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• W2004014022 abstract "The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5′-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756–2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1–1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis. The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5′-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756–2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1–1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis. Many viruses that replicate in the cytoplasm synthesize capped mRNAs by their own transcription systems (reviewed in Refs. 1Banerjee A.K. Microbiol. Rev. 1980; 44: 175-205Crossref PubMed Google Scholar and 2Furuichi Y. Shatkin A.J. Adv. Virus Res. 2000; 55: 135-184Crossref PubMed Google Scholar). As in the case of eukaryotic cells (reviewed in Refs. 3Mizumoto K. Kaziro Y. Prog. Nucleic Acids Res. Mol. Biol. 1987; 34: 1-28Crossref PubMed Scopus (62) Google Scholar and 4Shuman S. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 1-40Crossref PubMed Google Scholar), for cytoplasmic DNA viruses such as vaccinia virus and double-stranded RNA viruses such as reovirus, a γ-phosphoryl group is removed from the 5′-end of a nascent mRNA chain by viral RNA 5′-triphosphatase to generate a 5′-diphosphoryl-terminated acceptor RNA (ppN-) (1Banerjee A.K. Microbiol. Rev. 1980; 44: 175-205Crossref PubMed Google Scholar, 2Furuichi Y. Shatkin A.J. Adv. Virus Res. 2000; 55: 135-184Crossref PubMed Google Scholar). Then a GMP moiety from GTP is transferred to the 5′-diphosphate end of the RNA by viral mRNA guanylyltransferase to produce the cap core structure (GpppN-) (1Banerjee A.K. Microbiol. Rev. 1980; 44: 175-205Crossref PubMed Google Scholar, 2Furuichi Y. Shatkin A.J. Adv. Virus Res. 2000; 55: 135-184Crossref PubMed Google Scholar). Next, the methyl group is transferred to the guanine-7N position in the cap core structure from S-adenosyl-l-methionine (AdoMet) 1The abbreviations used are: AdoMet, S-adenosyl-l-methionine; G-7-MTase, mRNA (guanine-7-)methyltransferase; SeV, Sendai virus; RNP, ribonucleoprotein; cap-ribose MTase, mRNA (nucleoside-2′-O-)methyltransferase; RdRp, RNA-dependent RNA polymerase; VSV, vesicular stomatitis virus; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; BSA, bovine serum albumin; Ni-NTA, nickel-nitrilotriacetic acid; PEI, polyethyleneimine; MOPS, 4-morpholinepropanesulfonic acid. by mRNA (guanine-7-)methyltransferase (G-7-MTase) and also to the penultimate ribose-2′-OH position by mRNA (nucleoside-2′-O-)methyltransferase (cap-ribose MTase) (1Banerjee A.K. Microbiol. Rev. 1980; 44: 175-205Crossref PubMed Google Scholar, 2Furuichi Y. Shatkin A.J. Adv. Virus Res. 2000; 55: 135-184Crossref PubMed Google Scholar). Consequently, methylated cap structures, m7GpppN-(cap 0) or m7GpppNm-(cap I), are formed. Unlike cellular mRNA, the capping reactions of some viral mRNAs are carried out by unique mechanisms that differ from those of the cellular capping. For example, for plus strand RNA viruses within the alphavirus-like superfamily such as Semliki Forest virus, an m7GMP moiety of m7GTP, which is preformed by methylation of GTP, is transferred to the 5′-diphosphate end of the acceptor RNA to form a cap 0 structure (5Ahola T. Kääriäinen L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 507-511Crossref PubMed Scopus (239) Google Scholar). In vesicular stomatitis virus (VSV), which possesses a negative strand RNA genome and belongs to the Rhabdoviridae family of the order Mononegavirales, cap formation has been suggested to proceed through the transfer of a GDP moiety from GTP to the 5′-monophosphate end of the mRNA to form the cap core structure from the in vitro transcription studies with purified virions (6Abraham G. Rhodes D.P. Banerjee A.K. Cell. 1975; 5: 51-58Abstract Full Text PDF PubMed Scopus (161) Google Scholar). Finally, the cap core is methylated at the ribose-2′-OH position and then at the guanine-7N position to generate the cap I structure (7Moyer S.A. Abraham G. Adler R. Banerjee A.K. Cell. 1975; 5: 59-67Abstract Full Text PDF PubMed Scopus (72) Google Scholar, 8Testa D. Banerjee A.K. J. Virol. 