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Originally published In Press as doi:10.1074/jbc.M411167200 on November 30, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4429-4435, February 11, 2005
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Sendai Virus RNA-dependent RNA Polymerase L Protein Catalyzes Cap Methylation of Virus-specific mRNA*

Tomoaki Ogino{ddagger}§, Masaki Kobayashi{ddagger}, Minako Iwama{ddagger}, and Kiyohisa Mizumoto{ddagger}||

From the {ddagger}Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641 and §Research Center for Biologicals, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan

Received for publication, September 29, 2004 , and in revised form, November 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many viruses that replicate in the cytoplasm synthesize capped mRNAs by their own transcription systems (reviewed in Refs. 1 and 2). As in the case of eukaryotic cells (reviewed in Refs. 3 and 4), for cytoplasmic DNA viruses such as vaccinia virus and double-stranded RNA viruses such as reovirus, a {gamma}-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-) (1, 2). 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-) (1, 2). Next, the methyl group is transferred to the guanine-7N position in the cap core structure from S-adenosyl-L-methionine (AdoMet)1 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) (1, 2). 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 (5). 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 (6). 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 (7–9). 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. 10). The P gene also codes for C and V proteins (10). 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 (10). 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 (11–13). The RNP contains about 30 L, 300 P, and 2,600 N protein molecules (14). According to the 3'-entry model (10), 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 (10). The mononegavirus L protein is believed to possess all enzymatic activities required for RNA synthesis (11, 15, 16) and RNA processing including capping, cap methylation (17), and polyadenylation (18). Furthermore, a protein kinase activity is thought to reside in the L protein (19, 20). 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 (21–23). 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 (22). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (23). The N-RNA complex lacking the P and L proteins was prepared from the RNP essentially as described by Udem and Cook (24). 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 x 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 x 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 (25) derived from the SeV (Z strain) genome (GenBankTM accession numbers X00087 [GenBank] and X03614 [GenBank] ) 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 (25) 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 x 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 x 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 x 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) (GenBankTM accession number AF067791 [GenBank] ) (26) 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-{beta}-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 x 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 (27). 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 [{alpha}-32P]GTP (2 x 105 cpm/pmol) and 0.2 µg of purified yeast capping enzyme (28) 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.



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  		FIG. 2.
Detection of mRNA (guanine-7-)methyltransferase activity associated with the SeV RNP-M complex. A cap-labeled RNA containing the SeV mRNA 5'-end sequence ([32P]GpppAGGGU) was incubated with the SeV ribonucleoprotein-M protein complex (RNP-M) or cellular G-7-MTase under respective optimal conditions (see "Experimental Procedures"). After nuclease P1 digestion of the RNA products, the cap structures were analyzed by DE81-cellulose paper electrophoresis (A) or PEI-cellulose TLC (B). Lanes 1 indicate no enzyme. In lanes 2 and 3, the RNP-M complex (5.5 µg) was incubated without and with AdoMet, respectively. In lanes 4, G-7-MTase activity of recombinant human G-7-MTase (6 ng) was assayed as a positive control. The positions of standard m7GpppAm, m7GpppA, m7GpppG, GpppA, GpppG, and GTP visualized under UV light at wavelength of 254 nm are indicated on the right.

 



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  		FIG. 3.
The 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.

 



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  		FIG. 4.
Biochemical 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.

 



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  		FIG. 5.
Capped 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.

 



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  		FIG. 6.
The 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.

 
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 x 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 (29). 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. (21). 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 x 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 [{alpha}-32P]UTP (1.5 x 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. (21).

SDS-PAGE and Western Blotting—SDS-PAGE and Western blot analyses were carried out as described by Ogino et al. (23). Rabbit anti-L (23) and anti-P (30) polyclonal antibodies were used at dilutions of 1:2000 and 1:4000, respectively.

