virus-specific protein. This observation was confirmed with and extended to Sindbis virus-infected cells by Scheidel et al. (40). They showed an increased ...
Vol. 68, No. 11
JOURNAL OF VIROLOGY, Nov. 1994, p. 7418-7425
0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Expression of Semliki Forest Virus nsP1-Specific Methyltransferase in Insect Cells and in Escherichia coli PIRJO LAAKKONEN,* MARKO HYVONEN,t JOHAN PERANEN,t
AND
LEEVI KAARIAINEN
Institute of Biotechnology, University of Helsinki, Helsinki, Finland Received 13 June 1994/Accepted 15 August 1994
We have expressed the Semliki Forest virus (SFV)-specific nonstructural protein nsPl both in insect cells and in Escherichia coli in the absence of other viral proteins. A substantial amount of nsP1 was synthesized in Sf9 cells infected with the recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) AcNPVnsPl. These cells had a high level of guanine-7-methyltransferase activity compared with that of wild-type AcNPV-infected cells. The methyltransferase activity and nsPl were mostly in the mitochondrial pellet fraction (P15). The enzymatic activity was increased by treatment with deoxycholate (DOC), as in the case of SFV-infected BHK cells. The material released by DOC treatment from P15 of the AcNPV-nsPl-infected cells was analyzed by gel filtration and sucrose gradient centrifugation. Both the methyltransferase activity and nsPl were in aggregates. nsPi expressed in E. coli at 37°C sedimented at 15,000 x g, whereas after expression at 15°C, both nsPl and methyltransferase activity were in the supernatant fraction. Paradoxically, the activity from E. coli was completely inhibited by Triton X-100 and DOC. Sucrose gradient analysis showed that even the "soluble" nsPl-methyltransferase was in aggregates. The methyltransferase activities in the P15 fractions of SFV-infected BHK cells and AcNPV-nsPl-infected Sf9 cells and in E. coli catalyzed linear incorporation of the [3H]methyl group from S-adenosylmethionine to GTP for a 60-min period. The enzymes from the three sources had similar substrate specificities and Km values for S-adenosylmethionine. In addition to GTP, they all methylated dGTP and GpppG, but not m7GTP or GpppA, or in vitro-transcribed RNAs with GpppA and GpppG caps. The unique properties of SFV-specific nsPl methyltransferase are discussed.
Alphaviruses, which are enveloped positive-stranded RNA viruses, replicate in the cytoplasm of a broad range of eukaryotic cells. In addition to the 42S RNA genome of about 11.5 kb, a subgenomic 26S mRNA (4 kb), which codes for the structural proteins, is synthesized during the infection. Both of these RNA molecules are capped and methylated in the cytoplasm, evidently by virus-specific enzymes (for reviews, see 18, 42, and 48). About two-thirds of the RNA genome is translated into a nonstructural polyprotein P1234, which is processed autoproteolytically to yield nsPl, nsP2, nsP3, and nsP4. According to genetic criteria, all these proteins participate in the replication of alphavirus RNAs. Previously, Cross and Gomatos (7) showed a guanine-7methyltransferase activity in the P15 fraction of Semliki Forest virus (SFV)-infected cells: a radioactive methyl group of S-adenosylmethionine (SAM) was transferred to the 5' end (GpppA) of the 42S and 26S RNAs concomitantly with RNA synthesis, to form the cap 0 structure (m7GpppA) typical of SFV RNAs. Interestingly, the synthesis of the viral RNAs was not inhibited by the presence of S-adenosylhomocysteine, which inhibited the methylation reaction. This result would suggest that the initiation of RNA synthesis may not require all modifications at the 5' end. Cross (6) was able to uncouple RNA synthesis and methyltransferase activities in SFV-infected cells. She showed that GTP served as a substrate for the enzyme in SFV-infected cells, but not in mock-infected cells, resulting in the synthesis of m7GTP. This novel methyltransferase activity increased early in infection and remained stable for several hours, even when protein synthesis was inhibited by
cycloheximide, strongly suggesting that it was caused by a virus-specific protein. This observation was confirmed with and extended to Sindbis virus-infected cells by Scheidel et al. (40). They showed an increased methyltransferase activity in Sindbis virus mutant SVLM21-infected cells. This mutant had been obtained by serial passages in methionine-deprived Aedes albopictus cells. The mutations of SVLM21 were localized to nsPl (28). Direct evidence for the association of enhanced methyltransferase activity with nsPl was found by expressing this mutant nsPl in Escherichia coli (30). We are studying the functions of the SFV-specific nonstructural proteins. Here we report the expression of SFV nsPl both in insect cells and in E. coli in the absence of other virusspecific proteins. High yields of nsPl and associated methyltransferase activity were found both in Spodoptera frugiperda (Sf9) insect cells and in E. coli. The biochemical properties of the