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Journal of Virology, September 2001, p. 8031-8044, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8031-8044.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The Arginine-1493 Residue in QRRGRTGR1493G Motif IV of the Hepatitis C Virus NS3 Helicase Domain Is Essential for NS3 Protein Methylation by the Protein Arginine Methyltransferase 1

Jaerang Rho,1 Seeyoung Choi,1 Young Rim Seong,1 Joonho Choi,2 and Dong-Soo Im1,*

Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejeon 305-333,1 and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejeon,2 Republic of Korea

Received 20 October 2000/Accepted 23 May 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg1493 in the QRR1488GRTGR1493G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg1488 is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Hepatitis C virus (HCV) is a major inducer of chronic hepatitis (4, 15), and chronic HCV infection is associated with liver cirrhosis and, eventually, development of hepatocellular carcinoma (59). The technical development of a means to detect HCV in human serum substantially decreased the risk of infection through posttransfusion (4). Nevertheless, about 170 million people worldwide have already been infected with HCV (53), and yet, HCV infections still occur by unknown routes, resulting in ever-increasing worldwide health problems. Effective therapy against HCV infection, based on either immunological methods or small molecules, is not currently available except for alpha interferon and its combination with ribavirin (28, 43), which have limited efficacy.

HCV is assumed to be an enveloped virus containing a positive-stranded RNA genome of about 10 kb. The genome encodes a single, large polypeptide with about 3,010 amino acids, which is processed by a cellular signalase and the virally encoded proteases to produce core, E1, and E2/p7 as structural proteins and NS2, NS3, NS4A, NS4B, NS5A, and NS5B as nonstructural (NS) proteins (16; reviewed in references 17, 63, and 66). The NS3 protein is a multifunctional protein containing serine protease and RNA helicase activities. The protease domain is localized to the N terminus of NS3, which forms a stable complex with NS4A (27, 44). The C terminus of the NS3 protein was shown to contain an NTPase activity and to actively bind RNA substrates and unwind RNA-RNA, RNA-DNA, and DNA-DNA heteroduplexes (17).

While much effort has been given to enzymatic characterizations of the NS3 protein (11, 21, 27, 31, 35, 44, 54, 65, 69), few studies about the cellular functions of the NS3 protein associated with liver pathogenesis have been reported. NIH 3T3 mouse fibroblasts transfected with the N-terminal domain of NS3 become transformed and are tumorigenic in nude mice (61). The internal cleavage product of NS3 appears to have higher oncogenic potential than intact NS3 (71). Actinomycin D-induced apoptosis is suppressed in NIH 3T3 cells constitutively expressing NS3 with a truncation at the C-terminal end (18). Wild-type p53 enhances nuclear accumulation of full-size and carboxy-terminally truncated NS3 (29, 46). The NS3 protein (amino acids [aa] 1189 to 1525) with N- and C-terminal truncations and short peptides derived from the arginine-rich region of NS3 inhibited phosphorylation mediated by cyclic AMP (cAMP)-dependent protein kinase A (PKA) (6). Subsequently, NS3 (aa 1189 to 1525) was shown to inhibit the distribution of the free catalytic subunit of PKA (8) and to bind histone (7). The protein kinase C also recognizes the PKA-binding motif of the NS3 protein (9). These results suggest that a specific interaction of cellular factors with NS3 or their modulation by the NS3 protein partially correlate with cellular transformation or pathogenesis by HCV.

To search for cellular targets of the NS3 protein, we conducted a yeast two-hybrid screen. We found that the NS3 protein associates with protein arginine methyltransferase 5 (PRMT5) (S. Choi, J. Rho, Y. R. Seong, and D.-S. Im, unpublished data). This finding prompted us to examine whether arginine residues of the NS3 protein are methylated by PRMTs. PRMTs transfer the methyl group from S-adenosylmethionine to the guanidino nitrogen atoms of arginine residues (22). Protein arginine methylation is an irreversible and posttranslational covalent modification. Although much effort has been given to understanding the biological consequences of protein arginine methylation, the roles of protein arginine methylation in distinct cellular function are largely unknown. However, several recent reports have begun to clarify the biological roles for protein arginine methylation. For example, PRMT1 was shown to interact with the alpha/beta inteferon receptor (1). Subsequently, PRMT1 has been found to associate with and methylate STAT1 (signal transducer and activator of transcription) protein (45). The arginine methylation of STAT1 was required for transcriptional activation induced by alpha/beta interferon (45). The transcriptional coactivators of the p160 family enhanced the transcriptional activity of nuclear hormone receptors when coexpressed with a protein with PRMT activity (13). These findings give rise to a renewed interest in protein arginine methylation (22).

In this report, we present biochemical evidence that the NS3 protein of HCV is subjected to protein arginine methylation in vitro and that in vivo and that a cellular enzyme methylating the NS3 protein in vitro is PRMT1. We determined that the Arg1493 residue in QRRGRTGRG motif IV of the NS3 helicase domain is likely to be a major methylation site by using deletion and substitution point mutants of the NS3 protein.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell culture. 293 or 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin-streptomycin in an atmosphere of 5% CO2 at 37°C.