1977; 24: 786-793Crossref PubMed Google Scholar, 9Horikami S.M. Moyer S.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7694-7698Crossref PubMed Scopus (45) Google Scholar). However, direct biochemical studies on these reactions with purified viral component(s) have not been performed. Sendai virus (SeV), a prototype of the Paramyxoviridae family in the order Mononegavirales, possesses a monopartite negative strand RNA genome consisting of six genes encoding the nucleocapsid (N), phospho-(P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large (L) proteins (reviewed in Ref. 10Lamb R.A. Kolakofsky D. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott/Wlliams & Wilkins, Philadelphia2001: 1305-1340Google Scholar). The P gene also codes for C and V proteins (10Lamb R.A. Kolakofsky D. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott/Wlliams & Wilkins, Philadelphia2001: 1305-1340Google Scholar). N proteins wrap the 15.3-kb RNA genome to form a helical nucleocapsid called the N-RNA complex, which serves as a functional template for transcription as well as replication (10Lamb R.A. Kolakofsky D. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott/Wlliams & Wilkins, Philadelphia2001: 1305-1340Google Scholar). A viral RNA-dependent RNA polymerase (RdRp) complex consisting of L and cofactor P proteins binds to the N-RNA complex to form the ribonucleoprotein (RNP) complex, which functions as a basal transcription apparatus (11Hamaguchi M. Yoshida T. Nishikawa K. Naruse H. Nagai Y. Virology. 1983; 128: 105-117Crossref PubMed Scopus (166) Google Scholar, 12Curran J. Marq J.B. Kolakofsky D. Virology. 1992; 189: 647-656Crossref PubMed Scopus (151) Google Scholar, 13Horikami S.M. Curran J. Kolakofsky D. Moyer S.A. J. Virol. 1992; 66: 4901-4908Crossref PubMed Google Scholar). The RNP contains about 30 L, 300 P, and 2,600 N protein molecules (14Lamb R.A. Mahy B.W. Choppin P.W. Virology. 1976; 69: 116-131Crossref PubMed Scopus (167) Google Scholar). According to the 3′-entry model (10Lamb R.A. Kolakofsky D. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott/Wlliams & Wilkins, Philadelphia2001: 1305-1340Google Scholar), the RdRp enters at the 3′-end of the genomic RNA and then initiates the sequential synthesis of a positive-sense leader RNA of about 50 nucleotides and at least six capped and polyadenylated mRNA species. During replication, a full-length positive-sense anti-genomic RNA is first synthesized from the genomic RNA, which is in turn used as a template for the synthesis of progeny genomic RNA (10Lamb R.A. Kolakofsky D. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott/Wlliams & Wilkins, Philadelphia2001: 1305-1340Google Scholar). The mononegavirus L protein is believed to possess all enzymatic activities required for RNA synthesis (11Hamaguchi M. Yoshida T. Nishikawa K. Naruse H. Nagai Y. Virology. 1983; 128: 105-117Crossref PubMed Scopus (166) Google Scholar, 15Emerson S.U. Yu Y. J. Virol. 1975; 15: 1348-1356Crossref PubMed Google Scholar, 16De B.P. Banerjee A.K. Biochem. Biophys. Res. Commun. 1985; 126: 40-49Crossref PubMed Scopus (72) Google Scholar) and RNA processing including capping, cap methylation (17Hercyk N. Horikami S.M. Moyer S.A. Virology. 1988; 163: 222-225Crossref PubMed Scopus (84) Google Scholar), and polyadenylation (18Hunt D.M. Hutchinson K.L. Virology. 1993; 193: 786-793Crossref PubMed Scopus (39) Google Scholar). Furthermore, a protein kinase activity is thought to reside in the L protein (19Einberger H. Mertz R. Hofschneider P.H. Neubert W.J. J. Virol. 