guanine-7-methyltransferases, expressed in both cell types, were characterized. MATERIALS AND METHODS Cells and virus. The origin and cultivation of BHK 21 cells (clone 13) and SFV prototype strain, were described earlier (17, 19). The Autographa califomica nuclear polyhedrosis virus (AcNPV) and the Sf9 cell line from Spodoptera frugiperda were gifts from C. Oker-Blom (Abo Akademi, Turku, Finland). The cells were grown as monolayer cultures in plastic bottles (Falcon) at 28°C in TNM-FH medium (49) (Nordvacc Media) containing 10% fetal calf serum (PAA Labor- und Forschungsges M.B.H., Linz, Austria), 100 U of penicillin per ml, 100 ,ug of streptomycin per ml, and 300 ,ug of glutamine per ml. The propagation of AcNPV in Sf9 cells and the isolation of virus DNA was performed as previously described (49). Plasmid constructions. All DNA manipulations were done by the standard methods (38). Plasmid pPLH214, encoding the
Corresponding author. Mailing address: Institute of BiotechnolP.O. Box 45 (Valimotie 7), FIN-00014, University of Helsinki, Helsinki, Finland. Phone: 358-0-43461. Fax: 358-0-4346028. t Present address: European Molecular Biology Laboratory, D-69012 Heidelberg, Germany. *
ogy,
7418
VOL. 68, 1994
SEMLIKI FOREST VIRUS nsPl AS METHYLTRANSFERASE
SFV-specific nonstructural proteins (34), was linearized with Xhol and used as the template to amplify a fragment encoding nsPl by PCR. For this purpose, two oligonucleotides were synthesized: a 30-mer upstream primer 5'-AAT AGG ATC CAT GGC CGC CAA AGT GCA TGT-3', corresponding to nucleotides 86 to 105 with 10 additional nucleotides containing a BamHI recognition site, and a 34-mer downstream primer 5'-TCC ATC TAG ATT ATG CAC CTG CGT GAT ACT CTA G-3', corresponding to nucleotides 1676 to 1696 with 13 additional nucleotides containing an XbaI recognition site and a termination codon. Amplification was done by the aid of Thermus aquaticus (Taq) DNA polymerase (Promega). After PCR, the nsPl gene was cut with BamHI and XbaI and cloned into BglII-XbaI-treated transfer plasmid pVL1392 (gift from M. Summers, Texas A&M University; 23) to generate recombinant plasmid pBNS1. Plasmid pTSF1, encoding the SFV nsPl gene under the T7 promoter (31), was cleaved with NcoI and HindlIl. An approximately 1,700-bp fragment was cloned into the E. coli expression vector pBAT4 (33), which had been cleaved with NcoI and HindlIl. The resulting plasmid, pBATnsPl, contained nsPl under the T7 promoter inducible by isopropyl 1-thio-3-D-galactopyranoside (IPTG). Cloning of the recombinant virus. A cDNA molecule encoding nsPl was transferred into the AcNPV genome by homologous recombination in Sf9 cells. For this purpose, cells were cotransfected with AcNPV DNA and plasmid pBNS1 by lipofection (9, 12). Virus containing growth medium was collected after 1 week of incubation at 28°C, and the recombinant virus, AcNPV-nsPl, was isolated and subcloned by the method of Summers and Smith (49). Cell fractionation. Sf9 cells grown in plastic bottles (3 x 106 cells per 25-cm2 bottle) were infected with AcNPV or AcNPVnsPl or mock infected. At 48 h postinfection, the cells were collected and washed twice with cold phosphate-buffered saline and once with RS buffer (10 mM Tris-HCl [pH 8.0], 10 mM NaCl), containing 0.2 mM phenylmethylsulfonyl fluoride and 100 IU of Trasylol (Bayer AG, Leverkusen, Germany) per ml. After the washes, the cells were resuspended in 1 ml of RS buffer, left on ice for 15 min to swell, and disrupted in a Dounce homogenizer with 40 strokes. Nuclei were removed by centrifugation (500 x g for 10 min), and the postnuclear supernatant was centrifuged further (Biofuge maximum speed for 15 min at 4°C) to obtain P15 and S15 fractions. P15 fractions from SFV-infected and mock-infected BHK cells were prepared as previously described (36). Radioactive labeling. Control Sf9 cells and cells infected with AcNVP or AcNVP-nsPl were labeled with 14C-labeled amino acid mixture (Amersham) (33 puCi/75-cm2 bottle) from 24 h to 44 h postinfection in TNM-FH medium containing 1/10 of the normal amino acids. Labeling with [3H]palmitic acid (100 ,uCi per bottle) {[9, 10(n)-3H]palmitic acid, 54 Ci/mmol; Amersham} was for 2 h at 44 h postinfection. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the labeled proteins and fluorography were carried out as previously described (34, 50). Quantitation of the radioactive and Coomassie blue-stained protein bands was done by Ultroscan XL Enhanced Laser Densitometer (LKB). Immunoprecipitation, immunoblotting, and immunofluorescence. Immunoprecipitation was carried out as previously described (50), using anti-nsPl antiserum (32). For immunoblotting, proteins separated by SDS-PAGE were transferred to nitrocellulose membranes (Hybond-C-extra; Amersham). Anti-nsPl antiserum (1:10,000) was used as a primary antibody followed by goat anti-rabbit conjugated to horseradish peroxidase (DAKO, Copenhagen, Denmark). The filters were developed with enhanced chemiluminescence (ECL; Amer-
7419
