C型肝炎病毒非結構性蛋白質NS3對於Cystatin-A蛋白質表現的調控

Abstract

C型肝炎病毒具有單股正向RNA基因體，可轉譯出一多蛋白質前驅物，並由宿主和病毒蛋白酶切割成結構性與非結構性蛋白質。C型肝炎病毒的蛋白酶有2個，NS2/3半胱胺酸蛋白酶 (cysteine protease)及NS3絲胺酸蛋白酶 (serine protease)，兩者共同參與多蛋白質切割，以產生具功能性的非結構性蛋白質，協助病毒RNA的複製。先前研究指出NS3具有致癌性，表現NS3蛋白質可誘發裸鼠產生腫瘤。本實驗室先前的研究中發現基因型1b之病毒NS3蛋白質在輔助因子NS4A存在下具有自我內部截切及細胞轉型能力 ，而且截切產物NS3(1-402)較NS3全長具有更高的細胞轉型能力。當利用親和性管柱分析與NS3(1-402)會形成複合物的細胞因子時，發現Huh7細胞中的cystatin-A可與NS3(1-402)被共同純化出來，表示兩者間可能具有交互作用。而在共同免疫沉澱實驗中也證實了NS3全長與cystatin-A之間確實有交互作用。Cystatin-A為一半胱胺酸蛋白酶抑制蛋白 (cysteine protease inhibitor)，可調控細胞中半胱胺酸蛋白酶的活性。當細胞中cystatin-A表現量下降時，可能造成半胱胺酸蛋白酶活性不受調控而導致細胞癌變。本研究在探討NS3與cystatin-A之間交互作用的生物意義及cystatin-A是否參與在NS3造成癌化的過程。結果發現，在Huh7細胞中共同轉染cystatin-A和NS2C-3P蛋白酶表現質體時，並未偵測到NS2/3蛋白酶切割活性有改變，但將細胞中cystatin-A表現抑制時，NS3的表現量會下降。另一方面NS3表現時卻也會使cystatin-A表現量下降，並影響細胞黏著度 (adhesion)，而且NS3對細胞黏著度的負調控可藉由外送大量表現的cystatin-A而回復。另外也發現，NS3可以誘發細胞的自噬作用 (autophagy)，可能藉此降解cystatin-A。而在利用抑制劑抑制自噬作用後，在cystatin-A表現被抑制的細胞中NS3的表現及自噬作用的活性相較於對照組皆有下降的現象，表示自噬作用可能參與調控NS3蛋白質表現，而其中的機制需更進一步的探討。本研究對於cystatin-A在C型肝炎病毒感染中所扮演的角色提供一個新的研究方向。

Hepatitis C virus (HCV) has a positive single-stranded RNA genome. The viral genome can be translated into a polyprotein precursor that is further processed into structure and non-structure proteins by host and virus proteases. HCV possesses two viral proteases, NS2/3 cysteine protease and NS3 serine protease, which participate in the process to generate functional non-structure proteins. Our laboratory has previously demonstrated an internal NS3 cleavage activity of HCV genotype 1b. The major internal cleavage product, NS3(1-402), had a higher transforming activity than the full-length NS3. By performing tandem affinity purification, cystatin-A was co-purified with the NS3(1-402) protein, and its interaction with the full-lenth NS3 was confirmed by co-immunoprecipitation assay. Cystatin-A, as a cysteine protease inhibitor, can modulate cellular cysteine protease activity. The down-regulation of cystatin-A was suggested to be involved in tumorigenesis. In this study, the biological significance for the association between cystatin-A and NS3 and its implication in transformation induced by NS3 were examined. By co-expression NS2C-3P cysteine protease and exogenous cystatin-A in Huh7 cell, no inhibitory effect of cystatin-A to the NS2/3 cysteine protease was observed. On the other hand, NS3 down-regulated expression of cystatin-A protein in various cell types. In addition when cystatin-A as knocked-down, the expression level of NS3 was reduced. Further study demonstrated that NS3 may regulate cystatin-A expression by inducing autophagy. In the presence of inhibitor, NS3 expression was reduced and autophagy activity was significantly diminished in cystatin-A-knockdown cells, suggesting a possible involvement of autophagy in regulation NS3 stability. Furthermore, NS3-mediated reduction on cell adhesion could be rescued by overexpression of cystatin-A, implying a potential role of cystatin-A in HCV pathogenesis. The mechanisms involved in NS3-induced autophagy remain to be elucidated.