Mutagenesis of the Dengue Virus Type 2 NS5 Methyltransferase Domain
2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês
10.1074/jbc.m800613200
ISSN1083-351X
AutoresHelga Kroschewski, Siew Pheng Lim, R.E. Butcher, Thai Leong Yap, Julien Lescar, P.J. Wright, Subhash G. Vasudevan, Andrew D. Davidson,
Tópico(s)Malaria Research and Control
ResumoThe Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2′-O methyltransferase activities required for sequential methylation of the cap structure present at the 5′ end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-l-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2′-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-l-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-l-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-l-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-l-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation. The Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2′-O methyltransferase activities required for sequential methylation of the cap structure present at the 5′ end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-l-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2′-O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-l-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-l-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-l-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-l-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation. Cellular and many viral mRNAs contain a modified 5′-terminal guanosine "cap" structure covalently linked to the 5′ end of the mRNA. The study of mRNA cap formation using viral, eukaryotic, and protozoan systems has shown that the formation of the 5′-RNA cap structure requires three sequential enzymatic reactions. First, the 5′-terminal triphosphate of the nascent RNA is hydrolyzed to a diphosphate by the enzyme RNA triphosphatase. Second, the RNA is capped with GMP in a5′-5′ triphosphate linkage by mRNA guanyltransferase, and third, the guanosine is methylated at the N7 position by a (guanine-N7)-methyltransferase (N7 MTase) 2The abbreviations used are: MTase, methyltransferase; AdoHcy, S-adenosyl-l-homocysteine; AdoMet, S-adenosyl-l-methionine; DENV, dengue virus; DENV-2, dengue virus type 2; LB, Luria broth; N7 MTase, (guanine-N7)-methyltransferase; NGC, New Guinea C; NS, non-structural; OL-PCR, overlap PCR; pfu, plaque forming units; RT, reverse transcriptase; TAP, tobacco acid pyrophosphatase; TCID50, tissue culture infectious dose 50% end-point; VSV, Vesicular stomatis virus; WNV, West Nile virus; Bicine, N,N-bis(2-hydroxyethyl)glycine; UTR, untranslated region; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. 2The abbreviations used are: MTase, methyltransferase; AdoHcy, S-adenosyl-l-homocysteine; AdoMet, S-adenosyl-l-methionine; DENV, dengue virus; DENV-2, dengue virus type 2; LB, Luria broth; N7 MTase, (guanine-N7)-methyltransferase; NGC, New Guinea C; NS, non-structural; OL-PCR, overlap PCR; pfu, plaque forming units; RT, reverse transcriptase; TAP, tobacco acid pyrophosphatase; TCID50, tissue culture infectious dose 50% end-point; VSV, Vesicular stomatis virus; WNV, West Nile virus; Bicine, N,N-bis(2-hydroxyethyl)glycine; UTR, untranslated region; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. using S-adenosyl-l-methionine (AdoMet) as a methyl donor to form a type 0 (7MeG5′-ppp5′N) cap structure (1Shuman S. Prog. Nucleic Acid Res. Mol. Biol. 2001; 66: 1-40Crossref PubMed Google Scholar, 2Shuman S. Nat. Rev. Mol. Cell Biol. 2002; 3: 619-625Crossref PubMed Scopus (107) Google Scholar). Nucleotides adjacent to the cap structure may be further methylated by nucleoside-2′-O methyltransferases (2′-O MTase) to give type I (7MeG5′-ppp5′NMe) or type II cap structures (7MeG5′-ppp5′NMeNMe). In the simplest case, such as for yeast, each of the enzymatic activities required for RNA