I. C型肝炎病毒非結構性蛋白質NS5A對Ras-ERK訊息 傳遞路徑及下游分子的影響 II. C型肝炎病毒非結構性蛋白質NS4A與真核細胞轉譯 延伸因子的相互作用關係

I.The Effects of Hepatitis C Virus NS5A Protein on Ras-ERK Signaling Pathway and Its Downstream Molecules II.The Interaction between Hepatitis C Virus NS4A Protein and eEF1A

張雅姿(YA-TZU CHANG)

指導教授 ： 張鑫

國立臺灣大學/醫學院/微生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01758

摘要

C型肝炎病毒 (Hepatitis C virus) 為一單股正向RNA病毒，會感染人類造成C型肝炎。約有85% 的感染者會轉變成慢性肝炎，伴隨肝硬化的併發症，甚至導致肝細胞癌 (hepatocellular carcinoma)，臨床上多以干擾素合併ribavirin來治療患者。由於目前仍無法利用細胞培養的系統培養出C型肝炎病毒，對其致病機轉的了解還不十分清楚。近幾年的研究指出，HCV的非結構性蛋白質NS5A是造成干擾素抗藥性的可能因子之一；NS5A也會與細胞內的訊息蛋白質結合，干擾細胞内的訊息傳導路徑，例如MAPK (mitogen activated protein kinase) 與TNF-α(tumor necrosis factor-α) 的傳導路徑。MAPK傳導路徑可以調控細胞的生長與分化，而Ras-ERK 為其中的一條訊息傳遞路徑。在本實驗中利用受doxycycline調控表現NS5A的HeLa-tetoff-NS5A細胞株與HepG2-teton-NS5A細胞株，探討NS5A對Ras-ERK訊息傳遞路徑以及下游分子的影響。在HepG2-teton-NS5A細胞株中，當NS5A被誘導表現時，ERK1/2磷酸化的情形會受到抑制，但上游分子MEK1/2的磷酸化與MKP1蛋白質表現量皆不受NS5A影響。以流式細胞儀分析HepG2-teton-NS5A細胞株時，發現誘導NS5A表現並不會對細胞周期造成影響。因此，NS5A如何抑制ERK磷酸化以及ERK磷酸化減少後對細胞生長的影響仍需進一步探討。

目前已知HCV非結構性蛋白質NS4A為NS3的輔助因子 (cofactor)，NS4A會與NS3結合，增進NS3在serine protease的切割作用，對於HCV的非結構性蛋白質的成熟扮演重要的角色。本實驗室先前利用GST pull down的方法發現GST-NS4A與eEF1A有結合作用，更進一步利用活體外轉譯系統發現eEF1A是利用羧基端與 GST-NS4A結合。此外，最近的研究顯示NS4A會抑制蛋白質的合成，但其作用機制則不明瞭。本研究利用免疫沉澱，觀察細胞內兩個蛋白質互相作用的情形。結果發現NS4A會與細胞內全長的eEF1A互相結合，但卻無法與eEF1A氨基端(1-240)或羧基端(201-462)的次功能區域結合。由定點突變的實驗，發現NS4A胺基酸28的位置由arginine變異成alanine時會破壞其與eEF1A之間的結合能力。是否NS4A對細胞內蛋白質合成的抑制是因其與eEF1A結合導致，以及eEF1A在HCV複製時所扮演的角色，則需作進一步的探討。

關鍵字

C型肝炎病毒 ； 非結構性蛋白質 ； 轉譯延伸因子 ； 訊息傳遞路徑

並列摘要

Hepatitis C virus (HCV) is a positive, single-stranded RNA virus. Human infected by HCV causes type C hepatitis. Approximately 85% of persons infected with HCV develop chronic hepatitis and may lead to hepatocellular carcinoma. Because there is no efficient cell culture system for HCV infection and replication, the molecular mechanism of HCV pathogenesis is not well understood. NS5A is a non-structural protein of HCV. It participates in interferon resistance. NS5A has been shown to interact with a variety of cellular signaling proteins and perturb the signal transduction pathways such as MAPK and TNF-a signaling pathways. Ras-ERK is a MAPK signaling pathway involved in regulation of cell proliferation and cell growth. In this study, doxycycline-regulated HeLa-tetoff-NS5A and HepG2-teton-NS5A cell lines were used to examine the effect of NS5A on Ras-ERK signaling pathway and its downstream molecules. The induction of NS5A in HepG2-teton NS5A cells markedly reduced the level of phosphorylated ERK1/2, but no significant difference was detected on the level of phosphorylated MEK1/2 and the expression of MKP1. Flow cytometry analyses indicate that the NS5A has no significant effect on the regulation of cell cycle. Therefore, the mechanisms of NS5A involved in the down-regulation of ERK pathway and cell growths remain to be elucidated.

The HCV non-structural protein NS4A interacts with NS3 and is a cofactor of NS3 serine-type protease essential for the proteolytic processing of the viral polyprotein. By performing GST pull down assay, our laboratory has previously identified eEF1A that specifically interacted with GST-NS4A. In combination with in vitro translation system the C-terminal domain of eEF1A was identified to be involved in the interaction. NS4A was recently reported to inhibit protein synthesis, but the mechanism remains unclear. In this study, the interaction between NS4A and eEF1A was confirmed by immunoprecipitation assay following cotransfection of plasmids ecoding NS4A and eEF1A into culture cells. NS4A interacted with full length eEF1A, but not the N-terminal (amino acid 1-240) or C-terminal (amino acid 201-462) eEF1A. Furthermore, a mutation at amino acid Arg-28 disrupts the interaction between NS4A and eEF1A. Whether NS4A interferes with protein synthesis by forming complex with eEF1A and what’s the role of eEF1A on HCV replication remain to be elucidated.

並列關鍵字

NS5A ； signaling pathway ； eEF1A ； NS4A ； HCV

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