Plasmids. pCMV2-NS3F (Flag-NS3F), a mammalian expression plasmid encoding full-size NS3, was constructed by inserting a HindIII-XhoI fragment (aa 1027 to 1657) of pBSns3/1027-1657 (29) into a pCMV2-Flag vector (Sigma). pCMV2-NS3Delta C (Flag-NS3Delta C), with a deletion of the C-terminal region of NS3, was constructed by inserting a HindIII-NotI fragment (aa 1027 to 1459) of pBSns3/1027-1459 (29) into the pCMV2-Flag vector. pCMV2-NS3H (Flag-NS3H) was generated by inserting a BamHI-NotI fragment (aa 1196 to 1657) of pGST-NS3H1196-1657 into pBluescript II KS (Stratagene) and then inserting a HindIII-NotI fragment of the resulting plasmid into pCMV2-Flag vector. To construct Flag-NS5A, NS5A DNA was amplified in the PCR by using pTHE1964-3011 as a template and primers 5'-GGAAGCTTTCCGGCTCGTGGCTA-3' and 5'-GGGTCGACGCAGCAGACGA-3', and a HindIII-SalI fragment of the amplified NS5A DNA was inserted into the pCMV2-Flag vector. To construct a maltose-binding protein (MBP)-NS3F (aa 1027 to 1657) plasmid, pTM-NS3 was digested with EcoRI and filled in by Klenow treatment and then digested with SalI. The EcoRI-SalI fragment was then inserted into the EcoRI (filled by Klenow enzyme) and SalI sites of the pMALcRI vector (NEB Inc.). The N terminus of the NS3 region (aa 1027 to 1299) was amplified by PCR using pTM-NS3 as a template and primers 5'-GGAATTCCTGCTCCCAT-3' and 5'-TTCTGCAGGGTAGAGTATGT-3'. The amplified fragment was digested with EcoRI and PstI and then cloned into pTM1 (pTM-NS3N). pTM-NS3N (aa 1027 to 1299) was digested with EcoRI, filled in by using Klenow enzyme, and treated with SalI. The EcoRI-SalI fragment was inserted into the EcoRI (filled in by Klenow) and SalI sites of the pMALcRI vector (pMAL-NS3N). To construct glutathione S-transferase (GST) fusion expression plasmids with N- or C-terminal deletions, the primers GST-NS3H1196-1657 (5'-CCGAATTCGTGGACTTCATACCCGTT-3' and 5'-CCCTCGAGGTCAGCTGACATGCATGC-3'), GST-NS3H1196-1547 (5'-CCGAATTCGTGGACTTCATACCCGTT-3' and 5'-CCCTCGAGCAGGTAAGCCCGCAACCT-3'), and GST-NS3H1468-1547 (5'-CCGAATTCTTTAGCTTGGATCCCACC-3' and 5'-CCCTCGAGCAGGTAAGCCCGCAACCT-3') were used in the PCRs to amplify the NS3 DNA fragment of HCV1b. The amplified PCR products were subcloned into pGEX4T-1 (Amersham-Pharmacia Biotech). GST-NS3H1468-1546-DM was constructed by PCR amplification using pET21bNS3H DNA as a template and primers 5'-CCGGATCCTTCAGCCTTGACCCTACCTTC-3', 5'-CCGCGGCCGCCTACATGTACGCTCGTAGCCTAAC-3', 5'-ACTGGTACCGGGAAGCCAGGCATCTAC-3', and 5'-CCCGGTACCAGTCCTGCCCAGACGTTG-3'. The amplified DNA was subcloned into pGEX4T-1 and sequenced. The wild-type NS3 helicase domain expression plasmid pET21bNS3H (His-NS3H) and its mutants were reported previously (31).

To construct a Flag-tagged PRMT1 expression plasmid, pCMV2-PRMT1 (Flag-PRMT1), PRMT1 cDNA was amplified from pGEX(SN)-PRMT1 (36) by using primers 5'-CCGGATCCACCATGGCGGCAGCCGAGGCCGCG-3' and 5'-CCGCGGCCGCTCAGCGCATCCGGTAGTCGG-3'. The amplified PRMT1 DNA was subcloned into the BamHI and NotI sites of pBluescript II KS. The resulting plasmid was digested with HindIII and NotI. The DNA fragment obtained was then inserted into the HindIII and NotI sites of pCMV2-Flag. To construct the His-tagged PRMT1 expression plasmid His-PRMT1, the amplified PRMT1 DNA was subcloned into a pET28a(+) vector (Novagen Co.). pGEX(SN)-PRMT3 (GST-PRMT3), which is an expression plasmid in Escherichia coli, was reported previously (67). The construction of pCMV2-PRMT5 (Flag-PRMT5) was reported previously (55). pET21SRP1alpha (70) and a plasmid encoding STAT3 were obtained from A. I. Lamond and J. E. Darnell, Jr., respectively.

Preparation of soluble human cell extract and sedimentation analysis. 293T monolayer cells (2 × 108) were washed with ice-cold phosphate-buffered saline (PBS) and resuspended in 3 ml of lysis buffer (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1% NP-40, plus 10 µg of aprotinin and 10 µg of leupeptin per ml). The cells were briefly sonicated for 1 min on ice and spun down at 35,000 rpm for 2 h at 4°C in a Beckman SW55Ti rotor. The supernatant was dialyzed against PRMT buffer (25 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM EGTA, 1 mM PMSF). The protein concentration was measured by using a protein assay kit (Bio-Rad). The soluble protein extract was laid over a 35-ml 5 to 45% sucrose gradient prepared in PRMT buffer. Ultracentrifugation was done at 25,000 rpm at 10°C for 24 h in a Beckman SW28 rotor. The gradient was fractionated into a volume of 600 µl from the top, and 40 µl of the gradient fractions was used for the PRMT assay.

Immunoprecipitation and immunoblotting. 293 cells (1 × 107 to 5 × 107 cells) were plated 18 to 24 h before transfections with expression plasmids tagged with the Flag epitope. DNA transfections were performed by a standard calcium phosphate method. The cells were washed with PBS buffer and lysed with lysis buffer at 36 h posttransfection. Cell lysates were precleared with protein A-Sepharose for 30 min and incubated with anti-Flag antibody (Sigma) or antimono- or dimethylarginine (anti-mono/dimethylarginine) antibody (Abcam) and protein A-Sepharose for 2 h at 4°C. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer onto polyvinylidene difluoride membranes, proteins were detected with anti-Flag or anti-mono/dimethylarginine or -dimethylarginine antibodies.

PRMT assay. PRMT assays were carried out as previously reported (36, 67). Briefly, a soluble cell extract or purified PRMT proteins were reacted with methyl acceptors in the presence of 0.25 µCi of S-adenosyl-[methyl-14C]-L-methionine ([14C]SAM) as a methyl donor for 2 h at 30°C. The reaction products were resolved by SDS-PAGE. The gels were fixed and treated with an Amplifier (Amersham-Pharmacia Biotech) for 15 min, dried, and then exposed to X-ray film at -80°C for 5 to 14 days. The gels were also analyzed by a Fujix BAS1000 phosphorimager.

Preparation of recombinant proteins. All the GST fusion proteins were expressed in E. coli and purified by standard procedures. In brief, cells harboring GST or GST fusion expression plasmids were induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 2 to 3 h at 30 or 37°C. Cells were washed with PBS buffer, resuspended in lysis buffer, and sonicated. Supernatants obtained by centrifugation were loaded into a glutathione-agarose column (1 by 10 cm). The column was washed with 5 column volumes of lysis buffer. The bead-bound proteins were eluted with lysis buffer containing 10 mM reduced glutathione. Purified proteins were stored at -70°C. The expression and purification of proteins tagged with 6× histidine were carried out as previously reported (31). MBP or MBP fusion proteins were expressed and purified as described by the manufacturer (NEB). A rabbit polyclonal antibody recognizing the HCV NS3 helicase was prepared by using an MBP-NS3N fusion protein as an antigen.

293T cells (2 × 108 cells) were transfected with expression plasmids tagged with Flag epitope by a standard calcium phosphate method. Cell lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Supernatants were applied to an anti-Flag affinity column (1 by 10 cm) equilibrated with lysis buffer. The column was washed twice with lysis buffer. Proteins bound to the column were eluted with 100 mM glycine-HCl buffer (pH 3.5). The eluates were neutralized by 1 M Tris buffer (pH 8.0). The eluates containing the Flag epitope-tagged proteins detected by Western blots with anti-Flag antibody were collected. Purified proteins were then dialyzed against PRMT buffer.

