染色質重塑蛋白CHD4在EB病毒溶裂期的調控

Abstract

EB病毒（EBV）是人類第四型皰疹病毒，感染後會引起傳染性單核細胞增多癥，並與各種惡性腫瘤高度相關。EBV的生命周期分為潛伏期和溶裂期，分別具有不同的基因表現。病毒從潛伏期被激活後，病毒DNA聚合?輔助因子BMRF1會穩定DNA聚合?BALF5與DNA的結合，以促進EBV的DNA復製。此外，BMRF1作為一個轉錄調節因子，會激活幾個病毒溶裂期早期和晚期基因的表達。在我們以前的研究中，通過免疫沈澱法結合質譜分析，染色質重塑復合物NuRD的成員CHD4和RBBP4被確定與BMRF1間存在相互作用。本實驗中我們會進一步探討CHD4在EBV從潛伏期到溶裂期轉變中的調控作用。在此，我們發現利用shRNA降低CHD4的表基因現，減少了TW01-EBV和AGS-BX1細胞中EBV溶裂期蛋白質表現量。用shRNA抗性的CHD4（rCHD4）進行回補實驗，可以拯救Rta誘導的TW01-EBV中CHD4的表達水平。此外，熒光素?報導基因檢測實驗顯示，CHD4以ATP?活性依賴的方式促進了Rta介導的Zta啟動子的反式激活和Zta，Rta介導的BMRF1啟動子的反式激活。此外，CHD4以劑量依賴的方式影響Zta介導的BMRF1啟動子的轉活性。最後，Rta、Zta和BMRF1分別與CHD4免疫共沈澱。總之，這些結果表明，CHD4通過促進Zta和BMRF1的啟動子活性來支持EB病毒溶裂期的基因表現。其他NuRD成員是否參與在其中則需要進一步研究。

Epstein-Barr virus (EBV) is a gamma-herpesvirus that causes infectious mononucleosis and is highly associated with various malignancies. The life cycle of EBV is divided into latent and lytic stages with different gene expression profiles. Upon lytic reactivation, BMRF1, the viral DNA polymerase processivity factor, stabilizes the DNA binding ability of DNA polymerase BALF5 to facilitate EBV DNA replication. In addition, BMRF1 functions as a transcriptional regulator to activate the expression of several viral early and late genes. In our previous study, CHD4 and RBBP4, the components of the chromatin remodeling complex NuRD, were identified as BMRF1-interacting proteins by IP-mass spectrometry analysis. We are interested in further exploring the regulatory role of CHD4 in the EBV lytic cycle switch. Here we found knockdown of CHD4 reduced the lytic protein expression in both EBV-reactivated TW01-EBV and AGS-BX1cells. Complementation with shRNA-resistant form of CHD4 (rCHD4) rescue the expression level of CHD4 in Rta-induced TW01-EBV. Furthermore, luciferase reporter assays reveal that CHD4 contributes to Rta-mediated transactivation of Zta promoter and Zta- and Rta-mediated transactivation on BMRF1 promoter in an ATPase activity dependent manner. In addition, CHD4 affect Zta-mediated transactivation on BMRF1 in a dose-dependent manner. Moreover, Rta, Zta and BMRF1 are co-immunoprecipitated with CHD4 respectively. Taken together, the results suggest that CHD4 supports EBV lytic cycle by promoting the promoter activity of Zta and BMRF1. The role of other NuRD components in EBV lytic cycle needs further investigation.