EB病毒BSLF1蛋白質的去泛素酶活性與E3泛素連接酶TRIM5α的拮抗

Abstract

Epstein-Barr virus (EB病毒) 又稱人類皰疹病毒第四型，隸屬於人類皰疹病毒科，是一種感染人類鼻咽上皮細胞及B淋巴細胞的致癌病毒。宿主細胞為了抵抗病毒，會透過胞內蛋白質對入侵病毒蛋白質進行修飾與降解，進而抑制病毒感染。在先前的研究發現，宿主細胞中的E3泛素連接酶TRIM5α與EB病毒顆粒的產生有關，並會將EB病毒溶裂期的極早期蛋白質Rta及外鞘蛋白質BORF1泛素化，此修飾會進而影響到EB病毒的基因表現與病毒組裝，使病毒生產效率下降。然而先前研究發現，作為引子合成酶的EB病毒溶裂期早期蛋白質BSLF1具有去泛素化酶的活性，並能將Rta與BORF1蛋白質去泛素化，顯示BSLF1除了參與EB病毒基因複製外甚至能抵禦宿主細胞對病毒蛋白質進行的修飾。因此，本研究對BSLF1是否與TRIM5α彼此之間具有拮抗關係進行驗證。首先利用免疫螢光染色法及GST pull-down分析證明BSLF1與TRIM5α之間的結合。接著以變性免疫沉澱法證實了BSLF1會去除TRIM5α在Rta及BORF1上增加的泛素化修飾。但在冷光報導基因分析實驗中則發現BSLF1並不能使Rta被TRIM5α抑制的轉錄活性恢復，顯示BSLF1與TRIM5α並非在抑制Rta的轉錄活性上具有拮抗關係。而在分析BORF1蛋白質穩定性後，發現BSLF1會延長其半衰期，但卻無法使BORF1蛋白質的表現顯著增加，顯示BORF1與Rta上的泛素化修飾可能具有除了穩定性與抑制轉錄活性外的影響，因此本研究進一步檢驗BSLF1與TRIM5α彼此拮抗的泛素化修飾形式，結果發現BSLF1主要去除BORF1上K63鏈結多泛素鏈。綜上所述，本研究發現BSLF1會拮抗TRIM5α對於Rta及BORF1的泛素化修飾，並能夠使Rta和BORF1出現修飾型態上的改變，雖然不同修飾型態對EB病毒生活史的影響有待進一步研究，但病毒與宿主轉譯後修飾之間的拮抗確實存在，並可能作為病毒抵禦宿主細胞抑制作用的重要機制。

EBV, also known as Human Herpesvirus 4 (HHV-4), is a member of Herpesviridae. EBV infects epithelial cells and B cells, since its association with several cancers, it is known as an oncovirus. To protect themselves from infection by viruses, host cells usually degrade viral proteins to inhibit viral replication. Earlier research showed that TRIM5α, an E3 ubiquitin ligase, is important to EBV virion assembly. Also, it has been found that immediate-early protein Rta and late protein BORF1, both important in replication of EBV, are ubiquitinated by TRIM5α. Ubiquitination of Rta and BORF1 affect EBV DNA replication and viral capsid assembly, which decrease efficiency of virus production. In a previous study, BSLF1 protein of EBV was found to have deubiquitinase activity although it was widely known as a primase. BSLF1 is able to deubiquitinate Rta and BORF1, suggesting that BSLF1 plays a role in defending host modification. The aim of this study is to elucidate the antagonism between BSLF1 and E3 ubiquitin ligase TRIM5α. First, GST pull-down assay and immunofluorescence analysis revealed that BSLF1 interacts directly with TRIM5α. To examine the deubiquitination of Rta and BORF1 by BSLF1, I used denature immunoprecipitation and demonstrated that BSLF1 deubiquitinates Rta and BORF1 that are ubiquitinated by TRIM5α. Moreover, this study found that overexpressing BSLF1 decreased the transcriptional activity of Rta in a transient transfection assay. By using a HEK293T cell clone that expresses BORF1, this study found that overexpressing of BSLF1 increases the half-life of BORF1, but overexpression of BSLF1 did not rescue the levels of BORF1 in cells. This study also showed that deubiquitination of Rta or BORF1 does not influence the transactivation activity or increase the stability. Therefore, I further investigated which types of poly-ubiquitin chain did BSLF1 counteract against TRIM5α. Through expressing of K63-only or K48-only types of HA-Ub, this study found that although TRIM5α added both K48 and K63 poly-ubiquitin chains to BORF1, BSLF1 mainly deubiquitinated K63 poly-ubiquitin chain. Together, this study reveals BSLF1 deubiquitination activity and the antagonistic relationship between BSLF1 and TRIM5α. Although more research on how different types of poly-ubiquitin chain affect EBV life cycle is needed, the counteraction between EBV viral protein and host’s post-translational modification is likely important for EBV to escape from inhibition of host cells.