Protein Concentration—Protein concentrations were determined by the methods of Bradford (31) using bovine serum albumin as a standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of G-7-MTase Activity in the SeV Ribonucleoprotein Complexes—In vitro mRNA products synthesized in the presence of AdoMet from SeV particles or RNP contain methylated cap structures, m7GpppA and m7GpppAm (21, 22). This suggests that the viral RNP contains two cap methyltransferases, G-7-MTase and mRNA cap-ribose MTase. These data prompted us to develop an in vitro assay system to detect viral MTase activities. As shown in Fig. 1, the six SeV genes begin with a conserved 10-nucleotide gene-start signal (3'-UCCC(A/U)(C/G/A)UU(U/A)C-5') by which transcription initiation might be controlled (reviewed in Refs. 10 and 31). The gene-start sequences are transcribed into 5'-terminal consensus sequences (5'-AGGG(U/A)(G/C/U)AA(A/U)G-3') of mRNAs, and their 5'-ends are capped and methylated. This suggests that the consensus sequences also may act as cis-acting signals for cap methylation as well as capping. Therefore, 5'-pppApGpGpGpU, which contains a most conserved 5-nucleotide sequence found in the 5' termini of SeV mRNAs, was synthesized by T7 RNA polymerase from a synthetic DNA template (27). It was then capped in the presence of [32P]GTP with yeast capping enzyme (28) and was used as a methyl acceptor. The SeV ribonucleoprotein-M protein complex (RNP-M), which is mainly composed of genomic RNA, N, P, L, and M proteins (23), was purified from virus particles and used as an enzyme source. 32P-Cap-labeled G(5')ppp(5')ApGpGpGpU was incubated with the RNP-M complex in the presence of AdoMet (Fig. 2). After digestion of the RNA products with nuclease P1, cap structures were analyzed by paper electrophoresis on DEAE-cellulose paper or by thin layer chromatography on a PEI-cellulose plate. As shown in Fig. 2, A and B, when the capped RNA was incubated with the RNP-M complex in the presence of AdoMet under optimal conditions (see "Experimental Procedures"), a product that comigrated with a standard guanine-7-methylated cap structure (m7GpppA) but not with m7GpppAm or GpppAm (marker not shown) on DEAE-cellulose paper (Fig. 2A, lane 3) or PEI-cellulose plate (Fig. 2B, lane 3) was observed. The methylation reaction with the RNP-M complex depended upon the presence of AdoMet (Fig. 2, A and B, lanes 2 and 3). As a positive control, recombinant cellular (human) G-7-MTase that produces the m7GpppA cap structure was assayed (Fig. 2, A and B, lanes 4). These results indicate that the SeV RNP-M complex contains AdoMet-dependent G-7-MTase activity that can be assayed independently of viral mRNA synthesis. Under these conditions, however, we could not detect cap-ribose MTase activity.



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  		FIG. 1.
Synthesis of the capped and polyadenylated mRNA from SeV genome. The gene organization of negative-sense SeV genome is shown in the 3' to 5' order. The terminal leader and trailer regions are shown by le and tr, respectively. The gene-start, gene-end, and intergenic sequences are indicated by S, E, and I, respectively. The viral RdRp transcribes the genomic RNA into at least six monocistronic mRNAs, which contain 5'-methylated cap structures (m7GpppAm-, cap I) and 3'-poly(A) tails. Each viral mRNA starts with the conserved 5'-end sequence complementary to the gene-start sequence and terminates with the conserved 3'-end sequence followed by the poly(A) tail. W is A or U; V is G, C, or A; and B is C, G, or U.

 
L Protein Is Responsible for Cap Methylation as Well as RNA Synthesis—To investigate the SeV protein(s) involved in mRNA cap methylation, RNP (genomic RNA-N-P-L) and N-RNA (genomic RNA-N) complexes were prepared from the RNP-M complex and subjected to a G-7-MTase assay. SDS-PAGE (Fig. 3A) and Western blot (Fig. 3, B and C) analyses showed that the RNP complex lacking the M protein contained more than 80% L and P proteins (lanes 2) compared with the RNP-M complex (lanes 1), whereas the N-RNA lacked almost all of the L and P proteins (lanes 3). The RNP retained about 60% of the G-7-MTase activity exhibited by the RNP-M (Fig. 3D, lanes 1 and 2), but the N-RNA did not (lane 3). Thus, it is possible that the P and/or L proteins participate in mRNA cap methylation, although we failed to detect G-7-MTase activity in the solubilized and diluted materials from the RNP complex (data not shown).

Therefore, the L and P proteins were expressed as recombinant His-tagged proteins in insect cells and were affinity-purified. SDS-PAGE and Western blot analyses indicated that purified recombinant L and P proteins have apparent Mr values of 250,000 and 80,000, respectively (Fig. 3, A–C, lanes 5 and 6), which are similar to those of native L and P proteins in the RNP (lanes 4). As reported previously (33), the recombinant His-tagged P protein migrated slower than would be expected from its calculated Mr (63,000) (Fig. 3A, lane 6). Because a single SeV RNP complex is thought to contain about 30 L and 300 P protein molecules (14), recombinant L and P proteins were added, separately or together, at a molecular ratio of 1:10 to the reaction mixtures for the G-7-MTase assay (Fig. 3D, lanes 5–7). As shown in Fig. 3D, lane 6, recombinant L protein alone catalyzed the cap G-7 methylation of GpppA-RNA, whereas P protein was inert (lane 5). No significant effect of simultaneous addition of recombinant P protein on G-7-MTase activity with recombinant L protein was observed (Fig. 3D, lane 7). Furthermore, no ribose MTase activity was detected in these assay conditions. Because the molar quantity of recombinant L protein (Fig. 3D, lane 6) is about 12-fold lower than that of native L protein in the RNP complex used here (Fig. 3D, lanes 2 and 4), the G-7-MTase activity of recombinant L protein is comparable with or more than that of the RNP complex.