In vitro interactions between NS3 and PRMT1 proteins. Two micrograms of the purified Flag-NS3F protein were incubated with 2 µg of GST or GST-PRMT1 proteins purified from E. coli in 1 ml of lysis buffer for 2 h at 4°C. After incubation, the mixtures were divided into equal portions. Either anti-Flag- or glutathione-agarose beads were added to the mixtures, which were then further incubated for 2 h at 4°C. The beads were washed five times with lysis buffer. The bead-bound proteins were analyzed by Western blots with anti-GST or anti-Flag antibodies.

One microgram of the NS3 helicase domain (His-NS3H) or His-SRP (suppressor of RNA polymerase) proteins was incubated with 1 µg of purified Flag-PRMT1 in 1 ml of lysis buffer. The reaction mixtures were divided into equal portions. The mixtures were immunoprecipitated with anti-Flag-conjugated agarose beads. The beads were washed five times with lysis buffer. The bead-bound proteins were analyzed by Western blotting with anti-His6 antibody. Portions of the reaction mixtures were analyzed by Western blotting with anti-Flag or anti-His6 antibodies.

Poly(U) column-binding assay. To determine an interaction between the NS3 helicase domain and the PRMT1 protein, a poly(U) column-binding assay was performed. His-NS3H and Flag-PRMT1 proteins (50 µg of each) in a 500-µl reaction volume were incubated with 5 µCi of [14C]SAM for 2 h at 30°C. RNA-binding buffer (25 mM MOPS [morpholinepropanesulfonic acid]-KOH [pH 7.5], 3 mM MgCl2, 2 mM dithiothreitol, and 1 mM PMSF) was added to the reaction mixture to make 5 ml total volume. The reaction mixture was applied to a poly(U)-Sepharose 4B (Amersham-Pharmacia Biotech) column (10 cm high by 0.5 cm inner diameter, 1-ml bed volume) equilibrated with the RNA-binding buffer. The column was washed with 5 ml of the RNA-binding buffer. Proteins bound to the column were eluted by step gradient RNA-binding buffers containing from 100 to 700 mM NaCl in 100 mM intervals (5 ml per step) and collected into about 0.7 to 1 ml of fractions. Flag-PRMT1 and the 14C-labeled NS3 in 20 µl of the fractions were detected by Western blotting with anti-Flag antibody and fluorography, respectively. His-NS3H (50 µg) or Flag-PRMT1 (50 µg) as a control was also loaded onto the same column. The proteins were eluted under the same conditions as described above. His-NS3H and Flag-PRMT1 in 20 µl of the fractions were detected by Western blotting with anti-NS3 and anti-Flag antibodies, respectively.

Effect of homoribopolymers on methylation of NS3 helicase protein. His-NS3H protein (5 µg) was preincubated without or with 0.05, 0.5, and 5 µg of poly(U), poly(A), poly(G), and poly(C) in PRMT buffer for 20 min at room temperature. The protein methyltransferase assays were carried out in 40-µl reaction volumes by addition of assay mixtures containing 1 µg of His-PRMT1 and 0.25 µCi of [14C]SAM to the preincubation samples for 1 h at 30°C. The reaction products were separated by SDS-10% PAGE and visualized by fluorography.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

NS3 protein of HCV contains potential arginine residues to be methylated by a cellular PRMT. Protein methylation occurs mostly in an arginine residue in the Arg-Gly-Gly (RGG) motif known as an RNA-binding domain (30, 38, 40, 47). The RGG motif is composed of a varied number of closely spaced RGG repeats interspaced with aromatic amino acids. Recently, arginine residues in an Arg-Xaa-Arg (RXR) motif in poly(A)-binding protein II, where Xaa is any amino acid, and in RG repeats in Sam68 (Src-associated protein during mitosis) were shown to be methylated by PRMT1, PRMT3, or yeast Rmt1 (5, 64). Since NS3 is an RNA-binding protein, we examined whether the NS3 protein contains RGG or RXR motifs or RG repeats. The NS3 protein does not contain a typical RGG motif interspaced with aromatic amino acids, but it does contain seven RG motifs (Fig. 1A). In particular, we noticed the presence of RGR and GRG boxes in the arginine-rich region, QRRGRTGRG motif IV of the NS3 helicase domain of HCV. The RGR and GRG boxes are conserved in all HCV genotypes and subgenotypes and in HGV (Fig. 1B). The RGR box but not the GRG is present in dengue-2, tick-borne encephalitis, Japanese encephalitis, yellow fever, bovine diarrhea, and classical swine fever viruses. Therefore, sequence alignment of motif IV in a family of viral RNA helicases suggests that an arginine residue(s) in motif IV can be methylated by PRMTs in general. We hypothesized that an arginine residue present in the RGR and GRG boxes of motif IV of HCV NS3 helicase is a potential methylation site if posttranslational arginine methylation of the NS3 protein occurs.


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  		FIG. 1.   NS3 protein of HCV contains potential arginine methylation sites. (A) Amino acid sequence of NS3 protein of HCV1b. Conserved motifs I, II, III, and IV in the DEAD box family of RNA helicases (62) are underlined. Potential RG motifs in the NS3 protein methylated by PRMTs are boxed and indicated by boldface type. The amino acid sequence of NS3 was obtained from GenBank (accession no. AJ238799). (B) Sequence alignment of motif IV in the RNA helicases of various HCV genotypes and other viruses. Motif IV is boxed. RGR, GRG, and RG motifs are indicated by boldface type. Sequence data were obtained from the references or GenBank as follows: HCV-1a (16); Con1 (accession no. AJ238799); NIHJ1 (3); BK (66); HC-J6 (51); HC-J8G (50); BEBE1 (48); NZL1 (60); TrKj (12); ED43 (10); HC-G9 (49); euhk2 (2); HGV (accession no. U44402); Den-2, dengue type 2 virus (25); TBE, tick-borne encephalitis virus (39); JEV, Japanese encephalitis virus (26); YFV, yellow fever virus (56); BVDV, bovine diarrhea virus (41); CSFV, classical swine fever virus (42).

