| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 90 === The Rta protein is an Epstein-Barr viral immediate-early transactivator encoded by the open reading frame BRLF1. Rta plays an important role during the switch from latency to lytic cycle. To characterize the nuclear localization signal of Rta, various fragments of EBV Rta sequences were fused to enhanced green fluorescence protein and/or beta-galactosidase. Results from sequential deletions of Rta indicated that residues 401-441 was sufficient to trap GFP-tagged protein and beta-galactosidase to the nucleus of transfected HEp-2 cells. A 7-a.a. motif was identified at residues 408-414 of Rta (408PVKRKKV414) that contains high residue similarity to the classical SV40 NLS. Mutational analyses and luciferase reporter assay showed that the Rta NLS mutant (pEGFP-Rm) failed to locate in the nucleus and consequently abolished its transactivational activity to EBV DNase promoter. Interestingly, this cytoplasmic Rta can still disrupt viral latency into lytic cycle in EBV-containing epithelial cells. Endogenous BRLF1 (Rta), BZLF1 (Zta) and BMRF1 (EA-D) can be activated at the same time by either wild type or mutated exogenous Rta. Expression of BGLF5 (DNase), BXLF1 (TK) and BALF2 (major DNA binding protein) were delayed by cytoplasmic Rta. The results clearly suggested that nuclear localization is important for the transactivation function of Rta when direct DNA binding is required, but is not necessary to induce viral lytic cycle progression. The mechanisms involved require further investigation.
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