.” Native crystals were found to belong to the space group P3221 with the properties a = b = 52.5 Å, c = 156.3 Å. The Matthew’s value (Vm) was 2.7, assuming one molecule in an asymmetric unit. SeMet crystals were found in the same space group with the properties a = b = 52.6 Å, c = 156.4 Å. The crystal structure of PB2 3/3 was then determined by MAD phasing guided by the six SeMet of the using SeMet crystals C.M. Methods Enzymol. 1997; 276: PubMed Scopus Google Scholar). The model structure for PB2 3/3 was constructed from residues (aa for the C-terminal and molecules and using data of to 2.7 Å. There were no residues found in the disallowed region of the Ramachandran The determined structure for PB2 3/3 is in and of and 1, a and The N-terminal region (aa is in the C-terminal is of 1, a and The structure of the PB2 3/3 (aa of three and one which is similar to the reported structure of the domain of PB2 (13Tarendeau F. Boudet J. Guilligay D. Mas P.J. Bougault C.M. Boulo S. Baudin F. Ruigrok R.W. Daigle N. Ellenberg J. Cusack S. Simorre J.P. Hart D.J. Nat. Struct. Mol. Biol. 2007; 14: 229-233Crossref PubMed Scopus (265) Google Scholar). The N-terminal which has not previously been of helix bundles and The C-terminal region and the are by a (aa Because the of PB2 3/3 is located in of the surface of its C-terminal region in the tertiary the binding of the C-terminal region of PB2 to (13Tarendeau F. Boudet J. Guilligay D. Mas P.J. Bougault C.M. Boulo S. Baudin F. Ruigrok R.W. Daigle N. Ellenberg J. Cusack S. Simorre J.P. Hart D.J. Nat. Struct. Mol. Biol. 2007; 14: 229-233Crossref PubMed Scopus (265) Google Scholar) requires its N-terminal to or the structure of the domain was reported by Tarendeau F. Crepin T. Guilligay D. Ruigrok R.W. Cusack S. Hart D.J. 2008; PubMed Scopus Google Scholar). There are amino acid the structures of the N-terminal this structure Protein and and are host range from to to to these the tertiary structure is to the Å. These host range the tertiary that with host or virus structures also not the residues in the loop. We identified a structure in PB2 3/3 in which one helix (aa is encircled by a loop (aa 1, We this the its this 1, b and In the encircled and one are the influenza and virus PB2 subunits important structural for this The residue at position 627 in PB2 has been to a of pathogenicity and host range restriction for the influenza A virus W. J. Virol. 1993; PubMed Google Scholar, K. S. M. H. T. Kawaoka Y. Virology. PubMed Scopus Google Scholar, M. P. P. Kawaoka Y. PubMed Scopus Google Scholar). lysine 627 is located in the φ loop 1, b and In the φ is also a a amino which to to the essential of the φ structure in the of the influenza A virus. Because the of are the on the φ to in with The of this structure further suggests important for the PB2 which the pathogenicity of this as in the To the of the PB2 3/3 we the electrostatic on its surface using the program surface of PB2 3/3 is highly basic with the that it an important role in the with RNA primers as described in a The φ loop a along with a basic groove the basic surface of PB2 of three a by the φ loop a of the φ loop and a basic groove by the φ loop This basic surface is also from by helix 4 our that the basic surface in RNA we tested the recombinant PB2 3/3 RNA primer promoter binding activities using surface plasmon resonance analysis. This method is also for analysis. The of various concentrations (0.015, 0.044, 0.13, 0.40, 1.2, and 3.6 μm) of the PB2 3/3 protein with RNA oligomers and non-capped primer and a 5′-vRNA were then primer non-capped primer or promoter were tested in a PB2 3/3 domain protein binding T.E. Brownlee G.G. J. Gen. Virol. 2006; 87: 679-688Crossref PubMed Scopus (42) Google Scholar, P. L. J. A. E. Brownlee G.G. J. Biol. Chem. 2003; PubMed Scopus Google Scholar). in the resonance an The PB2 3/3 protein was found to with of these RNA oligomers in a that it RNA binding activity. this is the first report to show that this domain of PB2 has RNA binding activity. To any for the binding of RNA to the PB2 3/3 domain the Kd values were determined for various RNA molecules using the BIAevaluation program The Kd value for 5′-vRNA promoter was as for capped and non-capped primers were found to and This suggests that PB2 3/3 has binding affinity for promoter in with capped and non-capped RNA and (9Honda A. Ishihama A. Biol. Chem. 1997; 378: 483-488PubMed Google Scholar) has that this domain has primer binding activity not show any data in of and Brownlee T.E. Brownlee G.G. J. Gen. Virol. 