Full-length NS3 is arginine methylated in vivo. We examined whether an arginine-methylated NS3 protein is present in the cells. Expression plasmids harboring full-length NS3, NS3 helicase, or NS3Delta C, with a deletion of the C-terminal portion including helicase domain IV, all of which are tagged with Flag epitope at their N termini, were transfected into 293 cells. Since the NS5A protein appears not to have an RG repeat, NS5A protein methylation is unlikely and was used as a negative control. STAT3 is likely to be arginine methylated like STAT1 in vivo (45) and was used as a positive control. The transfected cell lysates were immunoprecipitated with anti-Flag or anti-mono/dimethylarginine antibody. The methylated arginine-specific antibody was used successfully to detect the in vivo arginine methylation of STAT1 (45). The exogenously expressed proteins in the transfected cell lysates were detected by Western blotting with anti-Flag antibody (Fig. 2A). When proteins immunoprecipitated by anti-Flag antibody were analyzed by Western blotting with a mono/dimethylarginine-specific antibody, the methylated full-length NS3 protein was detected (Fig. 2B, lane 4). When proteins immunoprecipitated by the mono/dimethylarginine-specific antibody were analyzed by Western blotting with anti-Flag antibody, the methylated full-length NS3 and STAT3 proteins were detected (Fig. 2C, lanes 4 and 5). The results indicate that the full-length NS3 protein is arginine methylated in vivo.


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  		FIG. 2.   Full-length NS3 protein is arginine-methylated in vivo. Plasmids harboring full-length NS3 (Flag-NS3F), NS3 helicase domain (Flag-NS3H), NS3 protein with a C-terminal deletion including domain IV (Flag-NS3Delta C), NS5A (Flag-NS5A), and STAT3 (Flag-STAT3) were transfected into 293 cells. The cell lysates were divided into three portions. The portions were immunoprecipitated (IP) with anti-Flag or anti-mono/dimethylarginine antibodies. (A) The proteins immunoprecipitated by anti-Flag antibody were analyzed by Western blotting with anti-Flag antibody to detect expression of proteins tagged with Flag epitope in the transfected cell lysates. The expressed proteins are indicated by arrows. (B) The proteins immunoprecipitated by anti-Flag antibody were analyzed by Western blotting with anti-mono/dimethylarginine antibody. The methylated proteins are indicated by arrowheads and arrows. (C) The proteins immunoprecipitated by anti-mono/dimethylarginine antibody were analyzed by Western blotting with anti-Flag antibody. The methylated proteins are indicated by arrows.

Full-length NS3 and NS3 RNA helicase domain are methylated by soluble human cell extract. A soluble protein extract of 293T cells was incubated with increasing amounts of the full-length NS3 protein (NS3F) purified from human cells or the RNA helicase domain (His-NS3H) expressed in E. coli in the presence of [14C]SAM as a methyl donor. After incubation, the reaction products were separated by SDS-PAGE and visualized by fluorography (Fig. 3A). The full-length NS3 and the RNA helicase domain were labeled with 14C in a dose-dependent manner. Other protein bands labeled with 14C in the absence or presence of the NS3F or NS3 helicase domain presumably represent endogenous methyl acceptors. The result indicates that the NS3 protein is subjected to protein methylation by a SAM-dependent PRMT and that protein methylation occurred in the NS3 helicase domain.


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  		FIG. 3.   Full-length NS3 and the NS3 RNA helicase domain are methylated by a crude 293T cell extract. (A) Methylation of the full-length NS3 and the NS3 helicase domain by 293T cell extract. The protein methyltransferase assay was carried out with 100 µg of a soluble 293T cell extract (Cell Ext) and the indicated amounts of the full-length NS3 (Flag-NS3F) or the NS3 helicase domain (His-NS3H) in the presence of 0.25 µCi of [14C]SAM. The reaction products were separated by SDS-8% PAGE and visualized by fluorography. Flag-NS3F and His-NS3H bands are indicated by arrows. Molecular size markers are indicated at the left (in kilodaltons). (B) Partial purification of the NS3 protein-methylating enzyme from 293T cell extract. A soluble 293T cell extract was sedimented on a 5 to 45% sucrose gradient in PRMT buffer. The gradient was fractionated. Forty microliters of every other gradient fraction was incubated with a methylation mixture containing 1 µg of GST-NS3H1196-1657 and 0.25 µCi of [14C]SAM in a final volume of 50 µl. The reaction products separated by SDS-8% PAGE were visualized by fluorography. The GST-NS3H1196-1657 position is indicated by an arrow at the right. 14C incorporation quantified by a phosphorimager is shown at the bottom panel. Standard protein molecular size markers (Rocherst Co.), which ran in a parallel sucrose gradient, are indicated by arrowheads inside the bottom panel.

Four different kinds of mammalian PRMTs, PRMT1, -3, -4 (CARM1), and -5, have been reported (1, 13, 36, 55, 67). To find out which cellular PRMT methylates the NS3 protein, a soluble 293T cell extract was sedimented on a sucrose gradient. Proteins in every other gradient fraction were incubated with GST-NS3H1196-1657 protein containing the NS3 helicase domain in the presence of [14C]SAM. The reaction products were separated by SDS-PAGE, visualized by fluorography, and quantified by PhosphorImager (Fig. 3B). GST-NS3H1196-1657 protein and its early-termination or degradation product were methylated by certain gradient fractions, which apparently contained a cellular protein methyltransferase(s). Peak fraction 31, which showed the highest protein-methylating activity, also methylated the full-length NS3 (data not shown). The result suggests that the molecular mass of a cellular PRMT which methylates the NS3 protein is greater than 240 kDa. Rat PRMT1 in cells is present as an oligomer of 317 kDa (67). Human PRMT5 is also present as an oligomer of 288 kDa (55), while rat PRMT3 is present as a monomer of 37 to 59 kDa (67). The oligomerization of human PRMT1 or the human homolog of coactivator-associated arginine methyltransferase (CARM1) has not been reported. Thus, a cellular PRMT methylating the NS3 protein may be either PRMT1, PRMT5, or another PRMT, but not PRMT3. We note that rat PRMT1 is almost identical to human PRMT1 except for the 11 N-terminal amino acids and for Tyr-169 instead of His in IR1B4 (1).

NS3 protein is methylated by PRMT1. Next, we examined whether NS3 is methylated by rat PRMT1, PRMT3, or human PRMT5. GST-PRMT1 and -PRMT3 were prepared as previously reported (67). Human Flag-PRMT5 was purified from 293T cells as described in Materials and Methods. The enzymatic activities of the purified PRMTs were verified by the PRMT assay using nonspecific or artificial methyl acceptors such as myelin basic protein, histone, or GST-glycine- and arginine-rich region (GAR) (67) (data not shown). The purified PRMTs were incubated with either the full-length NS3 or the NS3 helicase domain in the presence of [14C]SAM. The reaction products were separated by SDS-PAGE and visualized by fluorography (Fig. 4A). PRMT1 but not PRMT3 or PRMT5 methylated full-length NS3 and the helicase domain.