capping resides in an individual protein. However, studies on viral capping systems have revealed many interesting variations on the order and the location of the enzymatic activities involved in capping (3Furuichi Y. Shatkin A.J. Adv. Virus Res. 2000; 55: 135-184Crossref PubMed Google Scholar). For vaccinia virus, the best characterized viral system, capping is performed by a heterodimeric enzyme. The larger D1 subunit has RNA triphosphatase and guanyltransferase activities, whereas full N7 MTase activity requires the formation of a complex with the smaller D12 subunit (4Shuman S. Prog. Nucleic Acid Res. Mol. Biol. 1995; 50: 101-129Crossref PubMed Scopus (128) Google Scholar). A third protein, VP39, possesses 2′-O MTase activity and converts the cap from a type 0 to a type I structure. For double-stranded RNA viruses of the family Reoviridae and negative strand RNA viruses of the order Mononegavirales, all of the enzymatic activities have been detected in single large multidomain proteins (5Hercyk N. Horikami S.M. Moyer S.A. Virology. 1988; 163: 222-225Crossref PubMed Scopus (81) Google Scholar, 6Ramadevi N. Burroughs N.J. Mertens P.P. Jones I.M. Roy P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13537-13542Crossref PubMed Scopus (68) Google Scholar, 7Ogino T. Kobayashi M. Iwama M. Mizumoto K. J. Biol. Chem. 2005; 280: 4429-4435Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Structural studies on Reoviridae proteins have assigned distinct enzymatic activities to individual protein domains (8Reinisch K.M. Nibert M.L. Harrison S.C. Nature. 2000; 404: 960-967Crossref PubMed Scopus (382) Google Scholar, 9Sutton G. Grimes J.M. Stuart D.I. Roy P. Nat. Struct. Mol. Biol. 2007; 14: 449-451Crossref PubMed Scopus (66) Google Scholar). By contrast, the N7 and 2′-O MTase activities contained in the L protein of vesicular stomatis virus (VSV) share a common binding site for AdoMet (10Li J. Fontaine-Rodriguez E.C. Whelan S.P. J. Virol. 2005; 79: 13373-13384Crossref PubMed Scopus (98) Google Scholar). This unusual MTase enzyme architecture has recently been proposed to be common to the flavivirus NS5 protein, which exhibits both N7 and 2′-O MTase activities but has only a single AdoMet binding site (11Ray D. Shah A. Tilgner M. Guo Y. Zhao Y. Dong H. Deas T.S. Zhou Y. Li H. Shi P.Y. J. Virol. 2006; 80: 8362-8370Crossref PubMed Scopus (286) Google Scholar, 12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (469) Google Scholar). The four serotypes of dengue virus (DENV types 1–4) are members of the Flavivirus genus in the family Flaviviridae along with a number of other medically important viruses such as West Nile virus (WNV), yellow fever virus, and Japanese encephalitis virus. The DENVs are transmitted by mosquitoes and infect up to 50 million individuals annually in subtropical and tropical regions of the world. Dengue is currently the most important arthropod-borne viral disease of humans and is described as an emerging disease because of its dramatic resurgence in recent years (13Gubler D.J. Trends Microbiol. 2002; 10: 100-103Abstract Full Text Full Text PDF PubMed Scopus (1127) Google Scholar). At present there is neither a safe and effective vaccine nor suitable anti-viral treatments to control dengue disease. Recent studies have shown that enzymes involved in viral capping have potential as anti-viral targets (14Benarroch D. Egloff M.P. Mulard L. Guerreiro C. Romette J.L. Canard B. J. Biol. Chem. 2004; 279: 35638-35643Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 15Li J. Chorba J.S. Whelan S.P. J. Virol. 