EB 病毒轉活化子Rta 會透過蛋白質激酶C-delta 促進細胞凋亡

Abstract

Epstein – Barr virus (EBV) R transactivator (Rta)是EBV進入裂解期時所表現的特早期蛋白 (immediate-early protein)。1999年，Swenson等人發現在Rta表現的人類纖維母細胞 (normal human fibroblast, NHF)及人類子宮頸癌上皮細胞株 (HeLa)中觀察到細胞凋亡現象的發生。此現象在本實驗室亦可觀察到，Rta蛋白之表現可促進死亡細胞增加。因此本研究希望探討EB病毒特早期活化子 Rta是否會導致細胞凋亡，並進一步了解其發生機轉及對於EB病毒複製與致病過程的重要性。 首先，我們在HeLa或HEp2細胞株中，證實Rta的確可以造成細胞凋亡。其次，在有Rta表現的情況下，則發現Rta可以使PKC-δ的cleavage form 上升，促使PKC-δ活化，進而導致細胞凋亡。接下來，利用Rottlerin或是 PKC-δ dominant negative突變蛋白可防止Rta所造成的細胞凋亡現象，證實Rta需要透過PKC-δ促成細胞凋亡。另外，我們也發現，不論是在pRTS15的細胞轉染實驗中，或是在EB病毒進入裂解期的NA細胞株中，皆可觀察到PKC- δ從細胞質進入細胞核中的量有增加的趨勢。進一步在免疫螢光染色的觀察及雙向免疫沉澱的實驗中，我們證明了Rta會和PKC-δ結合。除此之外，我們也發現抑制細胞凋亡並不會抑制EB病毒裂解期 (lytic cycle)的發生，而抑制PKC-δ的活性卻會抑制裂解期的進行。我們推論：PKC-δ也許是Rta促進EB病毒進入裂解期及導致細胞凋亡訊息傳遞發生的上游蛋白質。

Transactivator, Rta, encoded by the Epstein-Barr virus (EBV) immediate -early gene, BRLF1, mediates the switch from latency into lytic cycle. In 1999, Swenson et al observed that Rta expression can induce cell apoptosis in normal human fibroblast (NHF) and human cervical carcinoma cell line (HeLa). This phenomenon also can be observed in our laboratory. According to these findings, we intended to investigate whether Rta contributes to cell apoptosis in the human epithelial cells, and whether apoptosis is important to EBV lytic cycle progression. At first, EBV Rta induced cell apoptosis could be demonstrated in HeLa and HEp2 cell lines. Secondly, Rta increased cleavage form of PKC-δ could be detected in the presence of Rta. Using rottlerin or PKC-δ dominant negative mutant to inhibit cellular PKC-δ activity, the phenomena that Rta induced cell apoptosis was reduced. Additionally, PKC-δ translocated from cytosol to nucleus was observed both in pRTS15 transiently transfected HeLa cells or trichostatin A (TSA) induced NA cells. Co-immunoprecipitation assay and immunofluorescent staining demonstrated the interaction between Rta and PKC-δ. Furthermore, the EBV lytic cycle progession was suppressed by using PKC-.δ inhibitor, rottlerin. However, EBV lytic cycle was not affected by treating cell with caspase inhibitor, Z-VAD-Fmk. Taken together, these studies suggest that PKC-δ is the upstream protein of Rta-mediated apoptotic signaling pathway and EBV lytic cycle progression induced by Rta.