臺灣博碩士論文加值系統

D型肝炎病毒(HDV)為含有包膜的負股RNA病毒，其包膜由B型肝炎病毒表面抗原所組成。HDV不但為最小的動物病毒，且相對於其他動物病毒有其獨特性，包括其RNA基因體為含有ribozyme活性之不分支桿狀結構，且利用宿主細胞的RNA聚合酶來進行病毒之RNA複製。分析世界各地收集之HDV序列，可分成八型(HDV-1~8)，其中在台灣可分離出HDV-1、2、4三型。先前研究發現，無論是在綜合感染的病人檢體或RNA共同轉染細胞培養系統皆觀察到HDV RNA基因重組的現象；因此，論文中利用此細胞培養系統來探究HDV RNA基因重組的分子機制。首先，我建構屬於HDV-1之二病毒株間的重組圖譜，分析200 clones發現其中50 clones為重組分子，且多集中在桿狀中段(nt 190-746及nt 886-1306)(分別為40%及32%)及含ribozyme區域之桿狀一端(nt 564-1013)(40%)，而桿狀的另一端（nt 1182-256）發現重組分子的機會只有8%，分析這些區域附近之序列，發現可形成stem-loop(stem區>6bp)之二級結構，這個結構可能可增加模板轉移之機會。另一個支持模板轉移之實驗為當二HDV病毒序列以不同濃度存於細胞中時，我發現高濃度者模板轉移時為接收者(acceptor RNA)之機率較高。綜而言之，本論文之結果支持HDV RNA重組是藉由寄主細胞RNA polymerase 以template-switch機制來執行的。

Abstract
Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus, which provides the envelope proteins to form virus particles. HDV is the smallest animal virus and has an unique unbranched rod-like circular RNA genome with ribozyme activity. Its replication is carried out by host RNA polymerases via a double rolling circle mechanism. At least eight clades of HDV have been identified around the world. HDV-1,-2,and-4 have been isolated from Taiwan. RNA recombination occurs both in patients with mixed genotype infections, and also in an RNA co-transfection cultured cell system. Using the established cultured cell system, I attempted to investigate the molecular mechanism of HDV RNA recombination. First of all, I have established a recombination map between two HDV-1 strains. Among 200 clones analyzed, 50 clones are HDV recombinants. Sequencing analyses data indicated that recombination was frequently detected in the middle(nt 190-746) and onr end(nt 564-1013). In contrast, only 8% recombinants were mapped to the other end of the rod(nt 1182-256). Based on the RNA structure analyses around the recombination junctions, I have identified several potential stem-loop structures in which the stems contain >6bp. These structures might cause RNA polymerase pausing and thereby promote template-switching to generate recombinants. Furthermore, co-transfections with different concentrations of two HDV-1 RNAs into cells ,the dominant parental RNA species preferentially serves as the acceptor RNA in HDV RNA recombination . Taken together, these results support the hypothesis that HDV RNA recombination could be performed by cellular RNA polymerases via a template-switch mechanism.

目 錄
指導教授推薦書…………………………………………………………………
口試委員會審定書………………………………………………………………
授權書…………………………………………………………………………….iii
誌謝……………………………………………………………………………….iv
中文摘要……………………………………………………………………………vi
英文摘要………………………………………………………………………….vii
目錄……………………………………………………………………………….viii
第一章 序論 1
1.1 D型肝炎病毒的發現 1
1.2 D型肝炎病毒的病毒構造 2
1.3 D型肝炎病毒的基因體構造 2
1.4 D型肝炎抗原 (HEPATITIS DELTA ANTIGEN) 3
1.5 D型肝炎病毒抗原 (HDAG) 之結構 5
1.6 HDV基因體的複製 6
1.7 HDV的基因型 9
1.8 RNA 重組 10
1.9 HDV RNA 的重組 14
第二章 實驗設計 18
2.1 研究動機 18
2.2 實驗設計 18
2.3 材料與方法 19
第三章　 實驗結果 27
3.1 建構T/L重組圖譜(RECOMBINATION MAP) 27
3.2 接收模板 (ACCEPTOR TEMPLATE ) 濃度對重組的影響 31
3.2.1 當細胞內T病毒株濃度大於L時重組分之分析 31
3.2.2 當細胞內T病毒株濃度小於L時重組分之分析 33
第四章 討論 34
4.1 T/L RECOMBINATION MAP 34
4.1.1 T/L Recombination map 34
4.1.2 T/L recombination map 與T/Ⅱb recombination map比較 - 36 -
4.2 ACCEPTOR CONCENTRATION 43
參考文獻…………………………………………………………………….……79



圖表目錄
Fig.1 HDV的不分枝桿狀構造…………………………………………………….46
