TRIM5α 藉由細胞自噬作用調控 Epstein-Barr Virus 的 Rta蛋白質

Abstract

Rta 為Epstein-Barr virus (EBV) 溶裂極早期基因的產物，是溶裂期表現的轉錄因子。Tripartite-motif 5 alpha (TRIM5α) 具有泛素E3 連接酶 (ubiquitin E3 ligase)的活性， 為反轉錄病毒的限制因子 (restriction factor) ， 在免疫缺乏病毒 (immunodeficiency virus) 的物種特異性上扮演重要的角色。先前的研究發現TRIM5α 能夠促進自噬作用 (autophagy) ， 並和選擇性自噬作用 (selective autophagy) 的連接蛋白 (adaptor) sequestosome 1 (SQSTM1, p62) 直接結合，而減少TRIM5α 表現量會造成細胞自噬作用減少。根據本實驗室的研究，人類TRIM5α(huTRIM5α) 會與Rta 結合，並促進Rta 的泛素化，使Rta 被送入proteasome 進行降解，導致Rta 促使的轉錄活化作用減少。而將TRIM5α 基因抑制後，病毒顆粒的產量會上升，顯示TRIM5α 影響EBV 的溶裂循環。根據上述，本研究將證明TRIM5α 能促使Rta 被細胞自噬作用降解。首先，證明TRIM5α 和Rta 在細胞質作用，並能增加Rta 被K63-linked 泛素化修飾。由於p62 辨認待降解物質的根據之一是K63-linked 泛素化修飾，下一步便利用免疫共沈澱，發現Rta 會與p62 結合。免疫螢光染色的結果顯示Rta、TRIM5α 及p62 三者互相疊合。而利用有EBV 潛伏的TRIM5α-knockdown 細胞株進行免疫共沈澱及免疫螢光染色，發現TRIM5α 被抑制會造成Rta 與p62 的交互作用減少，證明TRIM5α 促進Rta 與p62的結合。此外，在TRIM5α 大量表現的細胞株中，Rta 能夠進入autolysosome 降解。最後，以藥物處理促使進入溶裂期的細胞進行自噬作用後，TRIM5α 未被抑制的細胞株其Rta 表現量明顯低於TRIM5α 被抑制的細胞株，顯示TRIM5α 能夠促使Rta 被自噬作用降解 。

The Epstein-Barr virus (EBV) Rta protein, known as a viral transcription activator, is one of the vital keys for activating EBV lytic cycle. Tripartite-motif 5 alpha (TRIM5α) a restriction factor with the activity of ubiquitin E3 ligase causes the species-specificity of immunodeficiency virus within primates. Previous studies have been showed that TRIM5α promotes autophagy and interacts with Sequestosome 1(SQSTM1, p62), a selective autophagy adaptor. Our recent studies indicated that Rta binds to TRIM5α and promotes ubiquitination of Rta, resulting in the suppression of Rta’s transactivation activity. Knockdown of the expression of TRIM5α also increased virion production, suggesting that TRIM5α affects the EBV lytic cycle. The aim of this study is to investigate the mechanism by which TRIM5α affects Rta function via autophagy. First of all, TRIM5α and Rta colocalize at cytoplasm. Moreover, TRIM5α promotes K63-linked ubiquitination of Rta, and K63-linked ubiquitin chain is a substrate recognaizad by p62. Furthermore, p62 interacts with Rta and TRIM5α. Inhibiting the expression of TRIM5α by lentivirus introduced P3HR1 cells reduced the interaction between p62 and Rta. Showing that TRIM5α promotes interaction of p62 and Rta. Additionally, expressing TRIM5α in the cells induces the colocalization of Rta and autolysosome. Finally, the levels of Rta are siginificantly reduced in P3HR1 cells after treatment of inducing autophagy, but not in TRIM5α-knockdown cells during the EBV lytic cycle. Overall, this study demonstrates that TRIM5α induces Rta degradation by autophagy.