The Hepatitis C virus (HCV) is a major inducer of chronic hepatitis, and its chronic infection is associated with liver cirrhosis, and eventually, development of hepatocellular carcinoma. To develop therapeutic tools against of hepatocellular carcinoma caused by chronic infection of HCV, it is important to understand molecular mechanism by which HCV causes liver pathogenesis and which cellular genes are involved in hepatocellular carcinogenesis. A number of studies suggest that the core protein of HCV is a major viral factor in in hepatocellular carcinogenesis. Also, NS3 protein of HCV has been shown to be implicated in the liver pathogenesis. To search for cellular targets of the NS3 protein, we conducted a yeast two-hybrid screen. We found that Janus kinase-binding protein 1 (JBP1), known as a human homologue of Shk1 kinase-binding protein 1 of the fission yeast (Skb1Hs), interacts with the nonstructural protein 3 of the hepatitis C virus by a yeast two-hybrid screen. Amino acid sequence analysis revealed that Skb1Hs/JBP1 contains conserved motifs of S-adenosylmethionine-dependent protein arginine methyltransferases (PRMTs). Here, we demonstrate that Skb1Hs/JBP1, named PRMT5, is a distinct member of the protein arginine methyltransferase family. Recombinant PRMT5 protein purified from human cells methylates myelin basic protein and histone. Myelin basic protein methylated by PRMT5 contains monomethylated and dimethylated arginine residues. Sedimentation analysis of the purified PRMT5 on a sucrose density gradient indicates that PRMT5 forms distinct homo-oligomeric complexes, including dimer and tetramer, which comigrate with the enzyme activity. Western blot analysis of sedimentation fractions suggests that endogenous PRMT5 is present as a homo-oligomer in a 293T cell extract.
This finding prompted us to examine the possibility of whether arginine residues of the NS3 protein are methylated by protein arginine methyltransferases (PRMTs). PRMTs transfer the methyl group from S-adenosylmethionine to the guanidino nitrogen atoms of arginine residues. The protein arginine methylation is an irreversible and posttranslational covalent modification. Here we present biochemical evidences that the NS3 protein of HCV is subjected to protein arginine methylation in vitro and in vivo, and a cellular enzyme methylating the NS3 protein in vitro is PRMT1. The NS3 protein of the hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. 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 purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. Mutational analyses indicate that the arg1493 in the QRRGRTGR1493G region of the NS3 RNA helicase is essential for the NS3 protein methylation. The NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in an RNA binding. The results suggest that arginine residue(s) in a QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the protein arginine methyltransferase 1. PRMT-NS3 interaction as enzyme-substrate may modulate posttranslational modification of cellular substrates by PRMTs in HCV-infected hepatocytes and aberrant arginine methylation of cellular proteins by the NS3 protein may partially account for liver pathogenesis by HCV.
The JAK-STAT pathway plays a crucial role in the signal transduction of many cytokines including IFNs. PRMT1 has been shown to associate with the IFNa/b receptor. In addition, a recent report described the isolation of a novel JAK binding protein 1 (JBP-1, PRMT5) with arginine methyl-transferase activity. Here we demonstrate arginine methylation of JAK kinases in vivo. PRMT5 associates with JAK2 in vivo. HCV NS3 decreases the binding of PRMT5 and JAK kinases. In addition, NS3 inhibits JAK-STAT-mediated IFN responses.. Thus, arginine methylation of JAK kinases is an additional posttranslational modification which may regulate transcription function, and HCV NS3 might be responsible for the down-regulation of interferon responsiveness.
Regulation of arginine methylation would presumably occur at the level of modulating methyltransferase activity toward unmethylated substrates, by sequentially increasing the amount of methylation over the lifetime of a protein, or by selective turnover of methylated proteins. Tyrosine phosphorylation of PRMT has not been established. PRMT1 is phosphorylated within 2 min of pervanadate treatment in PRMT1 transfected cells. Tyrosine phosphorylation was inhibited by PP2, a selective inhibitor of Src protein-tyrosine kinase (PTK) family members at concentrations consistent with inhibition of Src function. Finally, we demonstrate that PRMT can be phosphorylated directly both in cotransfection experiments using c-Src expression vector and in in vitro kinase assays with purified kinases. These data suggest that PRMT can be a target for phosphorylation by the Src family of PTKs. Tyrosine phosphorylated PRMT by Src can modulate the activity and substrate specificity of PRMT.
