C型肝炎病毒非結構性蛋白質NS5A負調控PKR訊息傳遞下游路徑之分子機制

Molecular Mechanisms of Hepatitis C Virus NS5A Protein Involved in the Down-Regulation of the Downstream PKR Signaling Pathway

吳舜祺(Shun-Chi Wu)

指導教授 ： 張明富

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.00754

摘要

C型肝炎病毒(HCV)感染後，大部分病人會演變成永久性帶原者並且可能引發肝癌發生。C型肝炎病毒非結構性蛋白質NS5A，目前已知會影響宿主細胞的生長和存活能力。最近的研究證明，NS5A蛋白質可能異常調控細胞週期中的有絲分裂階段，造成染色體不穩定，進而引發肝癌的發生。NS5A蛋白質已知會與宿主細胞中的PKR蛋白質形成異源雙體並抑制PKR蛋白質的活性，但NS5A對有絲分裂之影響仍不清楚。在本研究中，首先在老鼠系統以流體力學為基礎之體內轉染法進行實驗。成功表現出NS5A蛋白質的老鼠肝細胞，以雷射微切擷取方式分離出來，並且使用Affymetrix寡核苷酸微陣列系統，大規模地觀察有那些宿主細胞中基因的表現會被NS5A蛋白質所影響。結果發現，在34,000個基因中，有10個與細胞生長、發炎和細胞凋零相關的基因表現會被NS5A蛋白質所影響。其中一個受影響基因為Aspm (abnormal spindle-like, microcephaly associated)。反轉錄即時聚合酶連鎖反應及西方墨點法更進一步地確認在老鼠、C型肝炎病毒複製系統及細胞培養系統中，NS5A蛋白質均會負調控Aspm基因及ASPM蛋白質的表現。使用流式細胞儀分析，血清飢餓24小時再加入血清24小時，發現持續表現NS5A蛋白質之NIH3T3細胞，其細胞週期有較多比例停滯在G2/M階段。使用nocodazole處理24小時再將其移除48小時後，發現持續表現NS5A蛋白質之NIH3T3細胞，其細胞週期在sub-G1階段的比例，比控制組細胞明顯來得少，表示NS5A蛋白質具有抗細胞凋零的作用。另外，螢光素酶報導分析法證明NS5A蛋白質抑制Aspm基因啟動子的活性，但是將NS5A蛋白質之PKR結合區域重要的三個胺基酸突變，則失去此抑制效果。因為ASPM蛋白質為有絲分裂中紡錘體蛋白質，它可能是NS5A蛋白質在細胞週期調控中，一個嶄新的標靶基因。進一步使用MAPK和PKR的抑制劑，證明抑制Aspm基因的表現，表示NS5A蛋白質抑制Aspm基因是透過PKR-p38之訊息傳遞。而且，ASPM蛋白質的過度表現，能援救NS5A蛋白質所造成寄主細胞週期停滯在G2/M階段的表現型。實驗結果表明NS5A蛋白質抑制Aspm基因的表現，導致有絲分裂中紡錘體蛋白ASPM蛋白質的減少，使得有絲分裂異常，可能進而引起染色體不穩定和肝癌的發生。

關鍵字

C型肝炎病毒 ； 非結構性蛋白質 ； 雷射微切擷取 ； 微陣列 ； 紡錘體蛋白

並列摘要

Hepatitis C virus (HCV) often causes a persistent infection and virus-associated hepatocellular carcinoma. The nonstructural protein NS5A of HCV is likely to be involved in the control of cell growth and viability. Recent studies demonstrated that HCV NS5A protein may be directly involved in the induction of chromosome instability via mitotic cell cycle dysregulation and provided insights into the molecular mechanisms of HCV-associated hepatocarcinogenesis. Although NS5A forms heterodimer with PKR and down-regulates the PKR activity, the effects of NS5A on mitosis are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by Laser Capture Microdissection and gene expression profiles were examined by Affymetrix oligonucleotide microarray system. Among 34,000 genes, 10 genes including Aspm (abnormal spindle-like, microcephaly associated) associated with cell growth, inflammation and apoptosis were differentially regulated. The down-regulation of Aspm mRNA and protein was confirmed by real-time reverse transcription-polymerase chain reaction and Western blot analysis both in the mouse model system and in the viral subgenomic replicon and in vitro transfection culture system. By flowcytometer analysis, cell cycle G2/M arrest was demonstrated in NIH3T3 cells constitutively expressing the NS5A. After release from nocodazole treatment, the sub-G1 population was significantly decreased in the synchronized NIH3T3 cells expressing NS5A as compared with that of parental NIH3T3 cells, indicating that the anti-apoptotic effect of NS5A. In addition, a luciferase reporter assay demonstrated that NS5A down-regulated the promoter activity of Aspm gene. The regulatory effect was reduced in the NS5A protein with mutation of PKR interaction domain. Because ASPM is a mitotic spindle protein, it maybe a novel target of NS5A in the cell cycle regulation. Further studies demonstrated that MAPK and PKR inhibitors down-regulated Aspm gene, indicating that NS5A down-regulates Aspm gene via the PKR-p38 signaling pathway. Moreover, overexpression of ASPM could rescue the phenotype of NS5A-inducing cell cycle G2/M arrest. Results suggest that the down-regulation of ASPM by HCV NS5A protein induces aberrant cell mitosis associated with chromosome instability and hepatocellular carcinoma.

並列關鍵字

Hepatitis C virus ； NS5A ； Laser Capture Microdissection ； microarray ； ASPM

參考文獻

42. Hijikata, M., H. Mizushima, T. Akagi, S. Mori, N. Kakiuchi, N. Kato, T. Tanaka, K. Kimura, and K. Shimotohno. 1993. Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67:4665-75.

14. Chen, C. M., L. R. You, L. H. Hwang, and Y. H. Lee. 1997. Direct interaction of hepatitis C virus core protein with the cellular lymphotoxin-beta receptor modulates the signal pathway of the lymphotoxin-beta receptor. J Virol 71:9417-26.

20. Di Bisceglie, A. M., S. E. Order, J. L. Klein, J. G. Waggoner, M. H. Sjogren, G. Kuo, M. Houghton, Q. L. Choo, and J. H. Hoofnagle. 1991. The role of chronic viral hepatitis in hepatocellular carcinoma in the United States. Am J Gastroenterol 86:335-8.

71. Matsumoto, M., T. Y. Hsieh, N. Zhu, T. VanArsdale, S. B. Hwang, K. S. Jeng, A. E. Gorbalenya, S. Y. Lo, J. H. Ou, C. F. Ware, and M. M. Lai. 1997. Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor. J Virol 71:1301-9.

7. Bond, J., E. Roberts, K. Springell, S. B. Lizarraga, S. Scott, J. Higgins, D. J. Hampshire, E. E. Morrison, G. F. Leal, E. O. Silva, S. M. Costa, D. Baralle, M. Raponi, G. Karbani, Y. Rashid, H. Jafri, C. Bennett, P. Corry, C. A. Walsh, and C. G. Woods. 2005. A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size. Nat Genet 37:353-5.