sham). For immunofluorescence, Sf9 cells, grown on cover slips in 3.5-cm-diameter plastic dishes, were fixed with 3% paraformaldehyde and made permeable with 0.05% Triton X-100 (16). Anti-nsPl was used as the primary antibody, followed by swine anti-rabbit immunoglobulin G conjugated to tetramethylrhodamine isothiocyanate (DAKO). Expression of nsPl in E. coli. Expression plasmid pBATnsPl was transformed into E. coli BL21(DE3) (Novagen). For a control, we used cells transformed with the pBAT4 vector. Cells were grown in the presence of 100,ug of ampicillin per ml in 2x YT medium (in 1 liter, 8 g of Bacto Tryptone, 5 g of yeast extract, and 2.5 g of NaCl) at 37°C until the optical density at 600 nm was 0.7 to 1.0. The cultures were then transferred to 15°C, and protein expression was induced with 500,uM IPTG. After 20 h, the cells were collected by centrifugation (JA-14 rotor; Beckman) for 15 min at 5,000 rpm. Pelleted cells were resuspended in lysis buffer (50 mM Tris-HCl [pH 8.0], 0.1% Tween 20, 1 mM phenylmethylsulfonyl fluoride) at 1/30 of the original volume. The cell suspension was passed twice through a prechilled French press (French Pressure Cell Press; SLM Instruments, Inc.) at a cell pressure of 10,000 lb/in2. The lysate was centrifuged for 15 min at 15,000 x g at 4°C, and the resulting supernatant was used for further analysis. Sucrose gradient analysis and gel filtration. The P15 fraction from AcNPV-nsPl-infected Sf9 cells solubilized with 1% deoxycholate (DOC) or the supernatant from pBAT-nsPl transformed E. coli cell lysate was analyzed in a linear 10 to 40% (wt/wt) sucrose gradient in 50 mM Tris, pH 8.0. Centrifugation was done in SW 50.1 rotor (Beckman) for 4 h at 35,000 rpm at 4°C. [35S]methionine-labeled SFV nucleocapsids were used as markers for 140S (17, 19). Fractions were analyzed by SDS-PAGE and for the methyltransferase activity. For gel filtration, a DOC-treated P15 fraction from AcNVP-nsPlinfected cells in 50 mM Tris (pH 8.0)-0.5% DOC was chromatographed in a Superose 12 HR 10/30 column (Pharmacia) at a flow rate of 0.8 ml min-1. The exclusion size of the column is 300 kDa. Methyltransferase activity assays. The rapid assay for methyltransferase activity described by Barbosa and Moss (2) and Guo and Moss (13) was modified as follows. The P15 fractions were treated with 1% DOC for 30 min on ice before the assay (7). The reaction mixture (10 or 25 ,ul) contained 10 mM GTP, 4 mM MgCl2, 2 mM dithiothreitol, 2 to 20 ,uM SAM (Sigma), 0.5 to 2 ,uCi of S-adenosyl[methyl-3H]methionine (71 to 81 Ci/mmol; Amersham), and 0.5 to 5 ,ul of enzyme source. The samples were incubated at 30°C for 90 min, and the reaction was stopped by the addition of an equal volume of 0.2% SDS and 20 mM EDTA. The samples were applied to DEAEcellulose filters (DE81; Whatman). Unincorporated label was removed by washing the filters four times with 25 mM ammonium formate, once with H20, and twice with absolute ethanol. The filters were dried, and the radioactivity was measured by scintillation counting with OptiScint HiSafe solution in a Rackbeta Counter (LKB-Wallac). In order to obtain a better counting efficiency and signal-to-noise ratio, the rapid assay was adapted as follows: 1 ml of DEAE-Sephadex equilibrated with 10 mM ammonium acetate (AA) buffer, pH 8.5, was packed into a Pasteur pipette. After the reaction was stopped as described above, the sample was diluted to 1 ml with AA buffer and applied to the column. The column was washed with 5 ml of AA buffer containing 100 mM NaCl to remove all of the unreacted label. Labeled cap analogs were eluted from the column with 1.5 ml of AA buffer containing 500 mM NaCl. Radioactivity in the eluted sample was determined by liquid scintillation counting. Up to 25 samples could be easily processed at the same time. In some experiments, in which
7420
J. VIROL.
LAAKKONEN ET AL. 12
3455
12
3
1>, :.x,
1
A
2
C
FIG. 1. Production of SFV nsPl in insect cells. (A) "4C-amino acid labeling of Sf9 cells that were mock infected (lane 1) or infected with wild-type AcNPV (lane 2) or AcNPV-nsPl (lanes 3 to 5). Cells were labeled for 20 h, and postnuclear supernatant (PNS) (lane 3), P15 (lane 4), and S15 (lane 5) fractions were prepared as described in Materials and Methods. Samples were run on SDS-10% polyacrylamide gels, and the labeled proteins were visualized by fluorography. Only PNS fractions are shown from mock- and AcNPV-infected cells. The position of nsPl is marked with an asterisk. The positions of 14C-methylated molecular mass markers are shown to the left of the gel as follows: phosphorylase b, 92.5 kDa; bovine serum albumin, 68 kDa; ovalbumin, 46 kDa; and carbonic anhydrase, 30 kDa. (B) Immunoprecipitation with anti-nsPl antiserum of "4C-amino acidlabeled PNS fractions from Sf9 cells that were mock infected (lane 1) or infected with AcNPV (lane 2) or AcNPV-nsPl (lane 3). The aggregated form of nsPl is seen at the top of the resolving gel. (C) Immunoblotting of P15 (lane 1) and S15 (lane 2) fractions from AcNPV-nsPl-infected Sf9 cells.
