C型肝炎病毒非結構性蛋白質NS3-4A對於宿主細胞之細胞凋亡的調節作用

Abstract

C型肝炎病毒 (Hepatitis C virus, HCV) 非結構性蛋白質NS3具有serine protease和RNA helicase的功能。NS4A作為NS3 protease的輔因子。在NS4A的存在之下，NS3會將病毒的多蛋白質進行切割，同時也會進行NS3的內部切割。在先前的研究中指出，NS3-4A會存在於細胞的粒線體當中，並且利用protease的酵素活性去切割粒線體上的MAVS/IPS-1而去干擾type-I IFN-b 的產生，使宿主的免疫反應受到抑制。近來也有研究顯示，NS4A會累積在細胞粒線體中，進而活化caspase-3及引發細胞凋亡。本研究首先建立能經由doxycycline誘導表現NS3-4A的HepG22-NS3-4A穩定細胞株。在此細胞株觀察到NS3-4A表現時會自行切割得到NS3和NS4A蛋白質，NS3和NS4A在細胞中的分佈大致重疊，位於粒線體和內質網上。過去已知脫氧膽酸會造成粒線體膜電位的改變。在此研究中發現不論是誘導表現NS3-4A的穩定細胞株或在短暫轉染表現NS3-4A的細胞當中，在給予脫氧膽酸的情況下，相較於控制組，都會減緩脫氧膽酸對於粒線體膜電位的影響。當分析293細胞在表現NS3-4A時，對於脫氧膽酸所造成DNA fragmentation、cytochrome c釋放和caspase-3活化的情形，發現NS3-4A的表現能減緩脫氧膽酸所造成cytochrom c的釋放和caspase-3的活化，但是對於脫氧膽酸所造成的DNA fragmentation，則沒有太大的影響。進一步將帶有NS3、NS4A的表現質體分別轉染293細胞，發現NS3和NS4A單獨存在時都具有穩定粒線體膜電位的功能，減緩脫氧膽酸的效應。其中NS4A也能減緩脫氧膽酸所造成的cytochrome c釋放。已知位於粒線體的p53會促使細胞進行粒線體相關細胞凋亡。實驗亦發現，當NS3-4A表現時，p53在粒線體上的分佈量明顯下降。NS3-4A或許藉由調控粒線體上的p53，來達到抗細胞凋亡的效果。但其真正作用機制還有待進一步的確認。

Hepatitis C virus (HCV) NS3 is a nonstructural protein that possesses protease and RNA helicase activity. NS4A is a cofactor of NS3 protease, it promotes HCV polyprotein processing and NS3 internal cleavage. NS3-4A can localize at mitochondria to cleave the mitochondria protein MAVS/IPS-1 then to block the type-I IFN-b pathway. NS4A was also found to accumulate on mitochondria membrane and induce mitochondria-mediated apoptosis. In this study, HepG22-NS3-4A inducible cell line was established. Expression of NS3-4A protein in the stable line resulted in a cleavage of NS3-4A protein and partial colocalization of the NS3 and NS4A to the mitochondria. Expression of NS3-4A also restored the mitochondria membrane potential collapsed by DCA both in the inducible HepG22-NS3-4A cell line and in 293 transient transfection system. Both cytochrome c release and caspase-3 activity induced by DCA can be repressed by NS3-4A, but NS3-4A can not protect the DCA-induced DNA fragmentation. Further dissection indicated that NS3 or NS4A alone can restore the mitochondria membrane potential collapse by DCA. NS4A can decrease the DCA-induced cytochrome c release. The mechanisms involved in the NS3-4A mediated anti-apoptotic effects are not completely understood. Nevertheless, it was reported that mitochondria p53 can promote mitochondria-mediated apoptosis. The observation that the level of p53 at mitochondrial fraction was lower in the NS3-4A expressing cells than that of the control cells, suggesting that p53 maybe a target of the NS3-4A regulated anti-apoptotic effect.