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  		FIG. 4.   PRMT1 methylates full-length NS3 and the NS3 helicase domain. (A) PRMT1, but not PRMT3 or PRMT5, methylates the NS3 protein. The expression and purification of GST-PRMT1, GST-PRMT3, and Flag-PRMT5 proteins were carried out as described in Materials and Methods. In vitro methylation reactions were performed in 40 µl of PRMT buffer containing 10 pM purified PRMTs, 1 µg of full-length NS3 (Flag-NS3F) or the RNA helicase domain (His-NS3H), and 0.25 µCi of [14C]SAM. The reaction products were visualized by fluorography (upper panel). A 10 pM concentration of the purified PRMTs was separated by SDS-8% PAGE and stained with Coomassie blue (bottom panel). Size markers (lane M) are shown at the right (in kilodaltons). (B) The NS3 helicase domain but not the protease domain is methylated by PRMT1. In vitro protein methyltransferase assays were performed with 0.5 µg of His-PRMT1 purified from E. coli, 1 to 10 µg of MBP-NS3, MBP-NS3N, Flag-NS3F, or His-NS3H protein or MBP as a control in the presence of 0.25 µCi of [14C]SAM. The reaction products were separated by SDS-8% PAGE and either fluorographed (upper panel) or stained with Coomassie blue (bottom panel). Protein bands are indicated by arrows at the right. Size markers are indicated at the left.

To determine whether purified PRMT1 methylates only the NS3 helicase domain, an in vitro protein methyltransferase assay was carried out with PRMT1 and the NS3 deletion mutants purified from E. coli. The reaction products separated by SDS-PAGE were stained with Coomassie blue and then fluorographed (Fig. 4B). His-PRMT1 methylated the full-length NS3 (Flag-NS3F), the full-length NS3 fused to MBP-NS3F, and the NS3 helicase domain (His-NS3H), but not the NS3 protease domain fused to MBP (MBP-NS3N) or MBP as a control. The results indicate that PRMT1 methylates the NS3 protein and confirm the above result of NS3 protein methylation by the 293T cell extract that NS3 protein methylation occurs in the helicase domain but not in the protease domain.

NS3 helicase domain binds specifically to and comigrates with PRMT1. Since the NS3 helicase domain is a substrate for PRMT1, we examined whether the full-length NS3 and the helicase domain bind PRMT1. The partially purified GST-PRMT1 and Flag-NS3F proteins were incubated in lysis buffer. The mixture was divided into equal portions. One was immunoprecipitated with anti-Flag antibody. The other was pulled down with glutathione-agarose beads. The precipitates were separated by SDS-PAGE and analyzed by Western blotting with either anti-GST or anti-Flag antibodies. The full-length NS3 protein was found to be associated with PRMT1 and vice versa (Fig. 5A). To determine a specific interaction between the NS3 helicase domain and PRMT1, His-NS3H or His-SRP as a control was incubated with Flag-PRMT1. The mixtures were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were analyzed by Western blotting with anti-His6 antibody (Fig. 5B). The input proteins in the reaction mixtures were detected by Western blotting with either anti-Flag or anti-His6 antibodies (Fig. 5B). The His-NS3H but not the His-SRP protein was associated with Flag-PRMT1, indicating a specific interaction between the NS3 helicase domain and PRMT1.


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  		FIG. 5.   Full-length NS3 and the NS3 RNA helicase domain bind PRMT1 in vitro. (A) Binding of full-length NS3 protein to PRMT1. Two micrograms of purified full-length NS3 protein (Flag-NS3F) was incubated with 2 µg of GST or GST-PRMT1 protein in lysis buffer. The incubation mixtures were divided into equal portions. The mixtures were then pulled down with glutathione-agarose beads (GST pulldown) or immunoprecipitated with anti-Flag agarose beads (IP). The beads were then washed with lysis buffer. The bead-bound proteins were analyzed by Western blotting with anti-GST or anti-Flag monoclonal antibodies. GST-PRMT1 and Flag-NS3F bands are indicated by arrows at the right. (B) The NS3 helicase domain specifically binds Flag-PRMT1. One microgram of the NS3 helicase domain (His-NS3H) or His-SRP protein was incubated with 1 µg of purified Flag-PRMT1 in lysis buffer. The incubation mixtures were divided into equal portions. The mixtures were then immunoprecipitated with anti-Flag-conjugated agarose beads. After being washed with lysis buffer, the immunoprecipitates (IP) were subjected to Western blotting with anti-His6 antibody (alpha -6×His; Clontech). A part of the mixtures was analyzed by Western blotting with anti-Flag or anti-His6 antibodies (Input). Protein bands are indicated by arrows at the right.

To further demonstrate an interaction between the NS3 helicase domain and PRMT1, we determined whether the two proteins are eluted together from a poly(U) column. The NS3 helicase domain was methylated by PRMT1 in the presence of [14C]SAM, and the reaction mixture was applied to a poly(U) column. The proteins were eluted with step gradient buffers containing increasing amounts of salt. The eluted PRMT1 and NS3 proteins were analyzed by Western blotting with anti-Flag antibody and fluorography, respectively (Fig. 6A). The 14C-labeled NS3 helicase and PRMT1 proteins were detected in both low- and high-salt eluates. As controls, the NS3 helicase domain and Flag-PRMT1 loaded onto the same column were eluted under the same conditions. The NS3 helicase domain and PRMT1 in the fractions were detected by Western blotting with anti-NS3 and anti-Flag antibodies, respectively (Fig. 6B and C). The NS3 helicase domain alone was eluted from the column only at relatively high salt concentrations, while PRMT1 alone was eluted only at low salt concentrations. Taken together, the results suggest that the NS3 helicase domain comigrates with PRMT1.


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  		FIG. 6.   NS3 helicase domain comigrates with PRMT1. The purified His-NS3H protein (50 µg) was methylated by Flag-PRMT1 (50 µg) in 500 µl of PRMT buffer containing 5 µCi of [14C]SAM for 2 h at 30°C. After the incubation, the reaction mixture diluted in 4.5 ml of RNA-binding buffer was loaded onto a poly(U)-Sepharose column. The column was washed with 5 ml of RNA-binding buffer. Proteins bound to the column were eluted with RNA-binding buffer containing from 100 to 700 mM NaCl in 100 mM steps (5 ml per step) and collected into about 0.7- to 1-ml fractions. Flag-PRMT1 and the 14C-labeled NS3 in 20-µl fractions were detected by Western blotting with anti-Flag antibody (alpha -Flag) and fluorography (14C-methylation), respectively (A). His-NS3H and Flag-PRMT1 as controls were also loaded onto the same column. The proteins were eluted under the same conditions as described above. His-NS3H and Flag-PRMT1 in 20-µl fractions were detected by Western blots with an anti-NS3 antibody (alpha -NS3) (B) and with an anti-Flag antibody (C), respectively. Fraction numbers and NaCl concentrations are indicated on the top and at the bottom by arrowheads, respectively. The protein positions are indicated by arrows at the right. FT, flowthrough fraction; W, washed fraction.