1990; 64: 4274-4280Crossref PubMed Google Scholar, 20Hammond D.C. Haley B.E. Lesnaw J.A. J. Gen. Virol. 1992; 73: 67-75Crossref PubMed Scopus (28) Google Scholar). However, the precise enzymatic functions of the L protein remain to be studied. We established an accurate and efficient in vitro mRNA synthesizing system using purified SeV particles or RNP complexes (21Mizumoto K. Muroya K. Takagi T. Omata-Yamada T. Shibuta H. Iwasaki K. J. Biochem. (Tokyo). 1995; 117: 527-534Crossref PubMed Scopus (18) Google Scholar, 22Takagi T. Muroya K. Iwama M. Shioda T. Tsukamoto T. Mizumoto K. J. Biochem. (Tokyo). 1995; 118: 390-396Crossref PubMed Scopus (13) Google Scholar, 23Ogino T. Iwama M. Ohsawa Y. Mizumoto K. Biochem. Biophys. Res. Commun. 2003; 311: 283-293Crossref PubMed Scopus (29) Google Scholar). Polyadenylated mRNA products synthesized from the isolated RNP complex contained cap structures, GpppA-, m7GpppA-(cap 0), and m7GpppAm-(cap I), suggesting that the complex possesses a capping enzyme system including mRNA guanylyltransferase, G-7-MTase, and cap-ribose MTase (22Takagi T. Muroya K. Iwama M. Shioda T. Tsukamoto T. Mizumoto K. J. Biochem. (Tokyo). 1995; 118: 390-396Crossref PubMed Scopus (13) Google Scholar). Here we developed an assay system to detect G-7-MTase activity associated with the SeV RNP or purified viral proteins independently of transcription. Our findings show that the SeV L protein is a viral mRNA-specific G-7-MTase, in which the C-terminal domain is responsible for G-7-MTase activity. To our knowledge, this is the first direct biochemical evidence to show that the Mononegavirales polymerase L protein catalyzes cap G-7-MTase activity. Preparation of SeV RNP-M, RNP, and N-RNA Complexes—The RNP-M (genomic RNA-N-P-L-M) and RNP (genomic RNA-N-P-L) complexes were prepared from SeV (Z strain) particles as described previously (23Ogino T. Iwama M. Ohsawa Y. Mizumoto K. Biochem. Biophys. Res. Commun. 2003; 311: 283-293Crossref PubMed Scopus (29) Google Scholar). The N-RNA complex lacking the P and L proteins was prepared from the RNP essentially as described by Udem and Cook (24Udem S.A. Cook K.A. J. Virol. 1984; 49: 57-65Crossref PubMed Google Scholar). The RNP complex (1 mg of protein) was diluted in 5 ml of TE (10 mm Tris-HCl (pH 7.9), 1 mm EDTA) containing 0.5% sodium lauroylsarcosine (Sigma) and CsCl to give an initial density of 1.29 g/cm3, and was centrifuged to equilibrium in a Beckman MLS-50 rotor at 90,000 × gav for 40 h at 20 °C. The nucleocapsid peak fractions with a density of 1.29–1.30 g/cm3 were pooled and diluted 10-fold with TE containing 500 mm NaCl. Then the N-RNA complex (180 μg of protein) was pelleted by centrifugation in a Beckman type 65 rotor at 80,000 × gav for 1 h at 4 °C and resuspended in 50 μl of TE containing 100 mm NaCl and 30% (w/v) sucrose. Expression and Purification of Recombinant SeV L and P Proteins— A136 and A50M cDNA fragments (25Kato A. Sakai Y. Shioda T. Kondo T. Nakanishi M. Nagai Y. Genes Cells. 1996; 1: 569-579Crossref PubMed Scopus (214) Google Scholar) derived from the SeV (Z strain) genome (GenBank™ accession numbers X00087 and X03614) were assembled and cloned into the pBluescript II KS+ plasmid to create a template for amplification of the L gene by PCR using ExTaq polymerase (Takara, Japan). To amplify DNA fragments encoding the wild-type L protein (1–2,228 amino acids) and its deletion mutants (1–1,120, 1,121–1,755, 1,756–2,228, and 1,121–2,228), the following sense (F, SalI site underlined) and antisense (R, KpnI site underlined and stop codon double-underlined) primers were used in various combinations: L1-_F, 5′-AGC GTC GAC ATG GAT GGG CAG GAG T; L1121-_F, 5′-AGC GTC GAC CCG GTG AAA GAC AAC ATC GA; L1756-_F, 5′-AGC GTC GAC GGT CTG ACG TTA CCA TTC GA; L-1120_R, 5′-GTG GTA CC T TAT TTC CTG AGA GTT CTA GTC A; L-1755_R, 5′-GTG GTA