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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 - PubMed

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

J Rho et al. J Virol. 2001 Sep.

Abstract

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 Arg(1493) in the QRR(1488)GRTGR(1493)G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg(1488) 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.

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Figures

FIG. 1
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).

FIG. 2
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-NS3ΔC), 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.

FIG. 3
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.

FIG. 4
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.

FIG. 5
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 (α-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.

FIG. 6
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 (α-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 (α-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.

FIG. 7
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.

FIG. 8
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.

FIG. 9
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.

FIG. 10
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.

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