C型肝炎病毒非結構性蛋白質NS3在WRN參與調控之非同源性末端接合修復上具雙重作用

Abstract

C型肝炎病毒與肝細胞癌的形成相關，病毒NS3蛋白質具有絲胺酸蛋白酶及解旋酶雙重活性。過去的研究顯示NS3蛋白質會抑制DNA修復並與細胞轉型作用有關；然而，NS3蛋白質參與在細胞癌化的機制仍未完全瞭解。在哺乳動物細胞中，DNA的修復是經由許多複雜的機制相互調控構成的系統，在這些複雜的調控當中，WRN在許多修復機制上均扮演了重要的角色，包含非同源性末端接合修復 (NHEJ repair)。在以往的研究中已知Ku蛋白質參與在啟動NHEJ修復的機制中，且與WRN具交互作用，有助於相互的調控。在本研究中發現，NS3蛋白質會與WRN及Ku70蛋白質有交互作用，並且在C型肝炎病毒次基因體複製系統及表現NS3蛋白質的Huh7細胞當中，WRN所調控之修復機制有拖延的現象，NHEJ修復效率也呈現下降的趨勢。NS3蛋白質亦會改變細胞核內WRN與Ku70蛋白質的分布，並干擾Ku70結合到DNA雙股斷裂處，這樣的發現可用於解釋為何NS3蛋白質會抑制NHEJ修復。同時，本研究也發現WRN蛋白質不但會受到蛋白酶體降解，其存在量也與NS3-4A蛋白酶的活性有關。C型肝炎病毒NS3蛋白質對WRN參與的NHEJ修復作用及WRN蛋白質降解的雙重影響，可能導致DNA突變的累積、增加基因的不穩定性、甚至腫瘤的生成。

Hepatitis C virus (HCV) is associated with the development of hepatocellular carcinoma. The viral NS3 protein, acting as serine protease and helicase, was shown to inhibit DNA repair and involve in cell transformation. However, implications of the HCV NS3 in the mechanisms of oncogenesis are not fully understood. DNA repair pathways in mammalian cells are mediated by complicated mechanisms. Among these, Werner syndrome protein (WRN) plays a major role in multiple repair pathways, including non-homologous end joining (NHEJ) repair. Ku protein was previously demonstrated to interact with WRN and involve in the initiation of NHEJ repair. In this study, HCV NS3 protein was found to interact with WRN and Ku70. In addition, WRN-mediated DNA repair was prolonged and the efficiency of overall NHEJ repair was reduced in both an HCV subgenomic replicon system and Huh7 cells expressing NS3. NS3 alters the nuclear distribution of WRN and Ku70. It interferes with the recruitment of Ku70 to the DNA DSB sites. This may partially explain why NS3 inhibits NHEJ repair. In addition, the level of WRN is regulated by both the proteasome degradation and the activity of NS3-4A protease. The dual role of HCV NS3 in regulating the function and expression level of the WRN protein provides possible mechanisms of HCV in the impairment of DNA repair. The impairment may lead to an accumulation of DNA mutation and genome instability and, eventually, tumor development.