Structural analysis of the Epstein-Barr virus lytic transactivator Rta

• Main Authors: Hsiang-Ling Li, 李湘鈴
• Other Authors: 詹迺立
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/5m9sks

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 106 === Epstein–Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is one of the most common human viral pathogens. Primary EBV infection usually begins in the oral cavity. The main target cells are CD21+ B cells and epithelial cells. EBV infection is the cause of infectious mononucleosis (glandular fever) and is associated with various cancers, including Hodgkin''s lymphoma, Burkitt''s lymphoma, gastric cancer, and nasopharyngeal carcinoma. Upon entering a host cell, EBV may adopt the latent or lytic life cycle. Although EBV predominantly establishes latency in host cells, a few lytically-infected cells could be carcinogenic through the release of growth factors and oncogenic cytokines. Therefore, to better appreciate the risks associated with EBV infection, we need to understand how the virus switches from latency to the lytic cycle in a process termed EBV reactivation. The viral activation can be divided into three phases: Immediate early (IE), Early (E) and Late (L). The viral IE genes BZLF1 and BRLF1 are first transcribed to produce two transactivators, Zta and Rta, which are required for EBV genome replication. These proteins can act synergistically or independently to trigger EBV reactivation. Rta may regulate transcription via three mechanisms of action. Rta may autonomously activate the expression of a certain set of genes, such as BMLF1 which encodes a mRNA export factor. Moreover, Rta and Zta may act in synergy to activate some lytic cycle genes, such as BMRF1. Furthermore, Rta may interact with specificity protein (Sp1) through an intermediary protein, MCAF1, to form a regulatory complex on Sp1-binding sites. Although both Rta and Zta act as transcriptional activators, they exhibit distinct functional and DNA-binding properties. So far, only Zta is biochemically and struturally characterized, much less regarding Rta is known. Because lytic-induction therapy in tumor cells are being regarded as a potential therapy for EBV-positive tumors, and the Rta-mediated reactivation of EBV is the main cause of viral transmission, it would be helpful to understand the function of Rta in greater detail. We aim to determine the crystal structures of Rta, either alone or in complex with the EBV gene promoter element to elucidate its function. Toward this goal, we have successfully expressed 6x His-tagged Rta in BL21-star cells, and the recombinant protein may be purified by a combination of Nickel, heparin and gel filtration column. Purified Rta is homogenous and exists as a homodimer, corresponding to the expected configuration. Electrophoresis mobility shift assay (EMSA) indicates purified Rta may associate with an DNA duplex containing the BMLF1-RRE sequence. Crystallization trials for the Rta dimer and Rta-DNA complex are currently underway. A preliminary crystallization condition for Rta-DNA complex has been identified. This condition will be systematically optimized to produce crystals of higher quality for subsequent crystallographic analysis.