2007; 81: 4104-4115Crossref PubMed Scopus (30) Google Scholar, 16Luzhkov V.B. Selisko B. Nordqvist A. Peyrane F. Decroly E. Alvarez K. Karlen A. Canard B. Qvist J. Bioorg. Med. Chem. 2007; 15: 7795-7802Crossref PubMed Scopus (68) Google Scholar). DENV, like other flaviviruses, has a single, positive-stranded RNA genome of ∼11 kilobases that contains a type I cap structure at the 5′ end but lacks a 3′ polyadenylate tail. The viral RNA contains a single long open reading frame encoding a single polyprotein that is cleaved by a combination of cellular signal peptidase and the virally encoded two component serine protease NS2B/NS3. Proteolysis yields three structural (C, prM, and E) and seven nonstructural (NS) proteins; NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (17Lindenbach B.D. Rice C.M. Knipe D.M. Howley P.M. Fields Virology. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 991-1042Google Scholar). The C-terminal region of the flavivirus NS3 protein has been shown to possess triphosphatase activity (18Li H.T. Clum S. You S.H. Ebner K.E. Padmanabhan R. J. Virol. 1999; 73: 3108-3116Crossref PubMed Google Scholar, 19Wengler G. Virology. 1993; 197: 265-273Crossref PubMed Scopus (174) Google Scholar) and is presumed to act in concert with the NS5 protein, which contains both N7 and 2′-O MTase activities (11Ray D. Shah A. Tilgner M. Guo Y. Zhao Y. Dong H. Deas T.S. Zhou Y. Li H. Shi P.Y. J. Virol. 2006; 80: 8362-8370Crossref PubMed Scopus (286) Google Scholar, 12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (469) Google Scholar) to cap the viral genome. The NS5 protein is 900 amino acids in size and contains two domains. Enzymatic activities involved in RNA capping are confined to an N-terminal domain, whereas a C-terminal domain contains RNA-dependent RNA polymerase activity (20Koonin E.V. J. Gen. Virol. 1993; 74: 733-740Crossref PubMed Scopus (199) Google Scholar, 21Koonin E.V. Dolja V.V. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 375-430Crossref PubMed Scopus (900) Google Scholar, 22Ackermann M. Padmanabhan R. J. Biol. Chem. 2001; 276: 39926-39937Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 23Guyatt K.J. Westaway E.G. Khromykh A.A. J. Virol. Method. 2001; 92: 37-44Crossref PubMed Scopus (94) Google Scholar, 24Selisko B. Dutartre H. Guillemot J.C. Debarnot C. Benarroch D. Khromykh A. Despres P. Egloff M.P. Canard B. Virology. 2006; 351: 145-158Crossref PubMed Scopus (104) Google Scholar, 25Tan B.H. Fu J. Sugrue R.J. Yap E.H. Chan Y.C. Tan Y.H. Virology. 1996; 216: 317-325Crossref PubMed Scopus (217) Google Scholar, 26Yap T.L. Xu T. Chen Y.L. Malet H. Egloff M.P. Canard B. Vasudevan S.G. Lescar J. J. Virol. 2007; 81: 4753-4765Crossref PubMed Scopus (331) Google Scholar). The N-terminal 296 amino acids of DENV-2 NS5 were initially shown to possess 2′-O MTase activity and the ability to bind GTP (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (469) Google Scholar). Subsequently, the MTase domains of WNV, DENV, and yellow fever virus NS5 were found to also possess N7 MTase activity (11Ray D. Shah A. Tilgner M. Guo Y. Zhao Y. Dong H. Deas T.S. Zhou Y. Li H. Shi P.Y. J. Virol. 2006; 80: 8362-8370Crossref PubMed Scopus (286) Google Scholar, 27Zhou Y. Ray D. Zhao Y. Dong H. Ren S. Li Z. Guo Y. Bernard K.A. Shi P.Y. Li H. J. Virol. 2007; 81: 3891-3903Crossref PubMed Scopus (269) Google Scholar). The x-ray crystal structure of the DENV-2 MTase was the first flavivirus MTase structure to be determined (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (469) Google Scholar). More recently, 15 flavivirus MTase x-ray structures, including those of WNV (27Zhou Y. Ray D. Zhao Y. Dong H. Ren S. Li Z. Guo Y. Bernard K.A. Shi P.Y. Li H. J. Virol. 2007; 81: 3891-3903Crossref PubMed Scopus (269) Google Scholar), Murray Valley encephalitis (28Assenberg R. Ren J. Verma A. Walter T.S. Alderton D. Hurrelbrink R.J. Fuller S.D. Bressanelli S. Owens R.J. Stuart D.I. Grimes J.M. J. Gen. Virol. 