C型肝炎病毒非結構性蛋白質NS4B對細胞基因FASTK與HRG表現之調控

Regulation of the gene expression of FASTK and HRG by the nonstructural protein 4B of hepatitis C virus

張巧伶(Chiao-Ling 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(2006年)

https://doi.org/10.6342/NTU.2006.02298

摘要

C型肝炎病毒 (Hepatitis C Virus，HCV) 是造成非A非B型肝炎的主要致病因子，屬於黃病毒科 (Flaviviridae)，具外套膜，為一單股正向RNA病毒。根據本實驗室對HCV subgenomic replicon細胞株和其母細胞株Huh7進行比較之cDNA microarray分析，以及其他實驗室比較肝癌病人癌細胞和正常細胞基因表現差異的研究結果，顯示在HCV感染時一些細胞基因的表現可能會受到調控，例如：SAA、DUSP1、GRB14、VEGF-A、integrin、FASTK、H-ras、HRG、SULT2A1等。為確認此調節作用並釐清造成此現象的病毒蛋白質，本研究利用DNA轉染的方式，分別將HCV非結構性蛋白質之表現質體送至培養細胞中表現，並以即時定量聚合酵素鏈鎖反應 (real-time quantitative PCR) 分析不同病毒蛋白質對於細胞基因表現的影響。結果發現NS4B會造成FASTK與HRG表現量的下降。NS4B為病毒非結構性蛋白質之一，分子量 27 kD，具疏水性，其在病毒生活史的功能目前尚不清楚，但有研究指出NS4B對於NS5A的磷酸化，和病毒RNA複製是重要的；另外NS4B具有GTPase活性，有抑制宿主細胞轉譯的能力，也會與Ha-ras基因共同造成細胞轉型，可能在HCV的致病機轉中扮演重要角色。已知FASTK為一survival protein，因此嘗試分析NS4B對細胞存活的影響，以西方墨點法偵測細胞中caspase3的含量，發現在受到anti-Fas抗體刺激誘導細胞凋亡的293細胞中，NS4B會使細胞凋亡的情形更為嚴重。不過在nucleosome ELISA assay和flow cytometry的分析上，NS4B對細胞凋亡的影響則不明顯。細胞凋亡為幫助病毒顆粒散播的方式之一，NS4B是否藉由細胞凋亡調節C型肝炎病毒的傳播過程，則需進ㄧ步的釐清。另一方面，HRG為一含量豐富的血漿蛋白質，主要在肝臟細胞中表現，本研究發現在表現NS4B的Huh7細胞中，HRG的mRNA量和蛋白質量均有下降的情形。將NS4B表現質體與帶有長度約450 bp的HRG promoter之luciferase reporter進行共同轉染時，發現NS4B並不會影響luciferase的活性，顯示NS4B不會藉由此段HRG promoter調控HRG mRNA的合成。其調控機制仍有待進一步的研究。

關鍵字

C型肝炎病毒 ； 非結構性蛋白質NS4B

並列摘要

Hepatitis C virus (HCV) is the major infectious agent leading to non-A, non-B hepatitis. HCV belongs to the family Flaviviridae and is an enveloped, single-stranded RNA virus. Previously, a cDNA microarray analysis with HCV subgenomic replicon and patients parental Huh7 cells was performed in our lab to identify differentially expressed genes. According to the results and data from RT-PCR analysis of hepatocytes of HCC patients by another group, it is likely that HCV proteins are involved in the regulation of expression of several genes including SAA, DUSP1, GRB14, VEGF-A, integrin, FASTK, H-ras, HRG, and SULT2A1. In this study, cultured cells were transfected with expression plasmids of HCV nonstructural proteins. RT-PCR was applied to analyze the effects of the viral proteins on the expression of the cellular genes. NS4B down-regulated the expression of both FASTK and HRG mRNA. Nonstructural protein 4B (NS4B) is a relatively hydrophobic 27 kDa protein of unknown function. Nevertheless, NS4B was demonstrated to be important for the NS5A hyperphosphorylation and replication of HCV. In addition, NS4B has a GTPase activity. It inhibits translation of host cells, and causes malignant transformation in cooperation with the Ha-ras gene. Therefore, NS4B may play an important role in the pathogenicity of HCV. FASTK was shown to be a survival protein. To understand possible effects of the HCV NS4B protein on cellular survivability, western blot analysis was performed to detect the level of caspase3 in both anti-Fas antibody-treated and untreated 293 cells. Although NS4B seems to increase the severity of cell apoptosis, only limited effect was detected by nucleosome ELISA assay and flow cytometry. Apoptosis is one of gateways for viral particle spreads, the role of NS4B involved in the apoptosis and in the process of viral diffusion need to be further elucidated. On the other hand, HRG is an abundant plasma protein. It expresses mainly in the liver tissue. In this study, NS4B was found to down-regulate HRG both at the RNA and protein levels. When NS4B expression plasmid was cotransfected with a luciferase reporter carrying a 450 bp HRG promoter, no effect on the luciferase activity was detected. These results suggested that the regulatory effect of NS4B on HRG is not through a direct binding of the NS4B protein to the 450 bp promoter sequences.

並列關鍵字

HCV ； NS4B ； FASTK ； HRG

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