C型肝炎病毒NS3與Integrin beta1的交互作用以及對細胞貼附和移動的影響

Abstract

C型肝炎病毒具有單股正向RNA基因體，可以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和非結構性蛋白質；非結構性蛋白質NS3具有serine protease, RNA helicase和 ATPase的功能。先前研究指出NS3具有oncogenic potential，表現NS3蛋白質能使細胞在沒有貼附以及低血清的環境下穩定生長，也可誘發裸鼠產生腫瘤。本實驗室研究也發現NS3具有內部截切以及細胞轉型的能力，另外，利用共同免疫沉澱法和GST-pull down assay 發現NS3蛋白質和細胞因子integrin β1 有交互作用。已知integrin 是一個細胞表面受器，參與調控細胞的貼附、生長、移動及凋亡，也與癌症的發生和轉移有極大的關聯性。因此，本研究進一步探討NS3對細胞貼附和細胞移動的影響並探討NS3與integrin β1在細胞中分布的位置及兩者間的調節作用。結果發現NS3表現時細胞初期貼附到collagen和fibronectin細胞外基質的能力明顯下降。同時在細胞傷口癒合(wound healing assay)和細胞散射到固定配體(scattering onto immobilized ligands assay)的實驗中，均觀察到NS3對細胞移動有增強的作用。另外，NS3和integrin beta1在細胞中有部分重疊的現象，西方墨點法分析結果顯示NS3蛋白質表現會造成integrin β1活化態比例增加。NS3是否參與integrin的recycling和degradation，藉此調節integrin的表現與活性，造成細胞型態的轉移，影響細胞貼附以及移動，均有待進一步探討。本研究對C型肝炎病毒 NS3蛋白質造成細胞癌化的可能機制提供一個研究的方向。

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 structural and non-structural proteins. The non-structural protein 3 (NS3) possesses serine protease, RNA helicase and ATPase activities. Previous studies have demonstrated a oncogenic potential of the NS3 protein. Expression of the NS3 protein allowed cells to grow in non-adhered environment and in low serum medium. NS3 can also induce tumor formation in nude mice. In addition, our laboratory has demonstrated an internal NS3 cleavage activity associated with cell transformation. By performing co-immunoprecipitation and GST-pull down assay, our laboratory also identified an interaction between NS3 and integrin β1. Integrin receptors are known to be involved in cell adhesion, growth, migration and apoptosis. Previous studies indicated that changes on the expression level and configuration of integrins have great correlation with cancer progression and metastasis. In this study, the effects of HCV NS3 on cell adhesion and migration and the interplay between the NS3 protein and integrin β1 were further investigated. The results demonstrated that expression of NS3 reduced initial adhesion of cells on fibronectin and collagen. Meanwhile, cell migration was enhanced by NS3 in wound healing and scattering onto immobilized ligands assay. Furthermore, NS3 partially co-localized with endogenous integrin beta1 and up-regulated the ratio of active to total form of integrin β1. Whether NS3 mediates cell adhesion and migration through integrin recycling and degradation remains to be elucidated. This study provides a direction in understanding the possible mechanisms of HCV NS3 protein involved in tumor formation.