Arginine-rich motif IV is a methylated region. We examined whether the QRRGRTGRG motif IV region of the NS3 helicase domain is methylated by PRMT1. We constructed three different kinds of GST-NS3 deletion mutants (Fig. 7B). The GST-NS3H1196-1657 and -1196-1547 mutants contain three RG motifs, including two RG motifs present in the arginine-rich region. The GST-NS3H1468-1547 mutant contains only the two RG motifs present in the arginine-rich QRRGRTGRG region. The protein methyltransferase assay was performed with His-PRMT1 and the purified deletion mutants or GST as a control. The reaction products separated by SDS-PAGE were stained with Coomassie blue and visualized by fluorography (Fig. 7A). PRMT1 methylated all the GST-NS3 deletion mutants, including GST-NS3H1468-1547, but not the GST protein. Thus, the result indicates that an arginine residue in the QRRGRTGR motif IV region is methylated by PRMT1.


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  		FIG. 7.   QRRGRTGRG motif IV region of NS3 helicase domain is methylated by PRMT1. (A) The QRRGRTGRG motif IV region is methylated by PRMT1. The purified GST-NS3 proteins containing motif IV with different flanking regions at the N or C terminus were incubated with 1 µg of His-PRMT1 protein in the presence of [14C]SAM. The reaction products were separated by SDS-10% PAGE and fluorographed (top) or stained with Coomassie blue (bottom). The protein positions are indicated by arrows at the right. Size markers are indicated at the left. (B) Schematic illustrations of the NS3 helicase domain deletion mutants. The mutants were constructed and purified as described in Materials and Methods. Gray rectangle, helicase domain IV; black rectangle, GST protein; +, presence of protein methylation; -, absence of protein methylation.

Arg1493 in the 1486-QRRGRTGRG-1494 motif is essential for NS3 protein methylation, and Arg1488 is likely methylated. We examined which arginine residues in the QRRGRTGR motif of NS3 helicase are essential for methylation by PRMT1. The protein methyltransferase assay was carried out with equal amounts of eight different point mutants, including four mutants with single arginine substitutions in the QRRGRTGRG motif and His-PRMT1 (Fig. 8A). The reaction products were separated by SDS-PAGE, visualized by fluorography, and quantified by a phosphorimager (Fig. 8B). The wild-type RNA helicase domain (His-NS3H) and its arginine substitution mutants R1487A, R1488L, and R1490A but not the R1493K mutant were methylated, indicating that Arg1493 is essential for NS3 methylation. The Q1486H, G1489A, T1491N, and G1492A mutants were also methylated. The Q1486H and R1487A mutants appeared to be better methyl acceptors than the wild-type NS3 helicase domain.


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  		FIG. 8.   Arg1493 residue is essential for methylation of QRRGRTGRG motif IV. (A) Amino acid sequence of motif IV with mutations. Amino acids mutated are indicated in boldface type and underlined. The methylated and unmethylated mutants were indicated as + and -, respectively. ++, mutant was methylated more than the wild type (WT). (B) Arg1493 residue is essential for NS3 helicase methylation. One microgram of wild-type RNA helicase (His-NS3H) and its substitution mutants was incubated with 0.1 µg of His-PRMT1 in the presence of 0.25 µCi of [14C]SAM. The reaction products were separated by SDS-8% PAGE and either fluorographed (top) or stained with Coomassie blue (bottom). The 14C incorporation was quantified by a phosphorimager and is shown in the middle panel. The protein bands are indicated by arrowheads at the right.

A protein methylation reaction may be dependent on concentrations of enzymes and substrates. The protein methyltransferase assay was performed in higher concentrations of wild-type His-NS3H and its R1488L and R1493K mutants and of His-PRMT1. The reaction products separated by SDS-PAGE were stained with Coomassie blue, visualized by fluorography, and quantified with a phosphorimager (Fig. 9A). The methylation of His-NS3H and the R1488L mutant was increased in an enzyme and substrate concentration-dependent manner. Although its methylation product was not detected in the presence of 0.1 µg of PRMT1 (Fig. 8B), the R1493K mutant was methylated in the presence of 0.5 or 15 µg of PRMT1. The methylation of the R1493K mutant was increased in a dose-dependent manner in the presence of 15 µg of PRMT1. The result indicates that another arginine residue(s) in the QRRGRTGRG region is methylated except Arg1493, even though the arginine methylation occurred inefficiently. Other potential arginine methylation sites may be Arg1488 or Arg1490, because they are in the RGR motif. To determine whether Arg1488 or Arg1490 is methylated, we constructed a double substitution mutant containing R1488L and R1493T (Fig. 9B). The purified double mutant was subjected to the protein methyltransferase assay. The reaction products separated by SDS-PAGE were stained with Coomassie blue, visualized by fluorography, and quantified with a phosphorimager (Fig. 9B). While methylation of wild-type GST-NS3H1468-1547 was increased in a dose-dependent manner, the mutant GST-NS3H1468-1547-DM was not methylated, suggesting that Arg1488 is a likely methylation site.


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  		FIG. 9.   Arg1488 in motif IV is potentially methylated by PRMT1. (A) An arginine residue, with the exception of Arg1493, is methylated. Increasing amounts (1 to 3 µg) of wild-type (His-NS3H) or mutant NS3 helicase domain proteins were incubated with 0.5 or 15 µg of His-PRMT1 in the presence of 0.25 µCi of [14C]SAM. The reaction products were visualized by SDS-8% PAGE and fluorography (top and second panels). The 14C incorporation was quantified by a phosphorimager and is shown in the third panel. Coomassie blue staining of methyl acceptors used in the assay is shown in the bottom panel. The NS3 helicase bands are indicated by arrowheads at the right. (B) The NS3 mutant containing R1488L and R1493T is not methylated. Double point mutations were introduced at Arg1488 and Arg1493 of the QRRGRTGRG region as described in Materials and Methods. A schematic representation of the amino acids present in motif IV of the wild type (WT) and the double mutant (DM) GST-NS3H1468-1547 is shown at the top. The mutated positions are indicated by arrowheads. Increasing amounts (1 to 3 µg) of the GST-NS3H1468-1547-WT and -DM proteins were incubated with 15 µg of His-PRMT1 in the presence of 0.25 µCi of [14C]SAM. The reaction products were analyzed by SDS-8% PAGE and fluorographed (second panel). The 14C incorporation was quantified by a phosphorimager and is shown in the third panel. Coomassie blue staining of proteins used in the assay is shown in the bottom panel. The protein bands are indicated by arrowheads at the right.

