C型肝炎病毒NS3-4A蛋白質對DNA損傷及修復的影響

Abstract

C型肝炎病毒具有一個單股正向的RNA基因體，感染宿主細胞時，會利用此RNA轉譯出一多蛋白質前驅物，再利用宿主細胞及病毒自身的蛋白酶進行切割形成有功能性的結構與非結構性蛋白質。非結構性蛋白質NS4A作為NS3 蛋白酶的輔助因子，幫助NS3進行病毒多蛋白質前驅物的切割，此外，NS4A存在下，亦能幫助NS3進行內部截切，提高NS3的細胞轉型能力。在本論文中，首先在C型肝炎病毒sub-genomic replicon系統，發現C型肝炎病毒非結構性蛋白質表現時會造成DNA雙股斷裂訊號γ-H2AX的增加，以及參與DNA修復的WRN蛋白質表現量降低，顯示C型肝炎病毒非結構性蛋白質可能會增加細胞DNA受損的情形。進一步表現NS3-4A蛋白質進行分析，結果顯示NS3-4A蛋白質亦能使γ-H2AX增加，表示細胞DNA有受損的情況。分析DNA修復系統，發現當病毒蛋白質NS3-4A存在下，會使修復DNA雙股斷裂的non-homologous end-joining修復能力降低。由這些結果推測，增加細胞DNA受損的情況以及降低DNA修復系統的修復能力可能是NS3-4A蛋白質造成細胞轉型的一種機制。

Hepatitis C virus (HCV) possesses a positive single-stranded RNA genome. Upon infection, the RNA genome is used as a template to encode a polyprotein precursor which is then processed by host and the viral proteases to form functional proteins. Nonstructural protein (NS) 4A is a cofactor of the NS3 protease involved in the polyprotein processing. In addition, NS4A facilitates the internal NS3 cleavage that produces products with higher transforming activity than the full-length NS3. In this study, an increased level of the DNA damage marker γ-H2AX and a reduced expression level of WRN which participates in DNA repair were detected in HCV sub-genomic replicon cells. The increased DNA damage was also detected in NS3-4A expressing cells. In addition, expression of NS3-4A protein reduced the activity of non-homologous end-joining involved in the repair of DNA double-strand breaks. The results suggest that an increased DNA damage coupled with a reduced DNA repair ability could be one of the mechanisms involved in the transforming activity of the HCV NS3-4A protein.