Yeh T S, Li S N, Wu C J, Liu S T, Meng C L, Chang Y S
Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Shih-pai, ROC.
DNA Cell Biol. 1997 Nov;16(11):1311-9. doi: 10.1089/dna.1997.16.1311.
Previously, we reported that the LMP 1 gene of Epstein-Barr virus (EBV) derived from nasopharyngeal carcinoma (NPC) tissues (i.e., NLMP 1 gene) was able to transform BALB/c3T3 cells. On the other hand, LMP 1 gene of B95-8 strain (i.e., BLMP 1 gene) was not able to transform these cells (Chen et aL, 1992). Further studies indicated that a 10-amino-acid deletion in the carboxyl terminus of NLMP 1 played an important role in transformation (Li et al., 1996). In this study, we tested if this 10-amino-acid deletion affected the induction of NF-kappaB activity by LMP 1. The long terminal repeat of the human immunodeficiency virus type 1 (HIV-1 LTR) contained two copies of NF-kappaB sites and was used to construct the Luc gene-based reporter plasmid, p kappaB-Luc. Plasmid p kappaB-Luc was co-transfected with plasmids containing the NLMP 1 gene, BLMP 1 gene, and their chimeric or deletion constructs, respectively, into C-33A and BALB/c3T3 cells. The activation was then measured by the luciferase activity. Results showed that the full-length proteins induced a similar level of NF-kappaB activity, the two 3' mutants (R15delta and D4delta) still induced a relatively high level of activity, and the two 5' deletion mutants (delta3058 and delta3243) of NLMP 1 gene did not show any significant activation in C-33A cells. However, none of these LMP 1 proteins induced NF-kappaB activity in BALB/c3T3 cells. Using subcellular fractionation analysis and an immunocytostaining method, the truncated proteins of delta3058 and delta3243 were detected in the cytoplasm of the cells whereas the full-length NLMP 1 protein was located at the cytoplasmic membrane. Stable BALB/c3T3 cell clones that expressed both truncated proteins were established and then their ability to induce tumors in nude mice was examined. Data showed that both truncated NLMP 1 proteins still maintained partial transformation activity. Our results suggested that there was no direct correlation between NF-kappaB activation and transformation activity of LMP 1 in BALB/c3T3 cell transformation and that the amino-terminal membrane-spanning domain was important for maintaining both functions of LMP 1.
此前，我们报道过源自鼻咽癌（NPC）组织的爱泼斯坦-巴尔病毒（EBV）的LMP 1基因（即NLMP 1基因）能够转化BALB/c3T3细胞。另一方面，B95-8株的LMP 1基因（即BLMP 1基因）则无法转化这些细胞（Chen等人，1992年）。进一步研究表明，NLMP 1基因羧基末端的10个氨基酸缺失在转化过程中起重要作用（Li等人，1996年）。在本研究中，我们测试了这10个氨基酸的缺失是否会影响LMP 1对NF-κB活性的诱导。人类免疫缺陷病毒1型（HIV-1）的长末端重复序列（LTR）含有两个NF-κB位点拷贝，被用于构建基于Luc基因的报告质粒pκB-Luc。分别将质粒pκB-Luc与含有NLMP 1基因、BLMP 1基因及其嵌合或缺失构建体的质粒共转染到C-33A和BALB/c3T3细胞中。然后通过荧光素酶活性来测量激活情况。结果显示，全长蛋白诱导的NF-κB活性水平相似，两个3'端突变体（R15δ和D4δ）仍能诱导相对较高水平的活性，而NLMP 1基因的两个5'端缺失突变体（δ3058和δ3243）在C-33A细胞中未显示出任何显著的激活。然而，这些LMP 1蛋白在BALB/c3T3细胞中均未诱导NF-κB活性。通过亚细胞分级分离分析和免疫细胞化学染色方法，在细胞的细胞质中检测到了δ3058和δ3243的截短蛋白，而全长NLMP 1蛋白位于细胞质膜上。建立了稳定表达两种截短蛋白的BALB/c3T3细胞克隆，然后检测它们在裸鼠中诱导肿瘤的能力。数据显示，两种截短的NLMP 1蛋白仍保持部分转化活性。我们的结果表明，在BALB/c3T3细胞转化过程中，NF-κB激活与LMP 1的转化活性之间没有直接关联，并且氨基末端跨膜结构域对于维持LMP 1的两种功能都很重要。
