Counteracting E3 Ubiquitin Ligase TRIM5α by the Deubiquitinase Activity of BSLF1 of Epstein-Barr Virus

• Main Authors: Tsun-Chieh Liang, 梁尊傑
• Other Authors: Li-Kwan Chang
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/rs565v

碩士 === 國立臺灣大學 === 生化科技學系 === 106 === 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.