guanylylimidodiphosphate (GIDP) (Sigma) was used as the substrate, m7GIDP was identified by thin-layer chromatography on polyethyleneimine cellulose plates (Merck), as previously described (6, 30). For the determination of Km values, the desired amount of unlabeled SAM was added to the reaction mixture. Unlabeled SAM was purified from contaminating S-adenosylhomocysteine (44). The Km values were calculated with the GraFit 3.0 program (Erithacus Software, Staines, United Kingdom). For the determination of acceptor specificities, GTP was substituted with 2 mM (each) dGTP, GpppG, GpppA, m7GTP, m7GpppG, or m7GpppA (Pharmacia). To study the methylation of mRNAs, plasmids pSP6SFV4 and pSP6-SFV5 were linearized with SacI, transcribed, and capped in vitro, to obtain mRNAs with 5' caps of GpppG and GpppA as previously described (22). The transcripts were purified by using the RNaid Kit (Bio 101, La Jolla, Calif.). RESULTS Expression of SFV nsPl in Sf9 cells. In order to study the putative methyltransferase activity of the SFV-specific nonstructural protein nsPl, we inserted the cDNA encoding authentic nsPl into the baculovirus genome by genetic recombination. The isolated AcNPV-nsPl recombinant virus was used to infect S. frugiperda (Sf9) cells. Analysis of the 14Camino-acid-labeled postnuclear supernatants by SDS-PAGE from mock-infected, wild-type AcNPV-infected and AcNPVnsPl-infected cells revealed a novel protein in the recombinant baculovirus-infected cells (Fig. 1A). This protein migrated
FIG. 2. Localization of nsPl in AcNPV-nsPl-infected Sf9 cells by indirect immunofluorescence (B). Mock-infected cells were used as a control (A).
slightly faster than the bovine serum albumin marker. This novel protein represented 15 to 20% of the Coomassie bluestained proteins, as estimated by laser densitometry. Centrifugation of the postnuclear supernatant at 15,000 x g gave pellet (P15) and supernatant (S15) fractions, which showed that most of the novel 14C-labeled protein was associated with the membranous P15 fraction. Essentially the same result was obtained with Coomassie blue staining (not shown). Immunoprecipitation and immunoblotting using anti-nsPl antiserum confirmed the identity of this protein as nsPl (Fig. 1B and C). Comparison of the patterns of 14C-amino-acid-labeled proteins from mock-infected and wild-type AcNPV-infected cells with that of AcNPV-nsPl-infected cells showed a significant inhibition of host protein synthesis both by wild-type AcNPV and AcNPV-nsPl infections. Interestingly, the expression of nsPl also seemed to inhibit the synthesis of baculovirusTABLE 1. Methyltransferase activity in mock- and SFV-infected BHK cells and AcNPV- and in AcNPV-nsPl-infected Sf9 cells
Cell, infecting in fction Cell, virus, and fraction
No. of expts No.to
Methyltransferase activity (10 cpm ofof[3H]methyl mn7GTP/mg protein)a
BHK cells Mock infected P15 S15
6 6
2.4 ± 0.4 4.1 +0.7
SFV P15 S15
10 11
18 2.7 5.6 ± 3.2
Sf9 cells AcNPV P15 S15
4 2
6.2 ± 4.3 8.9 + 6.4
6 7
560+95
AcNPV-nsPl P15 S15
Measured in a DE-81 filter binding assay, using 2
16±2.3
puM SAM.
SEMLIKI FOREST VIRUS nsP1 AS METHYLTRANSFERASE
VOL. 68, 1994
7421
25
Ij ., 20 w-
e
il
Ln
E
15
_
16 5
0 P
I :.I 1, I
,:
FIG. 3. Gel filtration of nsPl. DOC-treated P15 fraction from AcNVP-nsPl-infected cells in 50 mM Tris (pH 8.0)-0.5% DOC was chromatographed in a Superose 12 HR 10/30 column. The elution time of the excluded material under these conditions was 9.32 min. The bovine serum albumin marker eluted at 13 min (not shown). (Insert) Coomassie blue-stained 10% polyacrylamide gel. Lanes: MW, molecular mass markers as described in the legend to Fig. 1; P15, input material; 9-11', 11-13', and 13-15', collected fractions.