2007; 88: 2228-2236Crossref PubMed Scopus (46) Google Scholar), and Meaban virus (29Mastrangelo E. Bollati M. Milani M. Selisko B. Peyrane F. Canard B. Grard G. de Lamballerie X. Bolognesi M. Protein Sci. 2007; 16: 1133-1145Crossref PubMed Scopus (32) Google Scholar), in complex with AdoMet, S-adenosyl-l-homocysteine (AdoHcy), ribavirin (14Benarroch D. Egloff M.P. Mulard L. Guerreiro C. Romette J.L. Canard B. J. Biol. Chem. 2004; 279: 35638-35643Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and a range of other cap analogues (30Egloff M.P. Decroly E. Malet H. Selisko B. Benarroch D. Ferron F. Canard B. J. Mol. Biol. 2007; 372: 723-736Crossref PubMed Scopus (136) Google Scholar) have been determined. The crystal structures of both the DENV and WNV RNA-dependent RNA polymerase domains have also been recently solved (26Yap T.L. Xu T. Chen Y.L. Malet H. Egloff M.P. Canard B. Vasudevan S.G. Lescar J. J. Virol. 2007; 81: 4753-4765Crossref PubMed Scopus (331) Google Scholar, 31Malet H. Egloff M.P. Selisko B. Butcher R.E. Wright P.J. Roberts M. Gruez A. Sulzenbacher G. Vonrhein C. Bricogne G. Mackenzie J.M. Khromykh A.A. Davidson A.D. Canard B. J. Biol. Chem. 2007; 282: 10678-10689Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Based on genetic and structural data, the structures of the WNV MTase and RNA-dependent RNA polymerase domains were assembled in silico, providing a hypothetical overall structural view of the flavivirus NS5 protein (31Malet H. Egloff M.P. Selisko B. Butcher R.E. Wright P.J. Roberts M. Gruez A. Sulzenbacher G. Vonrhein C. Bricogne G. Mackenzie J.M. Khromykh A.A. Davidson A.D. Canard B. J. Biol. Chem. 2007; 282: 10678-10689Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Although the flavivirus MTase is now one of the best structurally characterized viral MTase structures, little is still known concerning the mechanisms by which the NS5 protein carries out its multifunctional roles in RNA capping. To obtain further insight into the regions of the flavivirus NS5 protein that participate in N7 and 2′-O MTase activities, we have introduced a number of mutations into the DENV NS5 MTase domain, targeting either residues involved directly in AdoMet binding or proposed to be involved in NS5 conformation and/or charged interactions. The effects of these mutations on (i) N7 and 2′-O MTase activities using biochemical assays based on bacterially expressed forms of the NS5 MTase domain and (ii) viral replication has been examined using a DENV-2 infectious cDNA clone. Using this approach we have identified a number of amino acid mutations that commonly and differentially affect N7 and 2′-O MTase activities and regions of the protein that are likely to be involved in protein-protein interactions or overall stability of the protein. Cells Lines and Virus Titration—The growth of BHK-21, Aedes albopictus C6/36, and Vero cells and the production of DENV-2 stocks in C6/36 cells have all been described previously (32Gualano R.C. Pryor M.J. Cauchi M.R. Wright P.J. Davidson A.D. J. Gen. Virol. 1998; 79: 437-446Crossref PubMed Scopus (132) Google Scholar, 33Pryor M.J. Carr J.M. Hocking H. Davidson A.D. Li P. Wright P.J. Am. J. Trop. Med. Hyg. 2001; 65: 427-434Crossref PubMed Scopus (92) Google Scholar). The titer of recombinant viruses in tissue culture media was determined either by (a) plaque assay in C6/36 cells at 28 °C or Vero cells at 37 °C as described previously (32Gualano R.C. Pryor M.J. Cauchi M.R. Wright P.J. Davidson A.D. J. Gen. Virol. 1998; 79: 437-446Crossref PubMed Scopus (132) Google Scholar) or (b) TCID50 assay in Vero cells as follows. Viral stocks were serially diluted 10-fold in Eagle's minimal essential medium supplemented with 2% (v/v) fetal calf serum. Aliquots of each dilution were added to 1 × 104 Vero cells in the same medium in each of 12 wells of a 96-well plate. Plates were incubated at 37 °C for 7–8 days and then examined for cytopathic effect. The TCID50 was calculated according to the method of Reed and Muench (34Reed L.J. Muench H. Am. J. Hyg. 1938; 27: 493-497Google Scholar). Viral titers were calculated either as plaque forming units (pfu) or TCID50/ml of initial virus inoculum, respectively. Introduction of NS5 Mutations into Genome Length DENV cDNA—Mutations encoding changes in NS3 and NS5 amino acids were introduced into the genomic length DENV-2 strain New Guinea C (NGC) cDNA clone pDVWS601, which yields the virus v601 (32Gualano R.C. Pryor M.J. Cauchi M.R. Wright P.J. Davidson A.D. J. Gen. Virol. 