| Summary: | 博士 === 慈濟大學 === 醫學科學研究所 === 107 === Epstein-Barr virus (EBV), a world-wide oncogenic γ-human herpesvirus, is highly associated with several types of malignancies. EBV establishes permanent infection in B lymphocytes, which is characterized by expression of latency-associated nuclear antigens (EBNA), integral membrane proteins (LMPs), and non-coding RNAs. EBNA1 is the only latent gene to be prevalently expressed in almost all EBV+ neoplasms, and dimeric EBNA1-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. Our first study aimed to determine Ribosome Protein L4 (RPL4) is essential for EBNA1 function. In this study, we found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP mediated episome binding and maintenance, whereas the RPL4 C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection. The next study demonstrated that GAP31 from an ancient medicinal plant exhibits anti-viral activity through targeting to EBNA1. Herein, we identify the EBNA1 DNA-binding domain as a core for GAP31 binding by performing affinity pulldown assays. Recombinant GAP31 (rGAP31) was shown to impair EBNA1-induced dimerization; consequently, it abrogated both EBNA1/oriP-mediated binding and transcription. Importantly, the therapeutic effects of GAP31 showed its capability to abrogate EBV-driven cell transformation and proliferation, and EBV-dependent tumorigenesis in xenograft animal models. Notably, the EBNA1 binding-mutant rGAP31 R166A/R169A simply exhibits defective phenotypes in the above-mentioned studies. Our data suggest rGAP31 is a potential anti-viral drug which can be applied to the development of therapeutic strategies against EBV-related malignancies.
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