specific proteins (Fig. 1A). Indirect immunofluorescence revealed that nsPl was localized in the cytoplasm of the insect cells, apparently in association with the cytoplasmic membranes (Fig. 2), in accordance with the above results of the crude cell fractionation. Methyltransferase activity associated with AcNPV-nsPl. A simple assay using GTP as the substrate and S-adenosyl [methyl-3H]methionine as the methyl donor has been described for the vaccinia virus capping enzyme (2). We used this assay to test whether nsPl, produced in insect cells, had methyltransferase activity. For controls, we used mock-infected and SFVinfected BHK cells, as well as wild-type AcNPV-infected Sf9 cells. A high level of methyltransferase activity was found in the P15 fraction of AcNPV-nsPl-infected cells. It was about 90-fold higher than that in wild-type AcNPV-infected cells (Table 1). Very little activity was associated with the S15 fraction. A clearly detectable methyltransferase activity was found in the P15 fraction of SFV-infected BHK cells, where nsPl is almost exclusively localized (35). This fraction also has virtually all of the SFV-specific RNA polymerase activity (3, 36). A similar distribution of methyltransferase activity was obtained when GIDP was used as the substrate. The product, m7GIDP, was identified by thin-layer chromatography as described in Materials and Methods. Aggregation of nsPl in Sf9 cells. Since nsPl was associated with the P15 fraction of the insect cells, the membranes had to be solubilized in order to release it. The best result was obtained with DOC. When the DOC-solubilized material was subjected to gel filtration in a Superose 12 column, most of
1
2
3
4
5
6
7
8
9
10
Top Fracton Number FIG. 4. Sucrose gradient analysis of SFV nsPl produced in insect cells. The P15 fraction of AcNPV-nsPl-infected Sf9 cells was treated with DOC and layered on the top of a continuous 10 to 40% (wt/wt) sucrose gradient. Centrifugation was done with a SW 50.1 rotor (Beckman) for 4 h at 35,000 rpm at 4°C. Fractions of 0.5 ml were assayed for methyltransferase activity, and m7GTP was purified by using DEAE columns (open bars). The total amount of radioactive m7GTP was taken as 100%. The amount of nsPl in each fraction was determined by laser densitometry from immunoblots (shaded bars). The total amount of nsPl was taken as 100%. [35S]methionine-labeled SFV nucleocapsids (140S) corresponding to fraction 2 were run in an separate gradient.
nsPl eluted in the void volume (Fig. 3, insert, lane 9-11'), suggesting that the material had a molecular mass of at least 300 kDa (Fig. 3). No nsPl was detected in fractions corresponding to monomeric nsPl (Fig. 3, insert, lane 13-15'). The methyltransferase activity was found in the void volume (not shown). The solubilized material from the P15 fraction was also analyzed by sucrose gradient centrifugation. Both methyltransferase activity and nsPl, detected by immunoblotting, cosedimented with a broad peak at about 70 to 90S (Fig. 4). The pellet contained about 25% of the immunologically reactive nsPl, but less than 10% of the methyltransferase activity, indicating that the heavily aggregated protein had lost most of its enzymatic activity. In Coomassie blue-stained gels, the fast-sedimenting structures seemed to consist mostly of a protein migrating at the position of nsPl. All attempts to release the nsPl from the P15 membranes or to break the aggregates after DOC solubilization, using high concentrations of salt (1 M NaCl), 50 mM EDTA, or ribonuclease treatment (50 ,ug/ml for 30 min at 37°C), were unsuccessful (not shown). We have recently shown that nsPl is acylated by [3H]palmitate both in SFV-infected BHK cells and in transfected HeLa cells, which expressed nsPl with the aid of a recombinant vaccinia virus, vTF7-3 (32). Labeling of AcNPV-nsPl-infected Sf9 cells with [3H]palmitic acid resulted in the incorporation of the label almost exclusively in nsPl, strongly suggesting that nsPl was acylated (Fig. 5). Thus, the membrane association and aggregation of nsPl, after release with DOC, might be due to the acylation. Production of nsPl in E. coli. For the production of nsPl in E. coli, we used a tightly controlled vector, pBAT-nsPl. The nsPl coding region was under the control of the T7 promoter, containing an inserted lacO region. In order to obtain tight regulation of the expression, the lacI repressor gene was included in the plasmid (33). IPTG induction at 37°C of E. coli cells, transformed with pBAT-nsPl, resulted in the synthesis of a new protein, which in SDS-polyacrylamide gels migrated at the position of nsPl (Fig. 6). This protein represented about 13% of the Coomassie blue-stained proteins, as determined by
7422
J. VIROL.
LAAKKONEN ET AL. AcNPVnsPl
25
P15 S15 P15 S15
20
AcNPV
E 15 0.
°
10
5
u----..
0
P
FIG. 5. Palmitoylation of SFV nsPl produced in insect cells. AcNPV- or AcNPV-nsPl-infected Sf9 cells were labeled with [3H] palmitic acid for 2 h. Proteins in the P15 and S15 fractions were analyzed by SDS-PAGE in a 10% polyacrylamide gel followed by fluorography. To the left of the gel, the positions of the '4C-methylated molecular mass markers described in the legend to Fig. 1 plus myosin (200 kDa) are shown.
laser densitometry. When the cell debris was sedimented at 15,000 x g, the induced protein was found exclusively in the pellet. When the induction was carried out at 15°C, the 63-kDa protein remained soluble (Fig. 6). Immunoblotting confirmed that the new protein was nsPl, which comigrated with nsPl from SFV-infected BHK cells (not shown). Methyltransferase assays revealed that the activity at 37°C was only about 15% of the activity detected at 15°C (not shown). These results suggested that nsPl, synthesized at 37°C, was heavily aggregated and mostly inactive. The 15,000 x g supernatant fraction from the 15°C incubation had essentially all the methyltransferase activity of the E. coli lysate. Sucrose gradient centrifugation showed that the methyltransferase activity was in fast-sedimenting structures (Fig. 7), in which nsPl was a stained major protein (not shown). This showed that even the "soluble" nsPl was in large aggregates. Anyhow, the presence of a methyltransferase activity in insect cells and in E. coli, after expression of the SFV-specific nsPl, showed clearly that this protein was responsible for the new enzymatic activity.