Fig.2 HDV的mRNA………………………………………………………………47
Fig.3 HDV基因體的複製和HDAg的產生………………………………………48
Fig.4 HDV基因體的複製機制…………………………………………………….49
Fig.5 HDV RNA重組的假想模式………………………………………………...50
Fig. 6 第一基因型和Ⅱb基因型的重組分佈圖……………………......................52
Fig. 7 第一基因型和IIb基因型co-transfected至細胞中，經由RFLP分析 HDV RNA重組分子……………………………………………………………………….53
Fig. 8 T/L recombination map的建構………………………………......................54
Fig.9 第一基因型的義大利株（T）和美國株（L）co-transfected至細胞中，經由RT-PCR-RFLP分析 HDV RNA重組分子…………………………………………55
Fig.10 T和L共同轉染細胞第六天後細胞RNA萃取物之A’、B’、C’及D’片段之 RT-PCR-RFLP 的分析……………………………………………………..57
Fig.11 T/L recombination map—A’、B’、C’及D’片段重組發生位置….58
Fig. 12 第一基因型T/L的重組分佈圖…………………………………………..60
Fig.13 ：重組分子產生的模擬圖………………………………………………….61
Fig.14 T和L共同轉染細胞第六天後細胞RNA萃取物之RT-PCR-RFLP 的分析…………………………………………………………………………………….62
Fig. 15 T/Ⅱb及T/L之recombination map比較圖……………………………..63
Fig. 16 T/Ⅱb及T/L之recombination 發生頻率及位置比較圖……………….64
Fig.17 第一基因型義大利株 (T)、美國株 (L)和Ⅱb在nt737-847區域的二級結構預測圖……………………………………………………………………………….67
Fig. 18 T、L及Ⅱb在B’片段的nt 737-886區域之序列比對………………..68
Fig. 19 T、L及Ⅱb在A’片段之序列比對……………………………………..69
Fig.20 第一基因型義大利株 (T)和美國株 (L)在1159-1207區域前的二級結構預測圖…………………………………………………………………………………..70
Fig.21 第一基因型義大利株 (T)和美國株 (L)在nt 435-486區域前的二級結構預測圖………………………………………………………………………………..71
Fig.22 HDV RNA 重組分子機制模式圖…………………………………………72
Table.1 建構T/L recombination map所使用的consensus primer，分別為A’片段、B’片段、C’片段及D’片段…………….…………………………………………....74
Table.2 T/L co-transfection之A’、C’片段經RT-PCR-RFLP分析之基因重組頻率（recombintation frequency）整理表………………………………………….75
Table.3 T/L 各個片段（A’、B’、C’及D’）的基因重組分子種類及其重組點 ( junction sites ) 分佈整理表……………………………………………………76
Table.4 T/L 各個片段（A’、B’、C’及D’）的基因重組分子重組點 ( junction sites ) 整理表………………………………………………………………………...77
Table.5 將不同比例的T和L (T>L及T<L) 共同轉染細胞後之 RT-PCR 產物，經 T-cloning 後，以 Pst I-和 Afl II-RFLP 分析的結果，百分比為單一種重組分子數佔總重組分子數的比例………………………………………………………..78

參考資料
Beard, M. R., MacNaughton, T. B. & Gowans, E. J. (1996). Identification and characterization of a hepatitis delta virus RNA transcriptional promoter. J Virol 70, 4986-4995.
Bergmann, K. F. & Gerin, J. L. (1986). Antigens of hepatitis delta virus in the liver and serum of humans and animals. J Infect Dis 154, 702-706.
Bichko, V. V. & Taylor, J. M. (1996). Redistribution of the delta antigens in cells replicating the genome of hepatitis delta virus. J Virol 70, 8064-8070.
Bonino, F., Heermann, K. H., Rizzetto, M. & Gerlich, W. H. (1986). Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope. J Virol 58, 945-950.
Branch, A. D. & Robertson, H. D. (1984). A replication cycle for viroids and other small infectious RNA's. Science 223, 450-455.
Cascone, P. J., Haydar, T. F. & Simon, A. E. (1993). Sequences and structures required for recombination between virus-associated RNAs. Science 260, 801-805.
Casey, J. L., Brown, T. L., Colan, E. J., Wignall, F. S. & Gerin, J. L. (1993). A genotype of hepatitis D virus that occurs in northern South America. Proc Nati Acad Sci U S A 90, 9016-9020.
Casey, J. L., Niro, G. A., Engle, R. E., Vega, A., Gomez, H., McCarthy, M., Watts, D. M., Hyams, K. C. & Gerin, J. L. (1996). Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F. Journal Infect Dis 174, 920-926.
Chang, J. & Taylor, J. (2002). In vivo RNA-directed transcription, with template switching, by a mammalian RNA polymerase. Embo J 21, 157-164.
