EB病毒溶裂期基因表現及BKRF3 DNA-尿嘧啶糖苷酶功能之探討

Abstract

Epstein-Barr virus (EBV) is a gamma herpesvirus which has been demonstrated to be associated with various human malignancies such as Burkitt’s lymphoma and nasopharynegeal carcinoma (NPC). Although EBV latent status was documented in most tumors, recent observations indicate that during the process of carcinogenesis in some malignancies EBV reactivation does occur. However, current knowledge regarding EBV lytic gene expression and function is far behind that of latent genes. In order to monitor simultaneously the genome-wide gene expression control, an EBV DNA array was generated to analyze the pattern of transcription of the entire EBV genome under various conditions. Firstly, a complete set of temporal expression clusters of EBV genes was displayed by analyzing the array data of anti-IgG induced Akata cells. A series of genes with unknown function were respectively assigned to various clusters, In addition, increasing expression of latent genes, including EBNA2, EBNA3A and EBNA 3C, was observed during virus replication. Secondly, gene expression independent of viral DNA replication was analyzed in PAA blocked Akata cells and in chemically induced Raji cells. Several genes with presumed late functions were unexepectedly found to be expressed with early kinetics and independent of viral DNA replication, suggesting possible novel functions for these genes. Finally, the EBV array was used to identify Rta responsive gene expression in Raji cells, and in the EBV positive epithelial cells NA, using a Zta siRNA strategy. The array data were confirmed by northern blotting, RT-PCR and reporter assays. All the information here thus provides a better understanding of the control of EBV lytic gene expression. According to microarray results, the virally encoded DNA replication associated enzymes were found to be catalogued into two clusters, suggesting their participation at early theta-form replication and late rolling-circle replication, respectively. Among the genes expressed in the second cluster, BKRF3 was previously demonstrated to enhance the oriLyt-dependent DNA replication in a co-transfection replication assay. However, the expression and function of BKRF3 have not yet been characterized. Based on its amino acid sequence, the putative Uracil-DNA glycosylase (UDG or UNG), BKRF3 belongs to the UNG family of proteins which are the primary DNA repair enzymes responsible for the removal of inappropriate uracil from DNA. Recent studies further suggested that the nuclear human UNG2, and the UDGs of large DNA viruses, may coordinate with their DNA polymerase accessory factors to enhance DNA replication. In the second part of this study, His-BKRF3 was expressed in bacteria and purified for biochemical analysis. Similar to the E. coli and human UNG enzymes, His-BKRF3 excised uracil from single-stranded DNA more efficiently than from double-stranded DNA and was inhibited by the purified bacteriophage PBS1 UNG inhibitor, Ugi. In addition, BKRF3 was able to complement an E. coli ung mutant in rifampicin and nalidixic acid resistance mutator assays. The expression kinetics and subcellular localization of BKRF3 products were detected in EBV positive lymphoid and epithelial cells using BKRF3 specific mouse antibodies. Expression of BKRF3 is mainly regulated by the immediate early transcription activator Rta. The efficiency of EBV lytic DNA replication was slightly affected by BKRF3 siRNA, whereas cellular UNG2 siRNA or inhibition of cellular and viral UNG activities by expressing Ugi repressed EBV lytic DNA replication. Taken together, I demonstrated the UNG activity of BKRF3 in vitro and in vivo and suggest that UNGs may participate in DNA replication or repair and thereby promote efficient production of viral DNA.