CC T TAA ACA CCT GAT CTG CCT ATC T; and L-2228_R, 5′-GTG GTA CC T TAC GAG CTG TCA TAT GGC T. The resulting PCR products were digested with SalI and KpnI and cloned into the SalI and KpnI sites of the pUC119 plasmid. The cloned PCR products were partially sequenced to ensure accuracy, and then their middle portions, which could not be sequenced, were exchanged for those from the original template cDNA by using restriction enzymes. The clones for the wild-type L protein, 1–1,120, and 1,121–2,228 were subcloned into the SalI and KpnI sites of a baculovirus transfer vector pBlueBacHis2 A-BSSK. This vector was constructed by inserting a double-strand oligonucleotide formed by annealing BSSK_F, 5′-GAT CCG TCG ACC CCG GGG GTA CCA (SalI and KpnI sites underlined), and BSSK_R, 5′-TCG ATG GTA CCC CCG GGG TCG ACG (KpnI and SalI sites underlined), into the BamHI and SalI sites of the baculovirus transfer vector pBlueBacHis2 A (MaxBac Baculovirus Expression System, Invitrogen) to alter its multicloning site. The clones for 1,121–1,755 and 1,756–2,228 were subcloned into the SalI and KpnI sites of the baculovirus transfer vector pFastBac-HTc (Bac-to-Bac Baculovirus Expression System, Invitrogen). To produce a template for amplification of the coding sequence of P gene, SeV A7 and A21 cDNA fragments (25Kato A. Sakai Y. Shioda T. Kondo T. Nakanishi M. Nagai Y. Genes Cells. 1996; 1: 569-579Crossref PubMed Scopus (214) Google Scholar) were assembled and cloned into the pUC119 plasmid. To construct a baculovirus transfer vector for C-terminal His-tagged P protein, we first constructed an Escherichia coli expression vector for C-terminal His-tagged P protein. The P gene was amplified by PCR using the following sense and antisense primers: P_F, 5′-CCA GGA TCC ATG GAT CAA GAT GCC TTC AT (BamHI and NcoI sites underlined) and P_R2, GGT AGA TCT GTT GGT CAG TGA CTC TAT GT (BglII site underlined). The PCR products were digested with the NcoI and BglII and inserted into NcoI and BglII sites of the pQE-60 plasmid (Qiagen) to create a pQE-P-H vector. Next, to construct a baculovirus transfer vector for nontagged P protein, the coding sequence of P gene was amplified using the sense primer, P_F, and an antisense primer, P_R1, GGT AAG CTT CTA GTT GGT CAG TGA CTC TA (HindIII site underlined and stop codon double-underlined). The resulting PCR products were digested by BamHI and HindIII and inserted into the BamHI and HindIII sites of the pFastBac1 plasmid (Bac-to-Bac Baculovirus Expression System, Invitrogen) to generate a pFastBac-P. Finally, a DNA fragment encoding a C-terminal portion of the P protein fused to a hexahistidine tag was obtained by digestion of the pQE-P-H vector with NdeI and HindIII. It was then inserted into the NdeI and HindIII sites of the above pFastBac-P vector to generate a baculovirus transfer vector for C-terminal His-tagged P protein (pFastBac-P-H). Recombinant baculoviruses expressing N-terminal His-tagged wild-type L protein (residues 1–2,228), its deletion mutants (residues 1–1,120, 1,121–2,228, 1,121–1,755, and 1,756–2,228), and C-terminal His-tagged wild-type P protein were generated using the respective transfer vectors for the MaxBac and Bac-to-Bac Baculovirus Expression Systems (Invitrogen). Sf9 insect cells (1 × 108 cells) were infected with the respective baculoviruses at a multiplicity of infection of five plaqueforming units per cell and cultured in Grace's insect cell culture medium (Invitrogen) supplemented with 10% fatal bovine serum (Invitrogen) and 10 μg/ml gentamicin (Invitrogen) at 27 °C for 72 h. All subsequent purification steps were carried out at 4 °C. The baculovirus-infected cells were harvested and washed twice with ice-cold phosphate-buffered saline. The packed cell pellets (1.2–1.5 ml) were suspended in 10 ml of TMG buffer (20 mm Tris-HCl (pH 7.9), 5 mm 2-mercaptoethanol, 20% glycerol) containing 