Chang, M. F., Chang, S. C., Chang, C. I., Wu, K. & Kang, H. Y. (1992). Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen. J Virol 66, 6019-6027.
Chang, M. F., Sun, C. Y., Chen, C. J. & Chang, S. C. (1993). Functional motifs of delta antigen essential for RNA binding and replication of hepatitis delta virus. J Virol 67, 2529-2536.
Chao, M. (2007). RNA recombination in hepatitis delta virus: implications regarding the abilities of mammalian RNA polymerases. Virus Res 127, 208-215.
Chao, M., Hsieh, S. Y. & Taylor, J. (1990a). Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J Virol 64, 5066-5069.
Chao, Y. C., Chang, M. F., Gust, I. & Lai, M. M. (1990b). Sequence conservation and divergence of hepatitis delta virus RNA. Virology 178, 384-392.
Chou, H. C., Hsieh, T. Y., Sheu, G. T. & Lai, M. M. (1998). Hepatitis delta antigen mediates the nuclear import of hepatitis delta virus RNA. J Virol 72, 3684-3690.
Copper, P. D., Steiner-Pryor, A., Scotti, P. D. & Delong, D. (1974). On the nature of poliovirus genetic recombinants. J Gen Virol 23, 41-49.
Filipovska, J. & Konarska, M. M. (2000). Specific HDV RNA-templated transcription by pol II in vitro. RNA 6, 41-54.
Glenn, J. S. & White, J. M. (1991). trans-dominant inhibition of human hepatitis delta virus genome replication. J Virol 65, 2357-2361.
Gudima, S. O., Chang, J. & Taylor, J. M. (2005). Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs. RNA 11, 90-98.
Gudima, S. O., Chang, J. & Taylor, J. M. (2006). Restoration in vivo of defective hepatitis delta virus RNA genomes. RNA 12, 1061-1073.
Hsieh, S. Y., Chao, M., Coates, L. & Taylor, J. (1990). Hepatitis delta virus genome replication: a polyadenylated mRNA for delta antigen. J Virol 64, 3192-3198.
Hsieh, S. Y. & Taylor, J. (1991). Regulation of polyadenylation of hepatitis delta virus antigenomic RNA. J Virol 65, 6438-6446.
Hsu, S. C., Syu, W. J., Sheen, I. J., Liu, H. T., Jeng, K. S. & Wu, J. C. (2002). Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications. Hepatology 35, 665-672.
Hwang, S. B., Lee, C. Z. & Lai, M. M. (1992). Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications between the large and small forms. Virology 190, 413-422.
Imazeki, F., Omata, M. & Ohto, M. (1991). Complete nucleotide sequence of hepatitis delta virus RNA in Japan. Nucleic Acids Res 19, 5439.
Jayan, G. C. & Casey, J. L. (2002). Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of ADAR1 but not ADAR2 expression. J Virol 76, 12399-12404.
Kirkegaard, K. & Baltimore, D. (1986). The mechanism of RNA recombination in poliovirus. Cell 47, 433-443.
Kuo, M. Y., Chao, M. & Taylor, J. (1989). Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen. J Virol 63, 1945-1950.
Kuo, M. Y., Goldberg, J., Coates, L., Mason, W., Gerin, J. & Taylor, J. (1988a). Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications. J Virol 62, 1855-1861.
Kuo, M. Y., Sharmeen, L., Dinter-Gottlieb, G. & Taylor, J. (1988b). Characterization of self-cleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J Virol 62, 4439-4444.
Lai, M. M. (1992). RNA recombination in animal and plant viruses. Microbiol Rev 56, 61-79.
Lai, M. M. (1995). The molecular biology of hepatitis delta virus. Annu Rev Biochem 64, 259-286.
Lang, W. H. & Reeder, R. H. (1995). Transcription termination of RNA polymerase I due to a T-rich element interacting with Reb1p. Proc Natl Acad Sci U S A 92, 9781-9785.
Lee, C. M., Changchien, C. S., Chung, J. C. & Liaw, Y. F. (1996). Characterization of a new genotype II hepatitis delta virus from Taiwan. J Med Virol 49, 145-154.
Lee, C. Z., Lin, J. H., Chao, M., McKnight, K. & Lai, M. M. (1993). RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication. J Virol 67, 2221-2227.
Lo, K., Hwang, S. B., Duncan, R., Trousdale, M. & Lai, M. M. (1998). Characterization of mRNA for hepatitis delta antigen: exclusion of the full-length antigenomic RNA as an mRNA. Virology 250, 94-105.
Macdonald, L. E., Zhou, Y. & McAllister, W. T. (1993). Termination and slippage by bacteriophage T7 RNA polymerase. J Mol Biol 232, 1030-1047.