37tC MW C
T
S
15'C P
T
S
P
10 Fraction Number
5
15
Top
FIG. 7. Sucrose gradient analysis of nsPl produced in E. coli. The supernatant fraction from pBAT-nsPl-transformed E. coli cells induced at 15°C was layered onto a continuous 10 to 40% (wt/wt) sucrose gradient as described in the legend to Fig. 4. Fractions (250 ,ul) were
assayed for methyltransferase activity, and the m7GTP was purified by using DEAE columns. The amount of m7GTP is shown by shaded bars.
Properties of nsPl methyltransferase. An interesting difference between the eukaryotic and prokaryotic nsPl products was seen in respect to detergents. As reported by Cross (6), the detection of methyltransferase activity in SFV-infected cells required treatment with DOC. The same was true for the AcNPV-nsPl-associated methyltransferase expressed in insect cells, whereas the activity of nsPl produced in E. coli was almost completely inhibited by both DOC and Triton X-100. Also, AcNPV-nsPl methyltransferase activity was partially inhibited by Triton X-100 (Table 2). The methyltransferases from E. coli and insect cells showed, for 60 min, a linear incorporation of a [3H]methyl group from SAM to GTP (Fig. 8A). The incorporation was also linear in SFV-infected BHK cells, after an initial period of 10 min (Fig. 8B). None of the methyltransferases required Mg2+ ions, and omission of dithiothreitol reduced the activity only slightly (data not shown). The apparent Km values for SAM were determined for the methyltransferases from the three different sources. They all were of the same order of magnitude: 3.1, 5.4, and 7.5 ,uM for BHK, Sf9, and E. coli cells, respectively. These values differ from those reported for P15 fractions from SFV (119 ,uM)- and Sindbis virus (125 ,uM)-infected cells (40). We assume that removal of the contaminating S-adenosylhomocysteine from the commercial preparations of SAM, might explain this (see Materials and Methods). TABLE 2. Effects of detergents on the methyltransferase from Sf9 cells and E. coli Methyltransferase activity Cell, fraction and/or treatment (cpm of [3H]methyl m7GTP [%J)Q
AcNPV-nsPl-infected Sf9 cells FIG. 6. Expression of SFV nsP1 in E. coli. Cells were transformed with pBAT-nsPl, and expression was induced by IPTG either at 37°C for 3 h and at 15°C for 20 h. After collection, the cells were lysed and separated into supernatant (S) and pellet (P) fractions. Cells transformed with plasmid pBAT4 served as a control (C). T, total cell lysate. Samples were analyzed by SDS-PAGE, and the proteins were visualized by Coomassie blue staining. Molecular mass markers (MW) are as described in the legend to Fig. 1. The position of nsPl in the total cell lysate is marked with an asterisk.
P15 .................................. P15 + 1% DOC .................................. P15 + 1% Triton X-100 ..................................
16,500 (42) 39,600 (100) 3,300 (8)
E. coli-pBAT-nsP1 S15 .................................. S15 + 1% DOC.................................. S15 + 1% Triton X-100 ..................................
81,000 (100) 3,300 (4) 1,000 (1.2)
a Measured in an assay with 10-,ul samples, using a DEAE column and 3 ,uM SAM.
VOL. 68, 1994
SEMLIKI FOREST VIRUS nsPl AS METHYLTRANSFERASE
TABLE 3. Methylation of CAP analogs % Maximum incorporation of [3H]CH3' (no. of expts)
5
Cap analog
GTP dGTP
E
m7GTP o0
G?PpG
m GpppG
G?ppA
m GpppA co E 0 I-
20
40
60
80
100
2.0
E
7423
Mock-infected
BHK cells (2) 0 0 0 100e 0 ND ND
SFV-infected BHK cells
(2) 61 100b 6.7 72 14 6.7 7.0
AcNPV-nsP(
E. col( nsPl
6(5 38 1OOC 1.7 59 5.7 1.8 1.6
51 100d 3.5 18 4.3 4.2 3.7
a Measured in an assay with 10-,ul samples, using a DEAE column and 2 ,uM SAM. b 23,402 cpm. c 120,382 cpm. d 54,613 cpm. e 7,206 cpm.
B
I -r
DISCUSSION
1.5 E
8CV,
1.0
so -~~~~~~~~~
0
/I
|
I
I
I~~~
0.5
20
60 40 Time (min)
80
100
FIG. 8. Kinetics of m7GTP production by nsPl methyltransferase. The methyltransferase reaction was performed at 30°C, and 25-,Ul samples were taken at the times indicated, and the radioactive m7GTP was purified in a DEAE-column. (A) DOC-treated P15 fraction from Sf9 cells infected with AcNPV-nsPl (-) or AcNPV (A). 0, supernatant fraction from pBAT-nsPl transformed E. coli cells induced at 15°C. (B) DOC-treated P15 fraction from SFV-infected BHK cells. After 60 min, 270, 93, and 11 ,umol of protein of [3H]methyl groups mg-' had been incorporated by Sf9 nsPl, E. coli nsPl, and SFVinfected BHK methyltransferase, respectively.