To confirm whether recombinant L protein is transcriptionally active, we reconstituted the mRNA synthesizing reaction with the N-RNA template and recombinant P and L proteins (Fig. 3E). The addition of recombinant P (Fig. 3E, lane 2) or L protein alone (lane 3) to the in vitro transcription mixture containing the N-RNA (lane 1) resulted in no transcription activity. However, combination of the P and L proteins catalyzed the formation of 18 S size transcripts in the presence of the N-RNA complex (Fig. 3E, lane 5), but not in the absence of the complex (lane 4). This is consistent with the notion that both P and L proteins are essential for transcription (11–13). These data indicate that recombinant L protein is active in synthesizing RNAs from the N-RNA template as well as catalyzing G-7 methylation.

Biochemical Properties of SeV Cap G-7-MTase—It is interesting to compare the biochemical properties of viral G-7-MTase with those of cellular G-7-MTase. Fig. 4 shows the optimal G-7 methylation reaction conditions for the RNP-M complex, recombinant L protein, and human G-7-MTase. Cap methylation by the recombinant human G-7-MTase was optimal at pH 8.5 (Fig. 4A, open squares), whereas with the RNP-M complex (open circles) or recombinant L protein (closed circles) the maximal activity was obtained at around pH 6. Human G-7-MTase activity exhibited temperature optimum from 15 to 25 °C, and thereafter it sharply dropped (Fig. 4B, open squares). On the other hand, the profiles of the RNP-M complex (Fig. 4B, open circles) or recombinant L protein (closed circles) showed a broad peak with an optimum at 30 °C, and about 50–80% of the activity was remained even at 45 °C. Na+ and Mg2+ ions were not required for the reaction, and increasing concentrations of these ions inhibited all G-7-MTase reactions (Fig. 4, C and D). However, inhibitory effects of these ions were prominent on cellular enzyme especially at low ion concentrations (Fig. 4, C and D, open squares). These differences in optimal conditions for the reaction between purified viral and cellular enzymes also support the notion that viral G-7-MTase activity resides in the L protein itself and not in contaminating cellular enzyme.

Sequence Specificities of G-7-MTases—To examine RNA sequence specificity in cap methylation, capped RNAs of five nucleotides in length with different sequences were synthesized from synthetic DNA templates by T7 RNA polymerase. Because T7 RNA polymerase produces not only the full-length runoff product but also prematurely terminated RNAs and longer RNAs with an extra 3'-terminal nucleotide(s) (27), RNAs with five nucleotides were purified from the gel. The purified RNAs were then capped and used as the substrates for viral and cellular G-7-MTases (Fig. 5). The RNP-M complex (Fig. 5A, solid bars 1 and 2) and recombinant L protein (shaded bars 1 and 2) effectively methylated capped RNAs with 5'-end sequences of SeV mRNAs such as GpppApGpGpGpU (N, P, M, HN, and L mRNAs) and GpppApGpGpGpA (F mRNA). GpppApGpGpApA, in which the third G residue was replaced by A, was also methylated by the viral enzyme (Fig. 5A, solid and shaded bars 3), whereas GpppApGpApApA and GpppApApApApA were poor substrates, and almost no methylation was observed (solid and shaded bars 4 and 5). When the first A residue in GpppApGpGpGpU was replaced by G, its substrate activity was markedly reduced (Fig. 5A, solid and shaded bars 6). Furthermore, substitution of the second G residue in GpppApGpGpGpU with A also led to a significant decrease in its substrate activity (Fig. 5A, solid and shaded bars 7). These results suggest that the 5'-end GpppApGpG sequence is required by the viral enzyme. The capped 5'-ACCAA, which corresponds to the 5'-end sequence of the uncapped positive sense leader and anti-genomic RNAs transcribed from the 3'-end of SeV genomic RNA (10), showed no methylation with SeV G-7-MTase (Fig. 5A, solid and shaded bars 8). As seen in Fig. 5A, the RNP-M complex (closed bars) and recombinant L protein (shaded bars) exhibited the same sequence specificity, indicating that the L protein is the entity responsible for G-7-MTase activity in the RNP-M complex. In contrast, recombinant human G-7-MTase methylated all capped RNAs, although the efficiency varied depending upon the sequence (Fig. 5A, open bars). In particular, the recombinant human enzyme effectively methylated GpppGpGpGpGpU (open bar 6), which was a poor substrate for the L protein.

