C型肝炎病毒非結構性蛋白質NS4A對宿主細胞clusterin基因表現和caspase-3活性的調節作用

Hepatitis C virus NS4A protein regulates clusterin expression and caspase-3 activity of host cells

黃裕煦(Yu-Hsu Huang)

指導教授 ： 張鑫

國立臺灣大學/醫學院/微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02227

摘要

C型肝炎病毒 (Hepatitis C virus, HCV) 非結構性蛋白質NS4A具有54個胺基酸，分子量為6 kDa。主要在作為NS3 serine protease的輔因子，促使多蛋白質前驅物的切割。NS4A也可利用自身中央區域和NS3作用，活化NS3 serine protease並促使NS3的內部截切。最近的研究發現，NS4A單獨存在時會累積在細胞內的粒線體中，並經由粒線體相關的路徑引發細胞凋亡以及活化caspase-3。先前本實驗室以cDNA microarray分析HCV subgenomic replicon細胞株，發現在HCV非結構性蛋白質表現時，clusterin (CLU) mRNA有上升的情形。CLU是一個廣泛分布於組織與體液的醣蛋白質。先前研究指出CLU和DNA修復以及細胞凋亡或存活等功能相關。全長的CLU mRNA經由選擇性剪修 (alternative splicing) 可產生不同長度mRNA並分別轉譯出兩種不同功能的同型體，secreted clusterin (sCLU) 以及 nuclear clusterin (nCLU)。
本研究首先將單一HCV非結構性蛋白質表現質體轉染293細胞，抽取RNA藉由即時定量聚合酶反應分析，發現NS4A和NS5B均會特異性地增加細胞內CLU mRNA的量。NS4A增加細胞內sCLU mRNA的量，而對nCLU mRNA的表現則沒有明顯的影響。當共轉染NS4A表現質體和含有801 bp CLU 啟動子長度的pCLU-luc報導質體到293細胞時，分析luciferase 的mRNA合成量，發現比起控制組並無明顯改變，顯示NS4A可能並非影響在CLU mRNA的合成上，或者NS4A作用位置不在801 bp CLU 啟動子的範圍內。以上結果也可推測：NS4A增加細胞內CLU mRNA的機制可能是經由抑制 CLU mRNA的分解，亦即增加CLU mRNA的穩定度。在蛋白質層次方面，表現NS4A的293細胞，細胞內的sCLU和precursor nCLU (pnCLU) 均有增加，但分泌至細胞培養液中的sCLU反而下降，推測NS4A降低293細胞中sCLU蛋白質的穩定性。而在可經doxycycline誘導表現NS3-4A的HepG22-NS3-4A穩定細胞株，其細胞內及細胞外的sCLU有微幅的增加，是否NS3的共同表現會影響NS4A對CLU表現之調控則有待進一步確認。另一方面，分析NS4A參與caspase-3活化的重要區域。刪除突變株NS4A(1-34) 則失去了促使caspase-3活化的特性。無論是NS4A(35-54) 刪除突變株或是NS4A中央區域的點突變株均仍保有活化caspase-3的能力，而NS4A(35-54) 活化caspase-3的能力略為下降。分析NS4A影響CLU基因表現以及活化caspase-3 的特性有助於了解HCV感染時，病毒與宿主之間的關係。

關鍵字

C型肝炎病毒 ； 非結構性蛋白質

並列摘要

Hepatitis C virus (HCV) NS4A is a nonstructural protein of about 6 kDa that consists of 54 amino acids. The well-known function of NS4A is the cofactor of NS3 serine protease that promotes the HCV polyprotein processing. NS4A also uses its central domain to interact with NS3 serine protease and to promote NS3 internal cleavage. A recent study demonstrated that NS4A accumulates on mitochondria membrane and induces mitochondria-mediated cell apoptosis and caspase-3 activation. cDNA microarray analysis from our laboratory revealed an increase of clusterin (CLU) mRNA in HCV subgenomic replicon cell line. CLU is a glycoprotein that expresses in virtually all tissue and fluid. CLU is associated with DNA repair, cell apoptosis and cell survival. It has two distinct isoforms: the secreted CLU (sCLU) and nuclear CLU (nCLU). sCLU and nCLU are translated from full-length CLU mRNA and alternatively spliced CLU mRNA, respectively.
In this study, HCV nonstructural protein expression plasmids were transfected into 293 cells. RNA was isolated for quantitative PCR analysis. Results showed that expression of NS4A or NS5B alone specifically increased cellular steady-state CLU mRNA. NS4A could increase cellular sCLU mRNA but it had little influence on nCLU expression. NS4A expression plasmid was co-transfected with pCLU-luc reporter plasmid containing 801 bp of CLU promoter into 293 cells. RNA was extracted for RT-PCR analysis. The synthesis of luciferase mRNA in NS4A-transfected cells was not significantly different from that in vector-transfected cells. These data suggest that NS4A has no influence on the promoter activity of clusterin or NS4A-responsive elements are beyond the cloned 801 bp region. These results indicate that the mechanism of NS4A-mediated upregulation of CLU mRNA may be through inhibition of CLU mRNA degradation. The level of sCLU protein in cell lysates was increased, but sCLU protein in medium was decreased in NS4A-transfected 293 cells. These results suggest that NS4A may decrease sCLU stability in 293 cells. Furthermore, sCLU protein was slightly increased both in the cell lysates and the culture medium of doxycycline-induced HepG22-NS3-4A stable cells. On the other hand, important sequences of NS4A involved in the enhancement of caspase-3 activity were analyzed. Deletion mutant NS4A(1-34) lost the ability to activate caspase-3 activity, indicating that NS4A(35-54) domain is important for caspase-3 activation. Understanding the NS4A-mediated CLU regulation and the important domain of NS4A involved in caspase-3 activation will help us to elucidate the interaction between host and virus during HCV infection.

並列關鍵字

Hepatitis C virus ； NS4A ； clusterin ； caspase-3

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