In addition to GTP, we tested some other cap analogs for the ability to serve as acceptors for the labeled methyl group from SAM. In each case, including SFV-infected BHK cells, dGTP was the most efficient acceptor for all forms of nsPl (Table 3). As expected, negligible incorporation of radioactivity was obtained with m7GTP as a substrate. The host enzyme in mock-infected BHK cells was not able to methylate GTP. GpppG was a poor substrate for the E. coli nsPl methyltransferase, in contrast to the enzyme expressed in eukaryotic cells. Some incorporation of label into m7GpppG was observed. This phenomenon has been reported previously for the vaccinia virus capping enzyme. It is evidently based on the symmetry of the GpppG molecule, which with a low frequency could accept a second methyl group when m7GpppG serves as the substrate (25). Interestingly, negligible incorporation of label was detected with GpppA, which is the 5' cap structure of both the 42S and 26S RNAs of SFV. Our attempts to methylate SFV-specific in vitro-generated transcripts, capped with either GpppG or GpppA, also gave negative results (not shown).
The best-characterized guanine-7-methyltransferase is that purified from vaccinia virions. The soluble enzyme has a mass of 127 kDa and consists of two polypeptides of 95 and 31 kDa (24-26). The complex has guanylyltransferase, RNA triphosphatase, and guanine-7-methyltransferase activities. The two polypeptides cannot be separated from each other without the loss of methyltransferase activity (5, 13, 15, 45-47). Another mRNA (nucleoside-02'-)methyltransferase, converting cap 0 to cap 1, is associated with VP39, which is also a component of the virus-specific poly(A) polymerase (2, 43). Unlike the host enzyme, vaccinia virus guanine-7-methyltransferase also methylates GTP and dGTP in the presence of SAM. Furthermore, this reaction does not require Mg2+, and in the absence of GTP, it can methylate Gppp(A)n (25, 26). As for cellular enzymes, a soluble guanine-7-methyltransferase has been purified from the postmitochondrial supernatant of HeLa cells (8). This 56-kDa protein methylated RNAs and polyribonucleotides with 5' GpppN. Free GpppG could also serve as a substrate, but not GTP. This enzyme did not have guanylyltransferase activity, which is associated with a 65-kDa protein devoid of triphosphatase and methyltransferase activities (51). Earlier studies with SFV have revealed a cytoplasmic guanine-7-methyltransferase activity in infected cells (6, 7). Since later studies with Sindbis virus indicated that the nonstructural protein nsPl was associated with the methyltransferase activity (29, 30, 40), we wanted to obtain direct evidence for the putative role of SFV-specific nsPl as a methyltransferase. To this end, the cDNA encoding all 537 amino acids of nsPl, was inserted into the baculovirus (AcNPV) genome by homologous recombination. S. frugiperda cells, infected with AcNPV-nsPl, produced a substantial amount of nsPl by SDS-PAGE, immunoblotting, and immunoprecipitation. The nsPl expressed in Sf9 cells was associated predominantly with the membranous P15 fraction, as is the case in SFV-infected BHK cells (35). AcNPV-nsP1-infected cells, and the P15 fraction isolated from them, had high levels of guanine-7-methyltransferase activity. This activity was evidently due to the expression of the SFV-specific nsPl for the following reasons. (i) The activity was very low in the wild-type baculovirus- and mock-infected cells. In fact, after we adopted the modified assay using the DEAE columns, negligible incorporation of [3H]methyl groups into GTP was observed when P15 from the AcNPV-infected cells was used as an enzyme source (Fig. 8A). (ii) The specificity of the enzyme was different from that of the host
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enzyme (Table 2) (8). The enzyme from the AcNPV-nsPlinfected cells could methylate GTP, unlike the AcNPV wildtype or host enzymes. (iii) The intracellular distribution of the new methyltransferase was also different. In the AcNPV-nsPlinfected cells, the activity was confined almost completely to the P15 fraction, whereas the host enzymes have been reported to be in the postmitochondrial supematant (8). (iv) Partial purification by sucrose gradient centrifugation showed cosedimentation of the enzymatic activity with nsPl. Only a fraction of the methyltransferase activity was detectable when DOC was omitted from the assay. This result suggests that the enzymatic activity is at least partly inhibited by the interaction of nsPl with the P15 membranes. The association with P15 membranes was very tight both in SFVinfected BHK cells and in AcNPV-nsPl-infected insect cells. nsPl could not be released from P15 membranes by high concentrations of salt, EDTA, or Na2CO3, which detach peripheral membrane proteins (4). The interaction of nsPl with membranes may be mediated by acylation, since we could label nsPl in the insect cells by [3H]palmitic acid. Palmityolation takes also place in SFV-infected BHK cells and in nsPl-expressing HeLa cells (32). After solubilization of the P15 membranes of AcNPV-nsPl-infected insect cells with DOC, nsPl and methyltransferase activity were released. However, attempts to purify the solubilized nsPl were unsuccessful, since nsPl was in large aggregates. A substantial proportion of nsPl did not enter the polyacrylamide gel (Fig. 5). Similar observations have been reported for other palmitoylated proteins (27). Expression of nsPl in E. coli resulted in the production of a normal-size nsPl and a high level of methyltransferase activity in the bacterial lysate. Attempts to purify the protein