EB病毒溶裂期基因表現及BKRF3 DNA-尿嘧啶糖苷酶功能之探討

Epstein-Barr Virus Genome-Wide Gene Expression Profile and the Function of BKRF3 Uracil-DNA Glycosylase in Viral Lytic Replication

呂志忠(Chih-Chung Lu)

指導教授 ： 陳美如

國立臺灣大學/醫學院/微生物學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.03025

摘要

EB病毒已被報導與淋巴瘤與鼻咽癌等人類惡性腫瘤有密切相關。過去認為EB病毒潛伏期基因產物與腫瘤的發生有關，而最近的研究顯示當EB病毒活化進入溶裂期時，可能參與了致病的過程。然而針對大多數EB病毒溶裂期基因的表現或功能的瞭解，仍相當有限。
為了進一步瞭解所有溶裂期基因的表現及其可能參與病毒DNA複製的機制。在本論文中，建立了EB病毒DNA微陣列，系統性的分析在Akata細胞利用抗G型免疫球蛋白，活化病毒進入溶裂期時，病毒基因在不同時間點表現的情形。並依表現模式將基因分為不同的表現群，發現潛伏期基因EB病毒核蛋白EBNA2、EBNA3A、EBNA3C，會隨著病毒活化進入溶裂期而增加。另外，在Akata細胞中加入DNA聚合酶抑制劑PAA，或利用化合物刺激Raji細胞，分析在EB病毒DNA無法複製時，病毒基因表現的情況，意外發現某些被推測為病毒DNA複製完成才會表現之晚期表現基因，卻在EB病毒活化進入溶裂期的早期開始表現，推測這些基因可能具有未知的生物功能。此外將EB病毒特早期表現之轉活化因子Rta轉染進Raji細胞，以及在帶有EB病毒之上皮細胞NA中，利用干擾性RNA抑制另一轉活化因子Zta的蛋白表現，藉以分析Rta轉活化之病毒基因。微陣列分析所得之結果，並經由北方墨漬法、轉錄聚合酶連鎖反應及報導基因表現分析得到證實。本論文分析並整理之資料，對於EB病毒溶裂期基因表現之調控，提供更進一步的瞭解。
根據微陣列之結果，EB病毒複製相關酵素分為兩大表現群，推測其分別參與了EB病毒早期以θ形式，及晚期以Rolling circle形式的複製過程。其中在第二表現群的基因中，BKRF3已被證明在共同轉染的實驗中，能促進帶有OriLyt片段質體的複製。然而對於BKRF3表現之調控及生物功能仍不清楚，經由對BKRF3胺基酸之分析，推測具有尿嘧啶糖苷酶之活性。尿嘧啶糖苷酶參與在DNA修復系統，負責移除DNA上不該存在之尿嘧啶，以確保DNA的正確性。在本論文中，由大腸桿菌所表現並純化之His-BKRF3的重組蛋白的生化特性和大腸桿菌及人類細胞表現的尿嘧啶糖苷酶均很相似。例如，His-BKRF3自單股DNA中移除尿嘧啶之效率高於自雙股DNA之移除，且噬菌體表現的尿嘧啶糖苷酶抑制蛋白(Ugi)能抑制BKRF3具有的尿嘧啶糖苷酶活性。另外在Rifampicin及Nalidixic acid的抗藥性突變分析中，BKRF3的表現能使得失去尿嘧啶糖苷酶基因的大腸桿菌突變種，恢復成具有野生型大腸桿菌之表現型。本論文也利用專一性偵測BKRF3的抗體，探討EB病毒在上皮細胞及B細胞活化進入溶裂期時，BKRF3所表現的時間點，及在宿主細胞內所表現的位置。其中BKRF3的表現，主要是透過轉活化因子Rta的調控，同時我們也發現，利用干擾性RNA抑制BKRF3蛋白的表現時，EB病毒DNA的合成受到一些的限制。當細胞或病毒所表現的尿嘧啶糖苷酶的活性被Ugi抑制時，或者細胞之UNG2的表現受到干擾性RNA抑制時，均嚴重影響EB病毒DNA的複製。經由上述的實驗結果，本論文證明了BKRF3不論在試管內或體內皆具有尿嘧啶糖苷酶的活性，並推測尿嘧啶糖苷酶可藉由參與DNA複製及修復機制，進而促進病毒DNA的合成。

關鍵字

EB病毒 ； 溶裂期基因 ； BKRF3 DNA-尿嘧啶糖苷酶

並列摘要

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.

並列關鍵字

Epstein-Barr Virus ； lytic gene expression ； BKRF3 Uracil-DNA Glycosylase

參考文獻

Lu, J., Chua, H. H., Chen, S. Y., Chen, J. Y. & Tsai, C. H. (2003). Regulation of matrix metalloproteinase-1 by Epstein-Barr virus proteins. Cancer Res 63, 256-62.

Chang, Y., Tung, C. H., Huang, Y. T., Lu, J., Chen, J. Y. & Tsai, C. H. (1999). Requirement for cell-to-cell contact in Epstein-Barr virus infection of nasopharyngeal carcinoma cells and keratinocytes. J Virol 73, 8857-66.

Ramiro, A. R., Jankovic, M., Callen, E., Difilippantonio, S., Chen, H. T., McBride, K. M., Eisenreich, T. R., Chen, J., Dickins, R. A., Lowe, S. W., Nussenzweig, A. & Nussenzweig, M. C. (2006). Role of genomic instability and p53 in AID-induced c-myc-Igh translocations. Nature 440, 105-9.

Rickinson, A. B. & Kieff, E. (2001). Epstin-Barr virus and its replication. In Fields virology, 4th edn, pp. 2575-2627. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Pa, Philadelphia.: Lippincott-Raven.

Roizman, B. & Sears, A. E. (2001). Herpes simplex viruses and their replication. In Fields virology, 4th edn, pp. 1795-1842. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Pa, Philadelphia.: Lippincott-Raven.