Methylation of NS3 helicase domain is inhibited by homoribopolymers. We investigated the effect of the RNA binding of the NS3 helicase domain on methylation. This experiment was performed in order to gain insight into how arginine residues in motif IV of the NS3 helicase domain contribute to RNA binding. If arginine residues of motif IV, in particular, Arg1493, could function as a direct RNA contact site or are subjected to a conformational change by their RNA binding, arginine methylation would be modulated. The NS3 helicase domain preincubated with increasing amounts of homoribopolymers, such as poly(U), poly(A), poly(G), and poly(C), was used for the protein methyltransferase assay. The reaction products were separated by SDS-PAGE, analyzed by Coomassie blue staining and fluorography, and quantified with a phosphorimager (Fig. 10). All the homoribopolymers inhibited the methylation of the NS3 helicase domain in a dose-dependent manner. The result suggests that arginines in the QRRGRTGRG motif are subjected to RNA binding or a conformational change, and thereby Arg1493 is not efficiently exposed to PRMT1 in the presence of homoribopolymers.


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  		FIG. 10.   Effects of homoribopolymers on NS3 helicase domain methylation. Five micrograms of the NS3 helicase domain (His-NS3H) was preincubated without homoribopolymers or with the indicated amounts of poly(U), poly(A), poly(G), and poly(C) for 20 min at room temperature. The enzyme assay in triplicate was carried out in a 40-µl reaction volume by addition of the assay mixtures containing 1 µg of His-PRMT1 and 0.25 µCi of [14C]SAM to the preincubation samples for 1 h at 30°C. The reaction products were resolved by SDS-10% PAGE. The gel was stained with Coomassie blue (top panel) or fluorographed (14C-methylation). A representative protein gel stained with Coomassie blue and a representative fluorograph are shown. The 14C incorporation was quantified by a phosphorimager. The mean value of 14C incorporation into the NS3 helicase domain without homoribopolymer was regarded as 100%, and mean values of 14C incorporations in the presence of homoribopolymers are presented as a percentage of that value.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

We found that HCV NS3 protein as an RNA-binding protein contains potential RG motifs to be methylated by a cellular PRMT. Indeed, we could detect an arginine-methylated NS3 protein in vivo (Fig. 2). We also demonstrated here that the full-size NS3 and the NS3 helicase domain are methylated by a human cell extract and by the purified PRMT1 (Fig. 3 and 4). PRMT1 has been shown to methylate arginine residues in many RNA-binding proteins such as histones, hnRNP1, poly(A) binding protein II, and Sam68 (5, 22, 36, 64, 67). PRMT1 is a predominant PRMT in mammalian cells and tissues, including liver (68). The NS3 helicase domain bound specifically and comigrated with PRMT1 in vitro (Fig. 5 and 6). Therefore, the findings suggest that NS3 of HCV, which has a liver tropism, is an in vivo substrate for PRMT1.

We determined that the methylated region is located in the RNA helicase domain, more specifically, in the two RG motifs in the RGR and GRG boxes present in arginine-rich motif IV by using deletion mutants of the NS3 helicase domain (Fig. 4 and 7). The result that the arginine substitution NS3 helicase mutants R1487A, R1488L, and R1490A but not R1493K were methylated (Fig. 8) indicates that the Arg1493 residue of the QRRGRTGR1493G motif IV is essential for NS3 methylation and that it is most likely a major methylation site. In particular, structural analysis of the NS3 helicase domain explains why the Arg1493 in motif IV is a preferential methylation site. The function of motif IV of the NS3 helicase with regard to its RNA-binding, unwinding, and NTPase activities is not clearly determined. Yao et al. (72) and Cho et al. (14) hypothesized that Arg1487, Arg1490, and Arg1493 are exposed to solvent and function as an RNA-binding motif. However, Kim et al. (33) suggested that Arg1490 and Arg1493 residues are solvent exposed in the interdomain cleft but that Arg1487 and Arg1488 are not. They hypothesized that Arg1490 and Arg1493 residues are involved not in RNA binding but in ATP binding. Whatever the function of motif IV of the NS3 helicase is, all the structural data are consistent with the supposition that Arg1493 is exposed to the surface of the NS3 helicase. Because of the exposure of Arg1493 to solvent, we are confident that PRMT1 can transfer the methyl group from SAM to Arg1493 of the NS3 protein efficiently. Therefore, our conclusion that Arg1493 is most likely a major methylation site by PRMT1 fits well with the structural data for the NS3 helicase.

The result that the R1493K mutant is methylated at high concentrations of PRMT1 (Fig. 9A) suggests that other arginine residues, with the exception of Arg1493, in motif IV are also potentially methylated. Structural analyses (14, 33, 72) suggest that other potential methylation sites are Arg1487 or Arg1490 or both, but not Arg1488, which is not exposed to the solvent. We expected that Arg1490, which is solvent exposed (33) and present in the RGR motif, is most likely methylated. However, the double point mutant with mutations at Arg1488 and Arg1493 was not methylated (Fig. 9B), suggesting that Arg1488 but not Arg1490 is methylated. The functional analyses with the arginine-rich motif mutants indicated that Arg1488 and Arg1490 are critical for binding to viral RNA, ATP hydrolysis, and RNA unwinding, but not for ATP binding (11). Therefore, it is likely that Arg1488 is exposed to a solvent and a contact site for binding to the viral RNA, and it is also susceptible to methylation by PRMT1. We used substitution mutants to determine an arginine methylation site. However, this method may not be conclusive, because amino acids adjacent to an arginine residue or a conformational change in motif IV with mutations may affect its efficiency as a methyl acceptor. Other means, such as matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (34), should be used to determine conclusively which arginine residues in motif IV of the NS3 helicase are methylated.

Since the Arg1493 residue is a major methylation site in the HCV NS3 helicase, analysis of the mutants (Fig. 8) suggests that efficiency of methyl acceptors is also determined by the amino acids flanking arginine residue. In particular, the G1492A mutant (QRRGRTARG) was methylated only slightly less than the wild-type NS3 helicase. This result suggests that an arginine residue methylated by PRMT1 should not necessarily be in the context of the GRG box. The Q1486H (HRRGRTGRG) and R1487A (QARGRTGRG) mutants were methylated about 1.5-fold more than the wild type. The T1491N (QRRGRNGRG) mutant was methylated slightly more than the wild type. These results indicate that flanking amino acids slightly distant from or adjacent to the RG motif determine efficiency as a methyl acceptor.