revealed that it also formed large aggregates. The aggregation could not be due to acylation, since proteins are not known to be acylated in E. coli. Our attempts to label nsPl with [3H]palmitate in E. coli, have also failed (1). The aggregation of nsPl expressed in insect cells and in E. coli may be due to improper folding, since in both cells, nsPl is synthesized in high amounts, ranging from 10 to 25% of the total proteins. In E. coli, the aggregation of foreign proteins is a common phenomenon, especially when they are produced in large amounts (20). The mechanism of aggregation of nsPl in insect cells may be different, since it is acylated and membrane associated. In SFV-infected cells, nsPl is synthesized as a part of a large nonstructural polyprotein, P1234. The assembly of the active RNA polymerase complex is evidently dependent on the cooperative folding of P1234 (21). Thus, massive expression of nsPl alone could be expected to result in aberrant folding and aggregation. In spite of their physical state, the nsPl proteins from insect cells and E. coli had preserved the capacity to function as guanine-7-methyltransferases. The two methyltransferase activities were similar to that from SFV-infected BHK cells in the following respects. (i) They showed linear kinetics of incorporation of [3H]methyl groups from labeled SAM to GTP. (ii) They had similar Km values. (iii) Like the vaccinia virus guanine-7-methyltransferase, they could use GTP and dGTP as substrates, unlike the host enzymes (25). The most striking difference between the nsPl methyltransferase from E. coli and AcNPV-nsPl-infected Sf9 cells was their behavior in respect to detergents. The enzyme from E. coli was virtually inactive in the presence of DOC or Triton X-100, whereas considerable activation was obtained for the AcNPV-nsPl enzyme in the presence of DOC (Table 2). Furthermore, Triton X-100 partly inhibited the AcNPV-nsPl enzyme. The reasons for these paradoxical properties of the
same enzyme from two different hosts are not possible to explain at present. We anticipate that these differences may be connected to the acylation of nsPl in the animal cells. Comparison with other guanine-7-methyltransferases revealed some interesting differences. (i) The natural cap analog of SFV RNAs, GpppA, could not serve as an acceptor in the in vitro assays. The same result was obtained with the methyltransferase of SFV-infected BHK cells (Table 3). Thus, it cannot be due to the special features of the nsPl methyltransferases from insect cells and E. coli. (ii) Our attempts to use SFV-specific transcripts, with 5' caps, of either GpppA or GpppG, failed. In this respect, SFV nsPl methyltransferase differs clearly from the guanine-7-methyltransferases of vaccinia virus (24), reovirus (11), and HeLa cells (8), which can readily methylate capped mRNAs. It may be possible that the aggregated form of nsPl is unable to accommodate the capped RNA properly. Another possibility would be that another SFV-specific protein is required for the methylation of the capped RNA. This could be expected, since the functions of many capping enzymes are split among two or more polypeptides. We have recently shown that purified nsP2 can bind RNA in vitro and could thus provide this function in vivo (37). nsPl is a multifunctional protein. It is required in the synthesis of the Sindbis virus 49S minus-strand RNA (14, 39, 52). It may anchor the replication complex to endosomes and lysosomes (32), which are the sites of virus RNA synthesis (10). Finally, nsPl is involved in the modification of the 5' termini of the viral RNAs. There is now direct evidence for both Sindbis virus and SFV that nsPl is a guanine-7-methyltransferase. Indirect evidence, suggesting that nsPl might also participate in the capping process, has been obtained by sequencing the cDNA encoding nsPl of Sindbis virus mutants resistant to mycophenolic acid (41). ACKNOWLEDGMENTS We thank Annikki Kallio and Tarja Valimaki for excellent technical assistance. We are grateful to Patrick Russo, Marja Makarow, and Tero Ahola for critically reading the manuscript. The help of Nisse Kalkkinen in protein analysis and Tapani Ronni in the computer calculation of Km values is gratefully acknowledged. The work was supported by Sigrid Juselius Foundation and the Academy of Finland. REFERENCES 1. Ahola, T., P. Laakkonen, and L. Kiariainen. Unpublished data. 2. Barbosa, E., and B. Moss. 1978. mRNA (nucleoside-2'-)methyltransferase from vaccinia virus. J. Biol. Chem. 253:7692-7697. 3. Barton, D. J., S. G. Sawicki, and D. L. Sawicki. 1991. Solubilization and immunoprecipitation of alphavirus replication complexes. J. Virol. 65:1496-1506. 4. Chong, L. D., and J. K. Rose. 1993. Membrane association of functional vesicular stomatitis virus matrix protein in vivo. J. Virol. 67:407-414. 5. Cong, P., and S. Shuman. 1992. Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme. J. Biol. Chem. 267:16424-16429. 6. Cross, R. K. 1983. Identification of a unique guanine-7-methyltransferase in Semliki Forest virus (SFV) infected cell extracts. Virology 130:452-463. 7. Cross, R. K., and P. J. Gomatos. 1981. Concomitant methylation and synthesis in vitro of Semliki Forest virus (SFV) ssRNAs by a fraction from infected cells. Virology 114:542-554. 8. Ensinger, M. J., and B. Moss. 1976. Modification of the 5' terminus of mRNA by an RNA (guanine-7-)methyltransferase from HeLa cells. J. Biol. Chem. 251:5283-5291. 9. Felgner, P. L., T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen. 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection
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