300 mm NaCl and 1 mm phenylmethanesulfonyl fluoride (PMSF), and the cells were disrupted by sonication. The disrupted cell suspensions were separated into supernatants (S15) and pellets (P15) by centrifugation at 15,000 × gav for 10 min at 4 °C. For purification of the His-tagged P protein, the supernatant fraction (S15) was mixed with 0.5 ml of nickel-nitrilotriacetic acid (Ni-NTA)-agarose resin (Qiagen) pre-equilibrated with TMG containing 300 mm NaCl. Following successive washes of the resin with 5 ml of TMG, 300 mm NaCl three times and with 1 ml of TMG, 300 mm NaCl, 20 mm imidazole three times, the protein was eluted from the resin with 0.5 ml of TMG, 300 mm NaCl, 250 mm imidazole four times. For solubilization of the His-tagged wild-type L protein and its deletion mutants, the respective pellets (P15) were resuspended in 5 ml of TMG buffer containing 500 mm NaCl, 0.5% Triton X-100, 10 mm imidazole, and 1 mm PMSF and then sonicated several times. After centrifugation at 15,000 × gav for 10 min at 4 °C, the resulting supernatants were incubated with 0.1 ml of Ni-NTA-agarose resins. The resins were washed with 1 ml of TMG, 500 mm NaCl, 10 mm imidazole three times, and then the proteins were eluted from the respective resins with 0.1 ml of TMG, 300 mm NaCl, 250 mm imidazole four times. The eluates containing the respective His-tagged proteins were pooled and then dialyzed against TEMG buffer (20 mm Tris-HCl (pH 7.9), 0.5 mm EDTA, 5mm 2-mercaptoethanol, 20% glycerol) containing 50 mm NaCl. Finally, 640 μg of the His-tagged P protein, 45 μg of the His-tagged wild-type L protein, and 20–70 μg each of the deletion mutants were obtained. Expression and Purification of Recombinant Human mRNA (Gua-nine-7-)methyltransferase—The coding sequence of human mRNA (gua-nine-7-)methyltransferase (G-7-MTase) (GenBank™ accession number AF067791) (26Pillutla R.C. Yue Z. Maldonado E. Shatkin A.J. J. Biol. Chem. 1998; 273: 21443-21446Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) was amplified from human leukocyte RNA by reverse transcription-PCR using the sense primer 5′-CCA GGA TCC ATG GCA AAT TCT GCA AAA G (BamHI site underlined) and antisense primer 5′-GGT GTC GAC TCA CTG CTG TTT CTC AAA G (SalI site underlined, stop codon double-underlined). The PCR products were digested with BamHI and SalI and inserted into the BamHI and SalI sites of the pQE-30 plasmid (Qiagen) to create a pQE-hCM vector. E. coli strain XL1-Blue harboring the pQE-hCM was grown in 800 ml of LB medium containing 100 μg/ml ampicillin at 28 °C until the A600 reached 0.6. After adding isopropyl-thio-β-d-galactoside to a final concentration of 1 mm, the cells were cultured for another 4 h. The cells were harvested by centrifugation, suspended in 20 ml of TMG, 300 mm NaCl, 1 mm PMSF, and then disrupted by sonication. Following centrifugation at 15,000 × gav for 20 min, the resulting supernatant (S15) was recovered and then incubated with 1 ml of Ni-NTA-agarose resin that had been equilibrated with TMG, 300 mm NaCl. After successive washes of the resin with 10 ml of TMG, 300 mm NaCl three times and with 2 ml of TMG, 300 mm NaCl, 20 mm imidazole twice, the protein was eluted from the resin with 1 ml of TMG, 300 mm NaCl, 250 mm imidazole four times. The eluates containing the protein were combined and then dialyzed against TEMG, 50 mm NaCl. Finally, 1.8 mg of the His-tagged human G-7-MTase was obtained. Preparation of Capped RNA Substrates—Triphosphate-ended RNAs with 5 nucleotides were synthesized by T7 RNA polymerase (Amersham Biosciences) from synthetic DNA templates as described by Milligan and Uhlenbeck (27Milligan J.F. Uhlenbeck O.C. Methods Enzymol. 