1998; 79: 437-446Crossref PubMed Scopus (132) Google Scholar, 33Pryor M.J. Carr J.M. Hocking H. Davidson A.D. Li P. Wright P.J. Am. J. Trop. Med. Hyg. 2001; 65: 427-434Crossref PubMed Scopus (92) Google Scholar). Mutations were first introduced into DENV-2 subgenomic cDNA fragments by overlap-PCR (OL-PCR) using specifically designed mutagenic primers (details are available from the author upon request). The OL-PCR fragments were then transferred into the pDVWS601 background either directly or via the intermediate vector pCR-Blunt II-TOPO (Invitrogen) using appropriate combinations of the unique restriction sites, NsiI4700, HpaI7406, StuI7874, and AatII8570 (the cleavage site in the DENV-2 genome (GenBank™ accession number AF038403) for each restriction enzyme is denoted in superscript). The strategies used to produce the mutant constructs are as follows. The mutations K46A/R47A/E49A, K76A/D79A and G81A/G83A/G85A (each NS5 amino acid targeted for mutagenesis is numbered followed by the substituted amino acid) were engineered into 1001-bp OL-PCR fragments (DENV-2 nt 7165–8165) that were HpaI7406/StuI7874-digested and introduced into the corresponding sites of pDVWS601 to produce pDVWS601-NS5K46A,R47A,E49A, pDVWS601-NS5K76A,D79A, and pDVWS601-NS5G81A,G83A,G85A, respectively. The mutations E138A/K139A/D141A, D146A/E149A and E192A/K193A/E195A were engineered into 1363-bp OL-PCR fragments (DENV-2 nt 7393–8755) that were StuI7874/AatII8570-digested and introduced into the corresponding sites of pDVWS601 to produce pDVWS601-NS5E138A,K139A,D141A, pDVWS601-NS5D146A,E149A, and pDVWS601-NS5E192A,K193A,E195A, respectively. The NS5 mutation K101I was engineered into a 1573-bp fragment (DENV-2 nt 7200–8772) that was HpaI7406/AatII8570-digested and introduced into the corresponding sites of pDVWS601 and pDVWS601-NS5E138A,K139A,D141A to produce pDVWS601-NS5K101I and pDVWS601-NS5K101I,E138A,K139A,D141A, respectively. The NS3 mutation A70G was introduced into a 3438-bp fragment (DENV-2 nt 4132–7569) that was NsiI4700/HpaI7406-digested and introduced into the corresponding sites of pDVWS601 and pDVWS601-NS5E192A,K193A,E195A to produce pDVWS601-NS3A70G and pDVWS601-NS3A70G-NS5E192A,K193A,E195A, respectively. The mutation D79A/L80I was engineered into a 3951-bp fragment (DENV-2 nt 6772–10723) contained in the vector pALTER-1 using the Altered Sites II in vitro mutagenesis system (Promega). The mutated plasmid was HpaI7406/StuI7874-digested, and the released fragment introduced into the corresponding sites of pDVWS601 to produce pDVWS601-NS5D79A,L80I. The mutations G48A, G48P, S56A, S56T, W87I, W87K, W87Y, D131A, D131E, and D131N were engineered into 2100-bp OL-PCR fragments (DENV-2 nt 7201–9100) that were blunt end-cloned into the vector pCR-Blunt II-TOPO. cDNA fragments containing the NS5 mutations G48A, G48P, S56A, S56T, W87I, W87K, W87Y or D131A, D131E, and D131N were digested with HpaI7406/AatII8570 or StuI7874/AatII8570, respectively, and cloned into the corresponding sites of pDVWS601 to produce pDVWS601-NS5G48A, pDVWS601-NS5G48P, pDVWS601-NS5S56A pDVWS601-NS5S56T, pDVWS601-NS5W87I, pDVWS601-NS5W87K, pDVWS601-NS5W87Y, pDVWS601-NS5D131A, pDVWS601-NS5D131E, and pDVWS601-NS5D131N. To confirm the introduction of the mutations and verify the integrity of the PCR derived regions, the region covering the introduced inserts was sequenced in the mutagenized pDVWS601 plasmid. Introduction of Mutated cDNA Fragments into pQE-30—cDNA fragments containing the NS5 mutations and encoding the first 296 amino