The enzymes in those experiments (Fig. 5A) were used in limiting amounts as follows: 1.4 µg of the RNP-M complex, 3 ng of the L protein, and 4 ng of human G-7-MTase. To further confirm the sequence specificity of the viral G-7-MTase, we carried out the reaction with saturating enzyme amounts (7 µg of the RNP-M complex, 15 ng of the L protein, and 20 ng of human G-7-MTase), the results of which are shown in Fig. 5B. Again, we found different sequence specificities between the viral and cellular enzymes. Enhanced methylation activity was observed with an elevated amount of human G-7-MTase in all cases (Fig. 5B, open bars 1–5). However, only poor methylation was observed for nonviral sequences, GpppAGAAA, GpppAAAAA, GpppAAGGU, and GpppACCAA, with elevated amounts of the RNP-M complex and L protein (Fig. 5B, closed and shaded bars 2–5). From these results, we concluded that the viral L protein is the G-7-MTase that specifically methylates the cap structure on the viral mRNA 5'-end sequences.

The C-terminal Part of the SeV L Protein Contains the G-7-MTase Domain—The L proteins from paramyxovirus, rhabdovirus, and filovirus families contain six conserved sequence blocks, I to VI (34, 35) (Fig. 6A). The N-terminal half that contains blocks I–IV has been suggested to include the P-binding site (36–38) as well as putative RdRp subdomains (blocks II–IV) (34). Although the function(s) of the C-terminal half is unknown, a C-terminal section containing block VI was recently proposed to be a cap-ribose MTase domain based upon its sequence (39, 40). To examine the domain(s) responsible for the G-7-MTase activity of the SeV L protein (2,228 amino acids), deletion mutants were prepared as recombinant His-tagged proteins (Fig. 6A) and tested for this activity. As seen in Fig. 6B, their purity was checked by SDS-PAGE. When the N-terminal (residues 1–1,120, Fig. 6B, lane 3) and C-terminal (residues 1,121–2,228, lane 4) halves were subjected to the SeV G-7-MTase assay (Fig. 6C), the C-terminal half but not the N-terminal half exhibited the activity. Furthermore, a shorter fragment containing block VI (residues 1,756–2,228, lane 6) but not a fragment containing block V without block VI (residues 1,121–1,755, lane 5) also catalyzed cap methylation, although to a lesser extent. These results suggest that the G-7-MTase domain as well as the putative cap-ribose MTase domain may be located within the furthest C-terminal region, although we failed to detect the latter activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although the mononegavirus L protein is most likely responsible for cap formation including guanylylation and methylation, no direct evidence has been obtained by using its purified protein form. The major obstacle in identifying the mononegavirus protein(s) involved in cap formation has been the lack of in vitro assay systems for respective enzymatic activities. It is also thought that activities with externally added RNA substrates are hard to detect as separate reactions due to tight coupling between transcription and cap formation (1). Initially, we could not detect mRNA (guanine-7-)methyltransferase activity associated with the SeV RNP-M or RNP complex when measured under conditions conducive to viral transcription or for cellular cap methylation using capped nonviral RNAs as methyl acceptors. However, in our efforts to examine various assay conditions for SeV G-7-MTase activity, we succeeded in detecting activity associated with the RNP-M and RNP complexes using a viral mRNA sequence GpppApGpGpGpU as a methyl acceptor (Figs. 2 and 3). Furthermore, we found that recombinant L protein catalyzes cap methylation as well as mRNA synthesis (Fig. 3). The optimal conditions for SeV cap methylation are distinct from those for SeV transcription or cellular cap methylation (Fig. 4).