2006; 87: 679-688Crossref PubMed Scopus (42) Google Scholar) have that the PB2 subunit binds to the promoter not show which domain to this activity. We show in our current that the PB2 3/3 domain has RNA binding activity with a Kd in the μm This affinity was found not to on our current data not the that the PB2 subunit or RNA polymerase binds to capped primers through this domain. The affinity of binding to the promoter is that found for capped primer that this domain promoter binding with the PB1 subunit T.E. Brownlee G.G. J. Gen. Virol. 2006; 87: 679-688Crossref PubMed Scopus (42) Google Scholar). We on the lysine residue at position which is located in the φ loop 1, b and and a and this residue has been to a of the pathogenicity and host range restriction of the influenza A virus (4De Clercq E. Nat. Rev. Drug Discov. 2006; 5: 1015-1025Crossref PubMed Scopus (596) Google Scholar, 5Hayden F.G. Atmar R.L. Schilling M. Johnson C. Poretz D. Paar D. Huson L. Ward P. Mills R.G. N. Engl. J. Med. 1999; 341: 1336-1343Crossref PubMed Scopus (471) Google Scholar, P.A. J. Med. Virol. 2007; 79: 1577-1586Crossref PubMed Scopus (96) Google Scholar). the residue at position 627 is glutamic acid, influenza A virus shows low in cells K. E. van der Werf S. Naffakh N. Virology. 2007; PubMed Scopus Google Scholar). this residue is the virus shows high pathogenicity in W. J. Virol. 1993; PubMed Google Scholar, M. P. P. Kawaoka Y. PubMed Scopus Google Scholar). is found in influenza virus glutamic acid is found in influenza virus at this This amino acid was also previously reported to involved in the with protein K. E. van der Werf S. Naffakh N. Virology. 2007; PubMed Scopus Google Scholar, Q. R.M. Nature. 2006; PubMed Scopus Google Scholar). on our data the basic surface of this we that the residue also to the binding of molecules as to lysine 627 to RNA binding we it with glutamic acid at this position in the PB2 3/3 protein 3/3 the purification of the PB2 3/3 and PB2 3/3 Glu627 proteins using column we found that PB2 3/3 to PB2 3/3 Glu627 not This suggests that the of one lysine at position 627 with glutamic acid has effects the surface of this are basic residues on the basic surface of the PB2 3/3 domain. Because the lysine at position 627 is located in the of the basic surface and within the φ loop it have effects surface To lysine 627 to RNA we the PB2 3/3 and PB2 3/3 Glu627 proteins for this activity using a surface plasmon resonance for of as primer/promoter RNA The RNA binding activity of PB2 3/3 Glu627 was significantly for RNA oligomers tested with that of PB2 3/3 3 and We thus that residue 627 is involved in RNA The in the RNA binding activity of PB2 therefore to the pathogenicity of influenza in Our current results are also not with for as it is not for a domain to have multiple and to our present thus that the PB2 surface is a for novel anti-influenza A virus drugs from the of pathogenicity and host range restriction To further the functional and structural relationships of the recombinant PB2 3/3 we for proteins with a similar tertiary structure using the Matras program T. Nucleic Acids Res. 2003; PubMed Scopus Google Scholar). The results of these that the helix bundles in the N-terminal of PB2 3/3 have with that of subunit from DNA clamp loader (replication factor The tertiary structure of DNA clamp loader was reported previously by and M. J. Nature. PubMed Scopus Google Scholar) The and at the helix as by the structures model that DNA clamp loader binds to RNA nuclear DNA and DNA polymerase at the initiation of DNA replication our analysis the structural and functional relationship between influenza A virus transcription and cellular DNA replication from the of RNA primer Because of the influenza RNA polymerase PB2 3/3 and DNA clamp have RNA binding it is that these helix bundles are involved with an RNA binding In the unique structure within the influenza A virus RNA-dependent RNA polymerase and the associated structure-function relationships between RNA binding and pathogenicity for this enzyme have the to to novel anti-influenza A virus The development of agents achieved by in in which the of these unique tested using molecule We N. N. Y. and S. for data using the The influenza RNA polymerase PB2 was by the DNA (Tsukuba, by with the of of the of Science and