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潛伏性膜蛋白(LMP1)是EB病毒所複製出來的一種致癌蛋白。在我們早先 的研究報告顯示來自鼻咽癌(NPC)組織的潛伏性膜蛋白(NLMP1)具有將BALB/c3T3細胞轉型的能力，而來自B95-8細胞株的潛伏性膜蛋白(BLMP1)則不然。進一步的研究指出這個膜蛋白基因3'端30個核甘酸的刪除對於細胞的轉型扮演相當重要的角色。本實驗中方當試探討遣兩種潛伏性膜蛋白及其混種(Chimera)對於活化NF-KB的能力。昔人將含有兩個NF-KB結合位的第一型人類免疫不全病毒(HIV typeⅠ)之LTR片段，插入不帶起動子活性的載體(pGL-2-Basic)中，構築了pKB-Luc質體。將其興LMP1衍生而來的Chimera或短缺蛋白共同轉染C33A細胞，藉由螢光酵素的活性(Luciferase activity)間接得知NF-KB被活化的程度。結果顯示兩個N端短缺的NLMP1蛋白對於．NF-KB活化的程度，相對於完整的NLMP1而言，大大的降低了許多。甚至於接近基底值。而兩個C端短缺的NLMP1及BLMP1，仍舊保有60%及80%的NF-KB活性。至於來自兩種不同病毒株的LMP1及它們的Chimera，彼此間並無明顯差異。為了探究這些不同大小的NLMPl蛋白在細胞內表現的位置，甚或是位置的差異性與NF-KB活性的啟動及致腫瘤能力之間有否關聯性，於是我們將這些會表現不同大小的NLMP1蛋白的基因，與含有neo基因的質體共同轉染BALB/c 3T3細胞。將這些用G418篩選出來的細胞株打到裸鼠皮下，藉此觀察形成腫瘤的能力。實驗結果顯示這兩個N端缺失的NLMP1蛋白仍具有稍弱的致腫瘤活性。利用免疫染色法，吾人亦觀察到這兩種N端缺失的NLMP1蛋白主要分佈在細胞質中，而完整的NLMP1則主要表現於細胞膜上。而利用次細胞分離法觀察所得的結果和免疫染色的結果是一致的。從以上的實驗得知LMP1所誘發的這兩種生物活性表現，兩者間並無任何關聯性存在。而 N端之transmembrane domain 對LMP1所引起的生物活性之完整與否，亦扮演了重要的角色。
Latent membrane protein 1 (LMP1) is an oncoprotein encoded by Epstein-Barr virus (EBV). Previously this laboratory had reported LMP1 derived from nasopharyngeal carcinoma (NPC) tissue (NLMP1) was able to transform BALB/c 3T3 cells. On the other hand, LMP1 of B95-8 strain (BLMP1) was not (Chen et al., 1992). Further studies indicated that a 30-bp deletion in the 3'terminus of the gene played an important role in the transformation activity (Li et al., 1996). In this study, we tested the ability of these two genes and their chimeric constructs in activation of NF-KB activity. The long terminal repeat (LTR) of the human immuno-defiency virus type I (HIV-1) contained two copies of NF-kB binding sites and was inserted in a promoterless luciferase gene plasmid, pGL-2- Basic. This construct was then co-transfected with plasmids containing genes coding for the NLMP1, BLMP1 or their chimeras into human epithelial cell line, C33A. The activation of NF-KB activity was determined by the luciferase activity. Results showed that the level of stimulation of NF-KB-mediated transcription by these constructs was similar. Two 3'deletion mutants (truncation of 150 aa from NLMP1 and 155 aa from BLMP1) still retained a high level of stimulation. We then further tested the activation of NF-KB activity by two 5'deletion NLMP1 mutants that encode two truncated proteins, tr-LMP1 and str-LMP1 (Chen et al., 1995). Results showed that the NF-KB activation by both proteins dropped to the basal level as compared to that by the full length protein. To correlate the NF-KB activation with the transformation ability, the plasmids which contained genes for tr-LMP1 and str-LMP1 were co-transfected with a neo-containing plasmid into BALB/c 3T3 cells. G418 cell clones were then tested for their ability to induce tumors in nude mice. Data showed that both tr-LMP1 and str-LMP1 still maintained partial transformation activity. The cellular localization of these three LMP1 proteins were determined by the immunofluroescence method and the subcellular fractionation analysis. Data indicated the LMP1 was detected at the cytoplasmic membrane, whereas two truncated proteins were mainly located in the cytoplasm of the cells. Our results suggested that there is no correlation between the transformation ability and stimulation of the NF-KB activity. However, the N-terminal amino acids including the membrane-spanning domains were important for maintaining the full functions of LMP1.
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