The most important finding is that SeV G-7-MTase selectively methylates capped RNAs containing the viral mRNA 5'-end sequence (GpppApGpG-) (Fig. 5). In addition, GpppApGpGpG was also methylated by the viral enzyme similar to GpppApGpGpGpU, whereas no activity was observed for GpppA (data not shown). This indicates that a four-nucleotide length is enough to act as a substrate and suggests that the conserved 5'-end sequence potentially serves as a cis-acting signal for guanine-7-methylation. In other words, the G-7-MTase domain of the SeV L protein recognizes the 5'-terminal AGG sequence in addition to the cap structure. The 5'-terminal AGG sequence is strictly conserved in the mRNAs of paramyxoviruses belonging to the Respirovirus (e.g. SeV, human parainfluenza virus types 1 and 3) and Morbillivirus (e.g. measles virus, rinderpest virus) genera of the Pramyxovirinae subfamily (reviewed in Refs. 10 and 32). On the other hand, 5'-terminal AG/AG and GGG sequences are found in the mRNAs of other paramyxoviruses belonging to the Rubulavirus genus (e.g. mumps virus) of the Pramyxovirinae subfamily and Pneumovirus genus (e.g. human respiratory syncytial virus) in the Pneumovirinae subfamily, respectively (10, 32). GpppApApG- and GpppGpGpG-containing RNAs were poor substrates for SeV G-7-MTase (Fig. 5), suggesting that the substrate specificities of paramyxoviral G-7-MTases (L proteins) may be different between virus groups. Stillman and Whitt (41) reported that introduction of some mutations into the gene-start sequence in a model VSV replicon inhibits 5'-end modification and elongation of the transcripts without affecting transcription initiation. They suggested that the conserved 5'-end sequence (5'-AAC) of VSV mRNA acts as a cis-acting signal(s) for capping and/or cap methylation, and that precise 5'-end modification is required for processive elongation of VSV mRNA. Their data and our findings suggest that mononegavirus L proteins specifically recognize the 5'-end sequences of nascent transcripts synthesized by themselves and modify their 5' termini to form methylated cap structures. In contrast, this study using purified cellular G-7-MTase indicates no specific sequence requirements for the RNA substrate (Fig. 5). Furthermore, cellular G-7-MTase methylates cap core structures such as GpppG, GpppA, GpppC, and GpppU without the RNA chain (3).

Comparative analyses of the L proteins from paramyxoviruses, rhabdoviruses, and filoviruses revealed that they contain the six conserved sequence blocks I–VI, which are interrupted by variable sequences (34, 35). These blocks are postulated to fold into functional domains associated with several enzymatic activities involved in RNA synthesis and processing. Blocks II–IV within the N-terminal half are putative RdRp subdomains that may correspond to fingers, palm, and thumb subdomains of positive-strand RNA virus RdRps and retroviral reverse transcriptases (34). The GDN motif in block III is thought to be a counterpart of the divalent cation coordination motif (GDD) of the positive-strand RNA virus RdRps and to be required for phosphodiester bond formation (42). Paramyxovirus L protein was suggested to bind to the P protein through its N-terminal portion containing block I (36–38). Although complex formation with the P protein is thought to be a prerequisite for stability of the SeV L protein in mammalian cells and for RdRp activity (12, 13), the recombinant SeV L protein was stably expressed in insect cells in the absence of the P protein and exhibited G-7-MTase as well as RdRp activity (Fig. 3). The observation that the C-terminal part alone (residues 1,756–2,228) of the SeV L protein exhibited G-7-MTase activity indicates that the binding sites for the P protein and the putative RdRp subdomains are dispensable for this activity (Fig. 6).

Recently, the C-terminal region (for SeV, residues 1,774–1,984) of the monegavirus L proteins was determined by computational analyses to be the putative cap-ribose MTase domain (39, 40). It includes an AdoMet-binding glycine-rich (GXGXG) motif and other characteristic motifs involved in ribose-2'-O-methylation as observed in 23 S rRNA 2'-O-ribose MTase (RrmJ) and vaccinia virus cap-ribose MTase (VP39). Although in the present study cap-ribose MTase activity could not be detected in the recombinant L protein, it is plausible that the C-terminal domain containing block VI possesses G-7-MTase and cap-ribose MTase activities. To ascertain whether the C-terminal domain is involved in ribose-2'-O-methylation, we need to develop an in vitro assay system for cap-ribose MTase activity.

In conclusion, the biochemical analyses using purified proteins presented here showed that the SeV L protein catalyzes guanine-7-methylation of virus-specific mRNA. Further characterization of the G-7-MTase domain in the L protein is in progress to evaluate its role in viral cap formation. The L protein plays a pivotal role in RNA processing as well as RNA synthesis as shown by this work. Identification of the functional domains of the L protein involved in these respective enzymatic activities would provide insight into the mechanism of mononegaviral gene expression and offer novel targets for developing specific anti-viral drugs.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and by the 21st Century COE Program, Ministry of Education, Culture, Sports, Science and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Present address: Virology Section, Dept. of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195. Back

|| To whom correspondence should be addressed. Tel.: 81-3-5791-6245; Fax: 81-3-3444-6198; E-mail: mizumotok{at}pharm.kitasato-u.ac.jp.

1 The 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. Back



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 ABSTRACT
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 RESULTS
 DISCUSSION
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