Abstract
EBNA1 (EBV Nuclear Antigent 1) is a critical EBV protein responsible for the maintenance of viral episome in host cell and for transactivation of latent viral genes. It is the only viral latency protein expressed in different types of EBV latency and is required in trans for the stable replication of viral genome. Hence, suppressing the normal functions of EBNA1 might be the key to alleviate or even eliminate EBV-associated diseases. It is conceivable that in the absence of EBNA1, EBV genome in the host cell may be lost during DNA replication and cell division. Therefore, this thesis mainly focuses on designing a protein molecule to target EBNA1 and at the same time to inhibit EBNA1’s ability to replicate, transactivate or segregate EBV genomic DNA.Our approach to design such a molecule is to fuse a strong transcriptional repressor to EBNA1. As EBNA1 functions as a dimer, it contains dimerization domain for intermolecular interaction, the dimerization domain may be manipulated to target endogenous, wild type EBNA1. On the other hand, a potent transcription repressor, KRAB domain, may be used to inhibit transactivation activity of a protein. By introducing the KRAB domain to EBNA1, a chimeric protein EBNA1-KRAB may dimerize with EBNA1 to form heterodimer, and such heterodimer may interfere the transactivation activity of EBNA1.Four chimeric proteins, EBNA1-repressor (KRAB) fusion proteins were constructed, and their ability to suppress an EBNA1-dependent enhancer reporter DNA, FR-luc, were assayed. The results demonstrate that the repression activity of EBNA1-KRAB chimeras are dose-dependent and the chimeras can compete with EBNA1.Abstract （English）………………………………………………...IIAcknowledge………………………………………………………….VIntroduction.…………………….……….…………………….….1~20I. Epstein-Barr Virus （EBV）………………………………..………1II. EBV nuclear antigen 1（EBNA1）………………...…………….. 8III. Kruppel-associated box（KRAB）domain…………………….....13IV. KRAB-associated protein-1（KAP-1）……………………...…...17V. Purpose of this study……………..…………………………………..20Material and Methods……………………………………..…..21~28I. Cell culture………………………………...…21II. DNA sequences analysis…………………………….21III. Electroporation of mammalian cells………………………..22IV. Luciferase activity assay………………...……………….22V. Plasmid construction…………………………….23VI. Immunoblot assay………..……………………….…………..26VII. Purification of plasmid DNA………………….………………..26Results……………………………………………………………29~34Discussion…………………………………………...……………35~39References………………………………………………………40~52Figures…………………………………………………………….53~68Tables………………………………………………………………69~71