acids of NS5 were amplified from the respective pCR-Blunt II-TOPO or pDVWS601 clones using the primers NS5-MT1 (5′-CACGGATCCGGAACTGGCAACATAGGAGAGACG-3′; DENV-2 nt 7571–7593 are underlined) and NS5-MT296 (5′-CTGCAGGTCGACTTATTGGTCATAGTGCCATGATGTTTC-3′; DENV-2 nt 8457–8434 are underlined, and the stop codon is in bold). The 890-bp cDNA fragments were then digested with BamHI/SalI and cloned into the corresponding sites of pQE30 (Qiagen). The parental clone was termed pQE30-NS5. The sequence of the mutated DENV-2 cDNA insert in each clone was confirmed by DNA sequencing. Production of Virus from Genomic Length DENV cDNA Clones—RNA was transcribed in vitro as described previously (32Gualano R.C. Pryor M.J. Cauchi M.R. Wright P.J. Davidson A.D. J. Gen. Virol. 1998; 79: 437-446Crossref PubMed Scopus (132) Google Scholar) and transfected into BHK-21 cells either by electroporation or using the TransMessenger Transfection kit (Qiagen). For the latter procedure two μg of RNA was used to transfect an 80% confluent cell monolayer contained in a single well of a 6-well tray following the manufacturer's protocol. After transfection, the cells were incubated at either 33 or 37 °C. Medium from BHK-21 cells incubated at both temperatures was passaged twice in C6/36 cells at 28 °C to produce virus stocks. Confirmation of the introduced mutations and the identification of second site mutations in the viral genome was performed by RT-PCR and sequencing as described previously (31Malet H. Egloff M.P. Selisko B. Butcher R.E. Wright P.J. Roberts M. Gruez A. Sulzenbacher G. Vonrhein C. Bricogne G. Mackenzie J.M. Khromykh A.A. Davidson A.D. Canard B. J. Biol. Chem. 2007; 282: 10678-10689Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Infectious Center Assay—BHK-21 cells were transfected with 2 μg of in vitro transcribed viral RNA transcripts as described above, detached after transfection, and adjusted to 1 × 105 cells/ml of culture medium. A 10-fold dilution series of the transfected BHK cells was prepared in minimal essential medium containing 2% fetal calf serum and 1 × 105 Vero cells. Aliquots of each dilution, containing 1 × 104 cells, were dispensed into 96-well plates, grown for 5–7 days, and examined for cytopathic effect. The TCID50 was determined and used to calculate the number of infectious centers produced per 105 transfected cells. Viral Competition Assay—Mutant and wild type recombinant viruses were mixed at a ratio of 10:1 (TCID50/ml) and used to infect Vero cells at a multiplicity of infection of 3. Each infection was allowed to proceed until there was extensive cytopathic effect. An aliquot of the culture supernatant was used to infect a second Vero cell monolayer. This procedure was repeated until the virus mixture had been passaged three times in Vero cells. RNA was extracted from the culture supernatants after each passage, and the presence of the introduced mutations was verified by RT-PCR and sequencing as described above. Expression and Purification of Wild Type and Mutant DENV-2 NGC NS5 MTase—Competent Escherichia coli strain M15(pREP4) (Qiagen) was transformed with wild type or mutant pQE30-NS5 MTase expression vectors and grown overnight on Luria broth (LB) agar plates containing 100 μg/ml ampicillin and 10 μg/ml kanamycin. A single colony was picked and grown overnight in 5 ml of LB medium. This was inoculated into 500 ml of LB media (100 μg/ml ampicillin, 10 μg/ml kanamycin), and the cells were incubated with shaking (220 rpm) at 37 °C until the A595 was ∼0.5. Cultures were induced with 0.4 mm isopropanol-d-thiogalactopyranoside, and growth was continued for a further 5 h at 30 °C for the wild type MTase and 12 h at 16 °C for the mutant MTase proteins. The cells were then pelleted by centrifugation and resuspended in 15 ml of cold buffer A (50 mm Bicine, pH 7.5, 300 mm NaCl, 5 mm imidazole, 10% (v/v) glycerol) per liter. Cells