The QRRGRTGRG motif IV of the HCV NS3 helicase, which is often represented by a QRXGRXXR or a QXXGRXXR motif, is one of the most conserved regions in helicase superfamily II (37, 52, 62). Because of the arginine richness of motif IV in the helicase family, it has been suggested that motif IV is involved in protein interactions with RNA (37). In fact, eukaryotic translation initiation factor 4A (eIF-4A) containing HRIGRGGRFGRKG motif IV was shown to have RNA helicase activity, and motif IV of eIF-4A is involved in RNA binding and ATP hydrolysis (52). However, the QRKGRVGRVNFG motif of vaccinia virus RNA helicase NPH-II has been shown to be required for ATP hydrolysis and RNA unwinding, but not for RNA binding (24). Arginine methylation of eIF-4A or NPH-II has not been reported. In particular, motif IV of eIF-4A is likely methylated by PRMTs, because it contains the GRG box in the arginine-glycine-rich region. The RGR or RG motif is conserved in the arginine-rich motif IV of certain viral RNA helicases (Fig. 1B). Therefore, our results suggest that arginine residues in the QRXGRXXR or QXXGRXXR motif in the RNA and DNA helicases of helicase superfamily II (23) are potentially methylated by a cellular PRMT either if arginine residues in motif IV are present in the context of RGG, GRG, or RXR or depending on the flanking amino acids adjacent to the RG motif.

To determine whether the NS3 protein is methylated in vivo, we originally carried out a metabolic labeling experiment by using cells expressing the NS3 protein. However, in vivo NS3 methylation was not detected by this method. This assay is based on the fact that the methyl group of S-adenosylmethionine, a methyl donor for protein methylation, is derived from free methionine in the cell. Thus, if protein synthesis is completely inhibited by treating cells with translation inhibitors, such as cycloheximide and chloramphenicol, methylated proteins can be specifically labeled by incubating cells in L-[methyl-14C]methionine. This method was successfully used to detect the in vivo methylation of RNA-binding proteins (38) and ICP27 of herpes simplex virus (40). However, this assay has a limitation in that methylation of certain proteins would not be detected if protein methylation were cotranslational. Furthermore, it is often impossible to block protein synthesis in cells completely, whereby the radioactively labeled methionine is incorporated into proteins. A monoclonal antibody which can specifically recognize proteins with mono- or dimethylated arginine residues (45) allowed us to detect in vivo arginine-methylation of the full-length NS3 (Fig. 2). However, we could not detect in vivo methylation of the NS3 helicase domain. This might result from inefficient in vivo methylation of the NS3 helicase domain in comparison to in vitro methylation. Alternatively, perhaps the mono/dimethylarginine-specific antibody could not detect an in vivo-methylated NS3 helicase domain efficiently. The full-length NS3 protein is likely to be more abundant than the NS3 helicase domain in HCV-infected cells. Therefore, the presence of an in vivo arginine-methylated full-length NS3 protein strongly reflects a biological significance of the in vitro methylation results of the NS3 protein presented here.

In general, it is believed that in vitro protein arginine methylation is plagued by poor efficiency. Furthermore, it has been shown that an arginine residue without glycine adjacent to the arginine in a protein can be methylated, like the arginine31 residue of STAT1 (45). Therefore, we cannot completely rule out the possibility that the NS3 protease domain or NS3Delta C protein containing a number of arginine residues whose arginine methylation could not be detected here in vitro and in vivo may be arginine methylated. In addition, although we detected in vitro methylation of the NS3 protein only by PRMT1 (Fig. 4), the NS3 protein can be methylated by other PRMTs in vivo, such as PRMT5, as we found that the NS3 protease domain interacts with PRMT5 (unpublished data).

Arginine methylation in protein is assumed to affect protein-RNA interaction by modulation of the affinity of nucleic acid-binding proteins, protein-protein interaction, signal transduction, protein stability, and the regulation of transcription (5, 13, 22). The inhibition of NS3 helicase methylation by PRMT1 in the presence of homoribopolymers (Fig. 10) suggests that arginine residues in motif IV are involved in RNA binding either directly or indirectly. It is unlikely that methylated NS3 protein affects RNA binding, because the RNA-binding domain of the NS3 protein appears to be dispersed over the protein (32) and because the R1493K mutant retained most of the RNA-binding activity (31). The fact that the R1493K mutant lost NTPase and RNA helicase activities (31) suggests that the methylated NS3 protein affects NTPase and RNA-unwinding activities more or less. This is consistent with the hypothesis that Arg1490 and Arg1493 interact with the gamma - and alpha -phosphate groups of ATP (35). We tried to prove this notion experimentally by using methylated and unmethylated NS3 helicases. However, this was not successful, presumably because only a small portion of NS3 helicase appeared to be methylated by PRMT1 in vitro. Neither was it possible to separate methylated from unmethylated NS3 helicase (Fig. 6). The biological consequences of protein methylation with regard to the enzymatic activities of the NS3 protein remain to be proved.

The full-length NS3 protein expressed in human cells and purified by anti-Flag immunoaffinity chromatography was methylated by crude cell lysate and PRMT1 in vitro (Fig. 3A and 4). These results indicate that only a portion of the NS3 protein expressed in human cells is methylated. Currently, we do not know why methylated and unmethylated full-length NS3 proteins are present in the cells. Rat PRMT1 was initially found as a protein interacting with the mammalian immediate-early TIS21 and the leukemia-associated BTG1 protein (36). BTG1 and TIS21 have been shown to be negative regulators of cell growth (57). Their overexpression in cells leads to cell growth arrest (58). Human PRMT1 was discovered as a protein interacting with the intracytoplasmic domain of INFAR1 chain in the type 1 interferon receptor (1). The arginine methylation of STAT1 by PRMT1 modulated alpha/beta-interferon-induced transcription (45). An interaction of NS3 with PRMT1 may affect the PRMT1-TIS21, -BTG1, or -INFAR1 interactions or STAT1 methylation by PRMT1. Interferon treatment for patients infected with HCV raises interferon resistance. NS5A of HCV has been shown to be responsible for interferon resistance (19, 20). NS3 may well be another inducer of interferon resistance by HCV. Therefore, the PRMT1-NS3 interaction as an enzyme substrate and its reaction products may affect cell physiology, antiviral defense of the host, or HCV replication. Further studies are required to address these questions. Our finding that HCV NS3 is subjected to posttranslational arginine methylation by PRMT1 may contribute to understanding of the molecular mechanisms of pathogenesis by HCV. Furthermore, it will be interesting and of importance to determine the protein methylation of other viral helicases by PRMTs and the biological consequences of arginine methylation.


    ACKNOWLEDGMENTS

Jaerang Rho and Seeyoung Choi contributed equally to this work.

We are grateful to H. R. Herschman for GST-PRMT1 and GST-PRMT3 plasmids; H. Hotta, S. K. Jang, and Y. C. Sung for HCV NS3 plasmids; and J. E. Darnell, Jr., for the STAT3 plasmid.

This work was supported by a grant from the Ministry of Science and Technology, Korea.


    FOOTNOTES

* Corresponding author. Mailing address: Cell Biology Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejeon 305-333, Republic of Korea. Phone: 82 42 860 4172. Fax: 82 42 860 4597. E-mail: imdongsu{at}mail.kribb.re.kr.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, September 2001, p. 8031-8044, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8031-8044.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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