간염 C형 바이러스(hepatitis C virus, HCV)는 만성 간염으로 진행하여, 간경화를 일으키고, 궁극적으로 간암으로 발전된다. 간염 C형 바이러스에 의해 유발된 간암에 대한 치료를 효과적으로 하기위해서는 간염 C형 바이러스의 간경변 진행과정에서의 분자적인 기전을 이해하는 것이 중요하다. 세포내 인자가 간염 C형 바이러스의 단백질과 결합하여 간암으로 발전될 가능성 이 높다. 간염 C형 바이러스 단백질중 NS3가 간경변 진행에 관련된 것으로 보고되었다. 따라서 간염 C형 바이러스의 NS3 단백질의 세포내 결합인자를 yeast-two hybrid 방법을 통해 탐색하였다. 우리는 NS3 단백질의 세포내 결합인자로 protein arginine methyltransferase 5 (PRMT 5)를 클로닝하였다. 여기서 293세포에서 정제되어진 재조합PRMT5는 효소활성을 갖고있었으며, myelin basic protein을 mono혹은 di-methylation 시켰다. 또한 정제한 PRMT5는 sedimentation 분석에서 homo-oligomer를 형성하였으며, dimer와 tetramer 복합체로 형성되어져 있었다. PRMT5의 효소활성은 tetramer 복합체로 형성되어져 있을때 가장 높게 측정되었다.
간염 C형 바이러스의 NS3 단백질이 protein arginine methyltransferase 5 (PRMT 5)에 의해 arginine 잔기에 methylation이 관찰되지는 않았으나, 간염 C형 바이러스의 NS3 단백질이 in vivo 에서 arginine 잔기에 methylation이 관찰되었으며, in vitro 에서 PRMT1에 의해 arginine 잔기에 methylation이 관찰되었다. 지금까지 여러 RNA결합 단백질의 arginine-glycine (RG) 부위가 PRMTs에 의해 arginine 잔기에서 methylation이 관찰되었다. 간염 C형 바이러스의 NS3 단백질의 아미노산분석 결과 7군데의 RG부위가 존재하였다. 그중 2군데는 RNA helicase motif IV인1486-QRRGRTGRG-1494에 존재하였다. 염기부위 치환 분석에 의해 arg1493 가 NS3 methylation에 필수적이다는 것을 알 수 있었다. NS3단백질의 PRMT1에 의한 methylation은 homopolymers에 의해 감소되었다. 이로써 NS3단백질의 RNA helicase motif IV는 RNA 결합에 관여한다고 간주할수 있다. 또한 NS3와 PRMT1과의 상호 결합으로 인하여 HCV 감염 세포에서 PRMT1의 세포내 기질변환을 일으킬수 있다. 이는 NS3 단백질에 의한 세포내 단백질의 arginine methylation 변이가 HCV에 의한 간경변으로의 진행을 일으킬 수 있다는 이론을 부분적으로 설명 할 수 있다.
최근에 interferon (IFN) 등을 포함한 사이토카인의 세포내 신호전달과정에서 중요한 단백질들이 PRMTs와 상호결합을 한다는 연구결과들이 발표되었다. IFN receptor가 PRMT1과 상호결합하고, STAT1이 PRMT1과 상호결합한다고 보고 되었다. 특히 Janus kinase2 (JAK2)와 PRMT5가 상호결합을 한다는 연구결과는 간염 C형 바이러스의 NS3 단백질이 JAK-STAT 세포내 신호전달과정에서 역할을 할것으로 예상하였다. 우리는 in vivo에서 PRMT5와 JAK2가 결합하고, 이들의 결합은 HCV NS3 단백질이 발현됨으로써 감소 되었다. 또한 in vivo에서 JAK2가 arginine methylation 되었다. 이로써 JAK kinase의 arginine methylation은 JAK-STAT 세포신호전달과정의 전사조절의 번역후 변환과정으로 간주되며, HCV NS3 단백질이 IFN 반응에 방해 기작으로 작용함을 보였다.
Arginine methylation의 조절 메커니즘은 아마도 unmethylated 기질에 대한 methyltransferase 활성의 변환 정도에서 일어날 것으로 간주된다. 지금까지 PRMT의 조절기작은 알려진 바 없다. PRMT의 tyrosine 인산화는 보고된 적이 없다. Pervanadate 처리시 PRMT1과 PRMT5는 tyrosine 인산화 되었다. 또한 Src 단백질-tyrosine kinase 저해제인 PP2에 의해 tyrosine 인산화가 방해 받았다. PRMT1 과 PRMT5는 c-Src와 cotransfection 실험에서와 in vitro kinase 실험에서 인산화가 되었다. 이로써 PRMT는 Src에 의해서 인산화가 되고, tyrosine 인산화가된 PRMT는 효소 활성과 기질특이성이 변환을 가져 온다는 사실을 증명하였다.