were sonicated, and the debris was removed by centrifugation at 50,000 g for 30 min. The protein solution was loaded onto a 5-ml HiTrap chelating HP (Amersham Biosciences) column, equilibrated, washed, and eluted with buffer A and a linear gradient concentration of imidazole from 5 to 500 mm. The eluted proteins were concentrated and stored in 50 mm Bicine, pH 7.5, 0.15 m NaCl, 1 mm dithiothreitol, and 10% (v/v) glycerol. N7 and 2′-O MTase Activity Assays—The determination of the N7 MTase activities of the wild type and mutant NS5 MTase proteins was performed in duplicate in a reaction mixture containing 1–5 μCi of S-adenosyl-l-[methyl-3H]methionine (64–72Ci/mmol; Amersham Biosciences), 1–5 μg of GTP-capped RNA comprising nt 1–211 of the DENV-2 5′-UTR (in vitro transcribed in the presence of biotinylated CTP using the T7 RiboMax Express kit from Promega), and 100 nm enzyme in assay buffer (50 mm Tris-HCl, pH 7.5, 20 mm NaCl, 2 mm dithiothreitol, 0.05% CHAPS, and 5 units of RNasin inhibitor (11Ray D. Shah A. Tilgner M. Guo Y. Zhao Y. Dong H. Deas T.S. Zhou Y. Li H. Shi P.Y. J. Virol. 2006; 80: 8362-8370Crossref PubMed Scopus (286) Google Scholar, 27Zhou Y. Ray D. Zhao Y. Dong H. Ren S. Li Z. Guo Y. Bernard K.A. Shi P.Y. Li H. J. Virol. 2007; 81: 3891-3903Crossref PubMed Scopus (269) Google Scholar)) at room temperature (22 °C). The extent of methylation at the N7 and 2′-O positions of the capped RNA transcript was determined by removal of the cap from the RNA by tobacco acid pyrophosphatase (TAP) digestion as follows. The RNA in the reaction mixtures was purified by phenol extraction and ethanol precipitation. Half of each purified RNA sample was then treated with TAP in a 50-μl reaction mixture containing 1× TAP reaction buffer and 10 units of TAP (Epicenter Biotechnologies, Madison, WI) for 2 h at 37 °C before the addition of 2× stop solution (200 mm Tris-HCl, pH 7, 100 mm EDTA, 600 mm NaCl, and 125 μm cold S-adenosyl-l-methionine) containing 8 mg/ml streptavidin scintillation proximity assay beads. As a control, the remaining half of the purified RNA sample was similarly treated but in the absence of TAP. All TAP-treated and untreated samples were transferred to a 96-well white opaque plate (Corning Costar, Acton, MA), shaken for 30 min at 750 rpm, and centrifuged for 2 min at 1200 rpm before analysis of 3H incorporation in a microbeta counter (PerkinElmer Life Sciences) with a counting time of 1 min/well. The determination of 2′-O MTase activity was performed in duplicate in a reaction mixture containing 1 μCi of S-adenosyl-l-[methyl-3H]methionine, 40 nm RNA substrate, GpppAGAACCUG-biotin (Trilink Biotechnologies, Inc.), and 20 nm enzyme in assay buffer containing 50 mm Tris-HCl, pH 7.0, 10 mm KCl, 2 mm MgCl2, 2 mm MnCl2, 0.05% (v/v) CHAPS, 2 mm dithiothreitol, and 5 units of RNasin inhibitor. 3S. P. Lim, D. Wen, T. L. Yap, K. Y. Chung, J. Lescar, and S. G. Vasudevan, manuscript in preparation. The reactions were performed directly in a 96-well plate at room temperature (22 °C). The reactions were stopped with 25 μl of 2× stop solution containing 8 mg/ml streptavidin scintillation proximity assay beads, and the plates treated and analyzed as described above. All data points were measured in triplicate. Selection of Amino Acid Mutations for Introduction into the NS5 MTase Domain—Structural analysis has revealed that the DENV-2 MTase contains three subdomains (12Egloff M.P. Benarroch D. Selisko B. Romette J.L. Canard B. EMBO J. 2002; 21: 2757-2768Crossref PubMed Scopus (469) Google Scholar). The core MTase domain (residues 55–222), responsible for AdoMet binding and catalytic activity, is comprised of a seven-stranded β sheet surrounded by four α helices (Fig. 1A). An N-terminal extension to the core domain (subdomain 1) has been shown to contain a GTP binding pocket, whereas the function of the C-terminal subdomain has not
Referência(s)