研究病毒寄主範圍基因CP77調控牛痘病毒生長的機制

牛痘病毒（vaccinia virus, VV）是一種痘病毒，整個病毒的生命週期是在宿主細胞的細胞質中完成。DNA 的基因體（genome）約有192Kb，可以表現出263個病毒蛋白。在多數的細胞中，病毒感染細胞之後會產生病毒後代;然而，在少數細胞中，病毒的生命週期被中止在post-entry的階段，顯示在特定的細胞株中，會有宿主限制的現象產生。對於牛痘病毒來說，在此病毒的其中一種會有宿主限制的細胞株CHO中，牛痘病毒要靠一個宿主範圍基因(host range gene) CP77來完成病毒的生命週期。我的論文主要著重於了解CP77在宿主限制的調控中所扮演的角色。我研究主題的第一部份是要探討CP77在結構及功能上的相關性。我們構築了能表現不同CP77截短片段的重組牛痘病毒，並且在HeLa及CHO細胞中監控這些截短蛋白的表現及測量其穩定性。實驗結果顯示，N端及C端具有保護作用，能在病毒感染的細胞中維持CP77蛋白的穩定性。第二部分是在研究CP77的後修飾轉譯作用。我們發現在病毒感染的HeLa及CHO細胞中，CP77都被ubiquitinated。並且CP77在細胞外也被sumoylated。最後，第三部份，根據之前我們實驗室的酵母菌雙雜交篩選實驗分析結果顯示，一個細胞內蛋白cytidine deaminase (CDA)會與CP77交互作用。在我研究報告中指出，CP77 以及CDA的確在GST-pull down實驗中有交互作用，顯示CDA可能參與調控牛痘病毒宿主限制。

關鍵字

牛痘病毒； CP77

並列摘要

Vaccinia virus (VV) is a poxvirus that replicates in cytoplasm of the host cells. The DNA genome of vv is 192 Kb in size and encodes 263 viral proteins. In most cells, VV infection leads to production of virus progenies; however, in few cell types, virus life cycle was blocked at post-entry step, indicating the presence of host restriction in specific cell types. In one of such restricting cell line, CHO, VV replication requires a host range (hr) gene CP77 to complete life cycle. My thesis focuses on understanding the role of CP77 in host restriction regulation. The first part of my project is to investigate the structural and functional relationship of CP77. We construct recombinant VV expressing different CP77 deletions, monitor deletion protein expression and measure protein stability in HeLa cells and CHO cells. The results showed that the N-and C-terminal regions have protection effect, maintaining CP77 stability in virus-infected cells. The second part is to investigate the post-translational modifications on CP77. We found that CP77 is ubiquitinated in both HeLa and CHO virus-infected cells. Besides, we also showed that CP77 sumoylated in vitro. Finally, the third part, our previous data indicated that a cellular protein cytidine deaminase (CDA) interacts with CP77 in yeast two-hybrid analysis. I have showed that indeed CP77 and CDA interact in GST-pull down experiments, indicating a role of CDA in vaccinia virus host restriction regulation.

並列關鍵字

vaccinia virus ； CP77

參考文獻

1. Cascalho, M. 2004. Advantages and disadvantages of cytidine deamination. J Immunol 172:6513-8.

2. Chung, C. S., I. A. Vasilevskaya, S. C. Wang, C. H. Bair, and W. Chang. 1997. Apoptosis and host restriction of vaccinia virus in RK13 cells. Virus Res 52:121-32.

3. Chung, S. J., J. C. Fromme, and G. L. Verdine. 2005. Structure of human cytidine deaminase bound to a potent inhibitor. J Med Chem 48:658-60.

4. Drillien, R., F. Koehren, and A. Kirn. 1981. Host range deletion mutant of vaccinia virus defective in human cells. Virology 111:488-99.

6. Gerber, A. P., and W. Keller. 2001. RNA editing by base deamination: more enzymes, more targets, new mysteries. Trends Biochem Sci 26:376-84.

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研究病毒寄主範圍基因CP77調控牛痘病毒生長的機制

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