1989; 180: 51-62Crossref PubMed Scopus (1022) Google Scholar). To prepare the templates for T7 RNA polymerase, an oligonucleotide T7P(+) (5′-TAATACGACTCACTATA) corresponding to the T7 promoter sequence (–17 to –1) was annealed to each template oligonucleotide at the promoter region. The template oligonucleotides used to synthesize 5′-AGGGU, 5′-AGGGA, 5′-AGGAA, 5′-AGAAA, 5′-AAAAA, 5′-GGGGU, 5′-AAGGU, and 5′-ACCAA were as follows: 5′-ACCCT-T7P(–), 5′-TCCCT-T7P(–), 5′-TTCCT-T7P(–), 5′-TTTCT-T7P(–), 5′-TTTTT-T7P(–), 5′-ACCCC-T7P(–), 5′-ACCTTT7P(–), and 5′-TTGGT-T7P(–) (T7P(–) indicates 5′-TATAGTGAGTCGTATTA). After the transcription reactions, the transcription mixtures were treated with deoxyribonuclease I (Roche Applied Science). Then the RNA products were extracted with phenol-chloroform and precipitated with ethanol. In Figs. 2, 3, 4, the transcripts containing RNAs with five nucleotides and unexpected sizes were used as capping substrates. In Figs. 5 and 6, the RNA products denatured with formamide were electrophoresed in a 20% polyacrylamide gel containing 8 m urea, and the RNAs with 5 nucleotides were eluted from gel slices in an elution buffer (1 m ammonium acetate, 10 mm magnesium acetate, 0.1% SDS). Then the eluted RNAs were recovered by ethanol precipitation and used as the capping substrates. The purified RNAs (20 pmol) were incubated for 2 h at 30 °C with 40 μm [α-32P]GTP (2 × 105 cpm/pmol) and 0.2 μg of purified yeast capping enzyme (28Itoh N. Yamada H. Kaziro Y. Mizumoto K. J. Biol. Chem. 1987; 262: 1989-1995Abstract Full Text PDF PubMed Google Scholar) in a reaction mixture (20 μl) containing 50 mm Tris-HCl (pH 7.9), 3 mm MgCl2, 8 mm DTT, 15% glycerol, 0.2 mg/ml bovine serum albumin (BSA), 5 units of ribonuclease inhibitor (Takara, Japan), and 0.2 units of inorganic pyrophosphatase (Sigma) to prepare [32P]cap-labeled RNA ([32P]GpppN-RNA). After the capping reaction, the RNAs were treated with calf intestinal alkaline phosphatase (Roche Applied Science) and extracted with phenol-chloroform. Finally, they were precipitated with ethanol and dissolved in H2O.Fig. 3The L protein is involved in cap methylation as well as RNA synthesis. A, the RNP-M complex (lane 1, 0.6 μg), RNP (lane 2, 0.5 μg; lane 4, 2.9 μg), N-RNA complex (lane 3, 0.4 μg), recombinant (r) L protein (lane 5, rL, 80 ng), and recombinant P protein (lane 6, rP, 0.4 μg) were analyzed by SDS-PAGE (lanes 1–3, 10%; lanes 4–6, 7.5%) followed by staining with Coomassie Brilliant Blue. The positions of marker proteins, viral proteins (L, P, N, and M), and recombinant proteins are shown on the left, middle, and right, respectively. B and C, samples corresponding to lanes 1–6 in A were immunoblotted with anti-L (B) or -P(C) polyclonal antibody. D, the RNP-M complex (lane 1, 5.5 μg), RNP (lanes 2 and 4, 4.3 μg), N-RNA complex (lane 3, 3.6 μg), rP (lanes 5 and 7, 25 ng), and rL (lanes 6 and 7, 10 ng) were subjected to in vitro SeV G-7-MTase reaction with [32P]GpppAGGGU. Cap structures were analyzed by PEI-cellulose TLC. The positions of m7GpppA and GpppA are indicated on the right. E, the N-RNA complex (lanes 1–3 and 5, 3.6 μg), rP (lanes 2, 4, and 5, 0.6 μg), and rL (lanes 3–5, 0.12 μg) were subjected to in vitro SeV transcription reaction. 32P-Labeled transcripts were analyzed by 1.2% agarose gel electrophoresis. The position of 18 S RNA is indicated on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Biochemical properties of SeV and human mRNA (guanine-7-)methyltransferases. G-7-MTase activities were measured in standard reactions (see “Experimental Procedures”) for the SeV RNP-M complex (open circles, 5.5 μg) and rL (closed circles, 10 ng) or for recombinant (r) human G-7-MTase (open squares, 6 ng) with [32P]GpppAGGGU RNA as the methyl acceptor. The pH (A), temperature (B), and concentrations of NaCl (C) and MgCl2 (D) varied as indicated. A, sodium acetate-acetic acid, MOPS-NaOH, and Tris-HCl buffers were used at pH 4.4–5.9, pH 6.2–7.4, and pH 7.5–9.0, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Capped RNA substrate specificity of SeV and human mRNA (guanine-7-) methyltransferases. A, G-7-MTase activities were measured in standard reactions (see “Experimental Procedures”) for the SeV RNP-M complex (solid bars, 1.4 μg) and recombinant (r) L (shaded bars, 3 ng) or for recombinant (r) human G-7-MTase (open bars, 4 ng) using purified capped RNAs with the indicated RNA sequences as methyl acceptors. B, reactions were carried out as in A except that 7 μg of the RNP-M complex (solid bars), 15 ng of recombinant L (shaded bars), and 20 ng of recombinant human G-7-MTase (open bars) were used.