TRIM5α 對 EB 病毒次外鞘殼體蛋白質 BORF1 的影響

Abstract

TRIM5α (Tripate motif 5 alpha) 為一反轉錄病毒的限制因子，能夠在恆河猴中限制愛滋病毒 (HIV-1) 的感染，並在人類中限制 N 型小鼠白血病病毒的感染 (N-MLV)。TRIM5α 屬於 TRIM 蛋白質家族，具有 E3 ubiquitin ligase 的活性。此外，TRIM5α 也能作為自噬作用受器 (Autophagy receptor) 直接結合目標蛋白質以調控自噬作用 (Autophagy)。近期的研究發現，除了限制反轉錄病毒之外，TRIM5α 也能限制人類第四型皰疹病毒Epstein-Barr virus (EBV) 的溶裂期發展，但是詳細的機制尚不清楚。BORF1 為 EB 病毒的外鞘殼體蛋白質 (Capsid protein)，具有入核序列 (Nuclear localization signal, NLS)，在病毒溶裂期時負責將其他外鞘殼體蛋白質 VCA、BDLF1 帶入核內完成殼體組裝 (Capsid assembly)。本研究首先確認 TRIM5α 為 BORF1 的泛素 E3 ligase，並且主要替 BORF1 修飾上 K63 鍵結的泛素化長鏈。此外，TRIM5α 無法利用蛋白酶體降解路徑降低 BORF1 的穩定性。本研究也以 BORF1 的全 lysine 突變株 (BORF1-6KR) 進行分析，結果發現 BORF1-6KR 不會受泛素化修飾，並且比 BORF1 穩定。若以氯喹 (chloroquine) 抑制自噬小體與溶酶體結合後，則能夠增加BORF1-6KR的穩定性，並且 TRIM5α 會降低 BORF1-6KR 的穩定性，而在共軛焦顯微鏡觀察下也發現 BORF1、TRIM5α 及自噬體相關蛋白質 LC3 的共點現象，顯示 TRIM5α 可能會直接結合 BORF1 將其引導至自噬體降解。此外，本研究也發現 TRIM5α 能夠增加 BORF1 的類泛素化修飾，並且能與 Ubc9 結合，顯示 TRIM5α 可能是一種 SUMO E3 ligase。綜合以上結果，本研究發現 TRIM5α 可以直接結合 BORF1 形成 TRIMosome 進行選擇性自噬作用降解 BORF1，此機制可能為 TRIM5α 限制 DNA 病毒溶裂期進程的其中一種方式，TRIM5α 也可以增加 BORF1 的類泛素化修飾，顯示 TRIM5α 作為一種 SUMO E3 ligase 的可能性，有助於更進一步了解 TRIM5α 對 DNA 病毒的限制作用機制。

TRIM5α (Tripate motif 5 alpha) is a retrovirus restriction factor that controls human immunodeficiency virus (HIV) infection in Rhesus monkey and N-tropic murine leukemia virus (N-MLV) infection in human. TRIM5α is a member of the TRIM protein family and possesses a RING domain with E3 ubiquitin ligase activity. TRIM5α is also known to be involved in selective autophagy regulation by acting as an autophagy receptor which binds the target protein directly. Recent studies also revealed that TRIM5α can restrict not only retrovirus but also DNA viruses such as Epstein-Barr virus (EBV). However, the mechanism remains elucive. BORF1 is a minor capsid protein of EBV with a nuclear localization signal (NLS), which carries EBV capsid proteins, including BDLF1 and VCA into the nucleus to facilitate capsid assembly. This study confirms that TRIM5α is a ubiquitin E3 ligase that promotes mainly the K63-linked polyubiquitination of BORF1 and does not affect the proteasomal degradation of BORF1. Mutation of all the six lysine residues on BORF1 (BORF1-6KR) abrogated its ubiquitination and increased the stability of BORF1. The amounts of BORF1-6KR were accumulated after chloroquine treatment although the half-life of BORF1-6KR was reduced if TRIM5α is present. Indirect immunofluorescence showed that TRIM5α, autophagy-related protein LC3, and BORF1 or BORF1-6KR colocalized as speckles in the cytoplasm. These results suggest that BORF1 is a substrate of TRIMosome which undergoes selective autophagy mediated by TRIM5α via direct recognition. The destabilization of BORF1 by TRIM5α suggests that TRIM5α interferes with EBV capsid assembly during lytic development. In addition, this study discovered that TRIM5α binds to SUMO E2 Ubc9 and increases the SUMOylation levels of BORF1, suggesting that TRIM5α may function as a SUMO E3 ligase. Overall, this study demostrated that TRIM5α promotes the selective autophagic degradation of EBV minor capsid protein BORF1 and showed the potentialitiy of TRIM5α serving as a SUMO E3 ligase, providing new aspects of TRIM5α-mediated DNA virus restriction strategy.