Rta regulates LMP1 expression during EBV lytic cycle

• Main Authors: Heng-Huan Lee, 李恆煥
• Other Authors: Ching-Hwa Tsai
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/02206025771379004947

碩士 === 國立臺灣大學 === 微生物學研究所 === 92 === Latent membrane protein (LMP)-1 is an Epstein-Barr virus latent protein and expressed dominantly in cytoplasmic membrane. Functionally, LMP1 is like a constitutively active CD40, which is a member of tumor necrosis factor receptor family. The oncogenic ability of LMP1 has been demonstrated by rodent fibroblasts transformation. As an EBV latent gene, all the studies about LMP1 gene regulation have been concentrated during the viral latency. However, little is known about the LMP1 gene regulation during the lytic cycle even though few studies indicated that the expression of LMP1 can be up regulated when EBV is entering into the lytic cycle. In our lab, ten sub clones have been obtained from 293A cells, which are 293 cells infected with a recombinant Akata strain of EBV. They can be classified into two groups: one is latent type, which only expresses EBNA1 protein, and the other is lytic type, which expresses lytic proteins. It is of interest that LMP1 are profoundly detected in five lytic clones but not in latent clones. We have further confirmed that lytic cycle progression is required for LMP1 expression by the Zta-targeted siRNA (si-Z1) blockage approach. Using different EBV-carrying epithelial cell lines, it is found that the induction amounts of LMP1 are more abundant in Rta-triggered lytic cycle than those in Zta-triggered lytic cycle. Furthermore, the results from transfection assay and si-Z1 blockage approach demonstrate that Zta is not required for Rta-mediated LMP1 induction, in our assay system. Data from Northern blotting assay demonstrate that LMP1 products can transcribe either from ED-L1 or from TR-L1 promoter in different epithelial cell lines. In addition, the results of luciferase reporter assay tell us that Rta can activate both LMP1 promoters in the absence of other viral genes. Experiments using a series of deletions on TR-L1 promoter indicate that the nucleotide sequences 170108 to170164 in TR-L1 are required for Rta activation. Also, the Western blot and luciferase assay using various of N-terminal, C-terminal deleted and nuclear localization signal (NLS)- mutant Rta constructs hint that transactivation domain, dimerization and NLS of Rta protein are all required for Rta-mediated LMP1 promoter activation. Finally, we also demonstrate Rta-initiated lytic cycle induces more abundant LMP1 expression than Zta in B cell lines, suggesting that Rta may also regulate LMP1 expression in B lymphocytes system. In this study, the new view for LMP1 regulation is explored, and the possible function of LMP1 expression during the lytic cycle will be discussed.