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6The C-terminal part of the L protein participates in mRNA cap methylation. A, schematic representation of the recombinant wild-type L protein, with amino acid positions as indicated, and its deletion mutants (1–1,120, 1,121–2,228, 1,121–1,755, and 1,756–2,228). The six blocks (I–VI) conserved in the mononegavirus L proteins are depicted as shaded boxes. B, the recombinant wild-type L protein (lane 1) and its deletion mutants (lanes 2–5) (0.3 μg each) were analyzed by 7.5% SDS-PAGE followed by Coomassie Brilliant Blue staining. The positions of marker proteins are shown on the left. C, the recombinant wild-type L protein (lane 2, 3 ng) and its deletion mutants (lanes 3–6, 20 ng each) were assayed for G-7-MTase activity under standard conditions with purified [32P]GpppAGGGU. Lane 1 indicates no enzyme. Cap structures were analyzed by DE81-cellulose paper electrophoresis. The positions of m7GpppA and GpppA are indicated on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Assays for mRNA (Guanine-7-)methyltransferases—SeV mRNA G-7-MTase activity was measured in a reaction mixture (10 μl) containing 40 mm sodium acetate buffer (pH 5.9), 10 mm NaCl, 2 mm DTT, 10% glycerol, 0.1 mg/ml BSA, 40 μm AdoMet (Roche Applied Science), 2 nm [32P]GpppN-RNA (2 × 105 cpm/pmol), and the appropriate amounts of viral enzyme. The reaction mixture was incubated at 30 °C for 2 h and then boiled at 100 °C for 5 min to inactivate the enzyme. The RNA products were digested with nuclease P1 (Yamasa Corp., Japan) to liberate the cap structures (29Mizumoto K. Lipmann F. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4961-4965Crossref PubMed Scopus (47) Google Scholar). The digests were analyzed by thin layer chromatography on a polyethyleneimine (PEI) cellulose plate (PEI-CEL UV254, Macherey-Nagel) with 0.65 m LiCl or by paper electrophoresis using DEAE-cellulose paper (DE81, Whatman) at pH 3.4 as described by Mizumoto et al. (21Mizumoto K. Muroya K. Takagi T. Omata-Yamada T. Shibuta H. Iwasaki K. J. Biochem. (Tokyo). 1995; 117: 527-534Crossref PubMed Scopus (18) Google Scholar). A human G-7-MTase assay was carried out for 15 min at 20 °C in a mixture (10 μl) containing 40 mm Tris-HCl (pH 8.5), 2.5 mm NaCl, 2 mm DTT, 0.1 mg/ml BSA, 40 μm AdoMet, 2 nm [32P]G-pppN-RNA (2 × 105 cpm/pmol), and the appropriate amounts of the recombinant human G-7-MTase. In Vitro SeV Transcription—In vitro mRNA synthesis was carried out for 2 h at 30 °Cina mixture (25 μl) containing 40 mm HEPES-KOH (pH 7.9), 6 mm MgCl2, 80 mm NaCl, 2 mm DTT, 40 μm AdoMet, 500 μm each of ATP, CTP, and GTP, 50 μm [α-32P]UTP (1.5 × 104 cpm/pmol), the N-RNA complex (3.6 μg of protein) as the template, and recombinant P (0.6 μg) and L (0.12 μg) proteins. After treatment of the transcription mixtures with proteinase K (Roche Applied Science), the transcripts were extracted with phenol-chloroform, precipitated with ethanol, glyoxylated, and electrophoresed in an 1.2% agarose gel as described by Mizumoto et al. (21Mizumoto K. Muroya K. Ta" @default.
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• W2004014022 title "Sendai Virus RNA-dependent RNA Polymerase L Protein Catalyzes Cap Methylation of Virus-specific mRNA" @default.
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