TRIM5α介導的TRIMosome對EB病毒BFRF3蛋白質的穩定性調控

Abstract

EB病毒 (Epstein-Barr virus) 隸屬於皰疹病毒γ亞科，不僅會感染全球近90%的人口，亦是第一個被發現具有致癌力的人類病毒。EB病毒的生活史包含潛伏期 (Latency) 以及溶裂期 (Lytic cycle)，而其在溶裂期時會大量表現病毒基因，並產生病毒顆粒，在此過程中外鞘殼體 (Capsid) 的組裝扮演相當關鍵的角色，其中小外鞘殼體蛋白質 (Small capsid protein) BFRF3已被證實在EB病毒的外鞘殼體組裝過程中是不可或缺的。TRIM5α (Tripartite motif 5 alpha) 是一種反轉錄病毒的限制因子，在恆河猴中可以抑制愛滋病毒 (HIV-1)的感染，其具有泛素連接酶E3的活性以及作為自噬作用受器 (Autophagy cargo receptor) 的能力。先前本實驗室已發現TRIM5α可以作為EBV次外鞘蛋白質 (Minor capsid protein) BORF1的泛素連接酶E3，且會形成TRIMosome並以精準性自噬作用 (Precision autophagy) 將BORF1降解。本研究首先確認TRIM5α是透過PRYSPRY domain與BFRF3結合，然後以變性免疫沉澱分析證實TRIM5α為BFRF3的泛素連接酶E3，且TRIM5α的RING domian與其促進BFRF3上的泛素化修飾有關。本研究建構了BFRF3的全lysine突變株 (BFRF3-5KR)，結果發現BFRF3-5KR不會被泛素化，其穩定性也比BFRF3高。接下來本研究發現TRIM5α不僅會降低BFRF3-5KR的穩定性，也會與BFRF3-5KR結合，此外TRIMosome的成員Beclin-1以及p62亦會與BFRF3-5KR結合，而在加入會抑制溶酶體 (Lysosome) 活性的氯喹 (Chloroquine, CQ) 後，會提升BFRF3與p62的結合，顯示TRIM5α可能藉由形成TRIMosome以精準性自噬作用降解BFRF3。未來將進一步探討BFRF3與TRIMosome的關係，得以更了解TRIM5α限制DNA病毒的機制。

Epstein-Barr virus (EBV) belongs to the herpesvirus gamma subfamily, which not only infects nearly 90% individuals worldwide, but also is the first human virus found to be carcinogenic. EBV has two life cycles, latency and lytic cycle. EBV expresses a number of viral genes and produces infectious virions during lytic cycle. During this process, capsid assembly plays a crucial role. The small capsid protein BFRF3 has been shown to be indispensable in the assembly of EBV capsid. TRIM5α (Tripartite motif 5 alpha) is a restriction factor that inhibits Human immunodeficiency virus type 1 (HIV-1) infection in rhesus macaques. The protein acts as an autophagy cargo receptor with the activity of ubiquitin E3 ligase. Our laboratory has previously found that TRIM5α not only is the ubiquitin E3 ligase of BORF1, but also degrades BORF1 via precision autophagy by forming the platform named TRIMosome. In this study, we firstly found that TRIM5α binds directly to BFRF3 through the conserved PRYSPRY domain. TRIM5α is the ubiquitin E3 ligase of BFRF3, and the RING domain of TRIM5α is required for the ubiquitination on BFRF3. Moreover, a mutant of BFRF3 (BFRF3-5KR) was generated whose whole lysine residues were mutated. The mutant BFRF3-5KR is not ubiquitinated and its stability is higher than that of BFRF3. Overexpressing TRIM5α reduced the stability of BFRF3-5KR. Finally, this study demonstrated that BFRF3-5KR binds to TRIMosome members including TRIM5α, Beclin-1 and p62. The interaction between BFRF3 and p62 was enhanced in the presence with chloroquine, revealing that TRIM5α may destabilize BFRF3 through precision autophagy. Overall, this study affords the potentiality of TRIM5α-mediated antiviral strategy against EBV. We will further explore the relationship between BFRF3 and TRIMosome to understand the antiviral mechanism of TRIMosome in this study.