C型肝炎病毒非結構性蛋白質NS3與WRN的交互作用及對WRN表現量的影響

Abstract

C型肝炎病毒(Hepatitis C virus, HCV) 是臨床上肝臟疾病常見的病源之一，感染後會造成肝纖維化和肝細胞癌等症狀。病毒的nonstructural protein 3 (NS3)具有protease及RNA helicase的功能，而NS4A可以作為NS3 protease的cofactor。過去研究顯示NS3除了會造成DNA的損傷外，它還會干擾參與DNA修補作用蛋白質的功能。Werner syndrome protein (WRN)是RecQ DNA helicases family的一員，在過去的研究中發現它可做為helicase或者是exonuclease參與在DNA修復作用中。為了探討NS3是否會透過WRN影響DNA修復作用，本研究首先探討NS3是否影響WRN表現量，結果顯示NS3及NS3/4A都會使WRN的表現量下降，其中以NS3/4A的影響較顯著。又進一步探討NS3/4A的內部截切產物NS3(1-369)、NS3(1-402)、NS3(1-462)及NS3(327-631)是否會影響WRN的表現量，結果顯示NS3(1-462)造成WRN表現量下降的幅度最為明顯。接著以GST pull-down 實驗證實了NS3與WRN的交互作用。將NS3依照蛋白質功能與結構區分為protease domain、NTPase domain、RNA binding domain及helical domain，並利用GST pull-down分析時，發現以C-terminal的helical domain與WRN的交互作用最為顯著。由本研究的結果推測NS3使WRN表現量下降，而NS3對WRN的功能及其所參與的DNA修補作用有何影響有待進一步研究。

Hepatitis C virus (HCV) is one of the main pathogens that infect human liver. HCV infection often causes liver fibrosis and develops to hepatocellular carcinoma. The viral nonstructural protein (NS) 3 possesses protease and RNA helicase activities and NS4A functions as a cofactor of the NS3 protease. In previous studies, NS3 was demonstrated to induce DNA damage and interfere DNA repair of host cells. Nevertheless, the molecular mechanisms are not fully understood. Werner syndrome protein (WRN) is classified as a member of the RecQ DNA family. It possesses helicase and exonuclease activities and participates in DNA repair. To learn whether NS3 affects DNA repair though WRN, the effects of NS3 and NS3/4A on WRN expression were first examined in this study. The results showed decrease on WRN expression when NS3 or NS3/4A was present. In addition, NS3/4A internal cleavage product NS3(1-462) also caused a decrease on WRN expression. GST pull-down assay was then performed to examine the interaction between WRN and NS3. The results showed that WRN interacted with the full-length NS3 and its various subdomains, and more notably, the GST-NS3(402-631) and GST-NS3(462-631) with the C-terminal helical domain. Taken together, this study detected the interaction between WRN and HCV NS3 protein and the effect of NS3 on WRN expression. Whether NS3 affects WRN functions and influences DNA repair needs to be further investigated.