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研究生:劉武明
論文名稱:EB病毒潛伏膜蛋白LMP1的轉抑制及轉活化功能
論文名稱(外文):The Transrepression and Transactivation Functions of EBV Latent Membrane protein 1
指導教授:王萬波
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:102
中文關鍵詞:EB病毒潛伏
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EB病毒 (Epstein-Barr virus,簡稱 EBV)為一種人類月太疹病毒(heroes virus),是含有外套 (envelope) 的正二十面體病毒,具有雙股線狀 (double-stranded, linear) DNA。EB 病毒一旦感染人體,即會終生潛伏於患者體內,目前已知和 EB 病毒感染有關的疾病有傳染性單核球增多症 (infectious mononucleosis),T 細胞淋巴瘤 (T cell lymphoma),慢性疲憊症候群 (chronic fatigue syndrome),淋巴上皮細胞瘤 (lymphoepithelioma),類風濕性關節炎 (rheumatoid arthritis),巴氏淋巴瘤 (Burkitt''s lymphoma) 及鼻咽癌 (nasopharyngeal carcinoma)等等多項疾病;由於鼻咽癌常見於中國南方,且為台灣地區十大癌症死因之一,故須對其致病因子加以研究探討。
EB 病毒轉錄的 LMP1 具有使不含 EB 病毒之 B 淋巴瘤細胞生長表現型改變的能力。另外,由於 LMP1 是鼻咽癌組織中少數表現的 EBV 蛋白之一,因此一般認為 LMP1 在 EB 病毒轉形鼻咽部上皮細胞的過程中,擔任主要的角色。
為了瞭解 LMP1 在上皮細胞中是否具有基因調控的功能,我們將 LMP1 表現質體及含有不同啟動子融合到 CAT 基因之報告質體,共同轉染至人類子宮頸部之上皮腫瘤細胞,C33A 細胞中。結果發現 LMP1 會抑制許多細胞及病毒啟動子的活性。同時,LMP1也可以活化 k B-TATA 啟動子。在 RHEK 及 HEp-2 細胞中,LMP1也同樣地具有轉抑制與轉活化的功能。而且,我們亦發現,抑制的程度隨 LMP1 的濃度而改變,即當我們利用較弱的啟動子表現LMP1時(例如:metallothionine 啟動子),其抑制能力會大大降低。
藉著使用 LMP1 突變株的分析,我們可以界定出 LMP1 的轉抑制與 NF-kB 轉活化的功能區。雖然結果顯示 LMP1 的穿越膜區域與 C 端區域對轉抑制與 NF-KB 轉活化都是需要的,但我們相信,LMP1的 C 端是傳遞轉抑制與轉活化訊息的重要區域,而穿越膜區域則負責讓 LMP1蛋白位處於正確的作用位置。由於功能區的相似,說明了 LMP1 的抑制與活化功能可能十分相關。為了瞭解 LMP1的抑制機轉,我們嘗試在 c-fos 啟動子上定位出 LMP1 的反應區域,我們發現,只含有 c-fos 啟動子上游 72bp 的報告質體,仍然能被LMP1所抑制,說明了 LMP1 的反應區可能住在 -72bp 的下游。最後,我們的結果顯示,LMP1 的轉抑制功能可以被 Bcl-2 或 BHRF1所回轉,目前此回轉效應的機制未明。
Epstein-Barr virus (EBV) is an enveloped herpes virus which contains a linear double-stranded DNA. After infects cells, it usually establishes latency in the cells. EBV has been reported to be associated with many diseases, including T-cell lymphoma, Burkitt''s lymphoma, chronic fatigue syndrome, lymphoepithelioma, rheumatoid arthritis, and nasopharyngeal carcinoma (NPC) etc. . Since NPC is one of the ten leading cancers in Taiwan, the elucidation of its mechanism in causing the disease thus becomes very important.
The Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) have previously been shown to cause EBV-negative B-Lymphoma cells to grow in large clumps and to alter expression of surface activation and adhesion molecules. It contributes to the immortalizating activity of the virus in human primary B lymphocytes. Since LMP1 is one of the few EBV proteins expressed in NPC tissues, we believed that LMP1 must play an important role in the carcinogenesis of NPC.
To study whether LMP1 have a regulatory function in epithelial cells, we cotransfected a reporter plasmid which contain various promoters fused to the CAT gene together with the LMP1 expression plasmid into C33A cells, a human cervical epidermoid carcinoma cell line. We found that LMP-1 can specifically inhibit the activities of many cellular and viral promoters while activate the activity of a synthetic promoter that contained five K B enhancer elements. This repression and NF-kB activation functions of LMP1 were observed not only in the C33A cells but also in the RHEK and HEp-2 cells. Moreover, we also found that the degree of repression was dependent on the concentration of LMP1. When LMP1 was expressed from a weak promoter (e.g. metallothionine promoter), its repression activity was greatly reduced.
By using various LMP1 mutants, we were able to locate the regions of LMP1 that are involved in the repression and NF-kB activation functions. Although both transmembrane and C-terminal regions of LMP1 are both required for proper repression and NF-kB activation functions of LMP1, we believe that the C-terminal region is the most important domain that transduces both repression and activation signals while the transmembrane region is required for proper localization of the LMP1 protein. The involvement of similar domain in both repression and NF-kB activation functions indicated that these two functions of LMP1 are closely related. To study the repression mechanism of LMP1, we have tried to locate the response elements within the c-fos promoter that might mediate LMP1 repression function. We found that the c-fos promoter mutant that contains only 72 bps upstream from the transcriptional start site could still be repressed by LMP1, mdicating that the response elements that mediate repression was located downstream from -72. Finally we showed that the repression function of LMP1 could be blocked by cotransfecting a plasmid that can express either Bcl-2 or BHRF-1 protein. The mechanism underlying this interesting effect is not clear at this moment.
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