Macnaughton, T. B., Beard, M. R., Chao, M., Gowans, E. J. & Lai, M. M. (1993). Endogenous promoters can direct the transcription of hepatitis delta virus RNA from a recircularized cDNA template. Virology 196, 629-636.
MacNaughton, T. B., Gowans, E. J., McNamara, S. P. & Burrell, C. J. (1991). Hepatitis delta antigen is necessary for access of hepatitis delta virus RNA to the cell transcriptional machinery but is not part of the transcriptional complex. Virology 184, 387-390.
Macnaughton, T. B. & Lai, M. M. (2002). Genomic but not antigenomic hepatitis delta virus RNA is preferentially exported from the nucleus immediately after synthesis and processing. J Virol 76, 3928-3935.
Makino, S., Chang, M. F., Shieh, C. K., Kamahora, T., Vannier, D. M., Govindarajan, S. & Lai, M. M. (1987). Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA. Nature 329, 343-346.
Modahl, L. E. & Lai, M. M. (1998). Transcription of hepatitis delta antigen mRNA continues throughout hepatitis delta virus (HDV) replication: a new model of HDV RNA transcription and replication. J Virol 72, 5449-5456.
Modahl, L. E., Macnaughton, T. B., Zhu, N., Johnson, D. L. & Lai, M. M. (2000). RNA-Dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases. Mol Cell Biol 20, 6030-6039.
Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L. & Gibbs, J. B. (1991). Sequence dependence of protein isoprenylation. J Biol Chem 266, 14603-14610.
Moraleda, G. & Taylor, J. (2001). Host RNA polymerase requirements for transcription of the human hepatitis delta virus genome. J Virol 75, 10161-10169.
Nagy, P. D. & Bujarski, J. J. (1995). Efficient system of homologous RNA recombination in brome mosaic virus: sequence and structure requirements and accuracy of crossovers. J Virol 69, 131-140.
Nie, X., Chang, J. & Taylor, J. M. (2004). Alternative processing of hepatitis delta virus antigenomic RNA transcripts. J Virol 78, 4517-4524.
Radjef, N., Gordien, E., Ivaniushina, V., Gault, E., Anais, P., Drugan, T., Trinchet, J. C., Roulot, D., Tamby, M., Milinkovitch, M. C. & Deny, P. (2004). Molecular phylogenetic analyses indicate a wide and ancient radiation of African hepatitis delta virus, suggesting a deltavirus genus of at least seven major clades. J Virol 78, 2537-2544.
Rizzetto, M. (1989). Hepatitis delta virus (HDV) infection and disease. Ric Clin Lab 19, 11-26.
Rizzetto, M., Canese, M. G., Gerin, J. L., London, W. T., Sly, D. L. & Purcell, R. H. (1980a). Transmission of the hepatitis B virus-associated delta antigen to chimpanzees. J Infect Dis 141, 590-602.
Rizzetto M, C. M., Arico S, Crivelli O, Trepo C, Bonino F, and Verme G (1977). Immunofluorescence detection of new antigen-antibody system (delta/anti- delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers Gut 18, 997-1003.
Rizzetto, M., Hoyer, B., Canese, M. G., Shih, J. W., Purcell, R. H. & Gerin, J. L. (1980b). delta Agent: association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infected chimpanzees. Proc Nati Acad Sci U S A 77, 6124-6128.
Sakugawa, H., Nakasone, H., Nakayoshi, T., Kawakami, Y., Miyazato, S., Kinjo, F., Saito, A., Ma, S. P., Hotta, H. & Kinoshita, M. (1999). Hepatitis delta virus genotype IIb predominates in an endemic area, Okinawa, Japan. J Med Virol 58, 366-372.
Tavanez, J. P., Cunha, C., Silva, M. C., David, E., Monjardino, J. & Carmo-Fonseca, M. (2002). Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm. RNA 8, 637-646.
Taylor, J. M. (1992). The structure and replication of hepatitis delta virus. Annu Rev Microbiol 46, 253-276.
Tolskaya, E. A., Romanova, L. I., Blinov, V. M., Viktorova, E. G., Sinyakov, A. N., Kolesnikova, M. S. & Agol, V. I. (1987). Studies on the recombination between RNA genomes of poliovirus: the primary structure and nonrandom distribution of crossover regions in the genomes of intertypic poliovirus recombinants. Virology 161, 54-61.
Wang, J. G. & Lemon, S. M. (1993). Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo. J Virol 67, 446-454.
Wang, K. S., Choo, Q. L., Weiner, A. J., Ou, J. H., Najarian, R. C., Thayer, R. M., Mullenbach, G. T., Denniston, K. J., Gerin, J. L. & Houghton, M. (1986). Structure, sequence and expression of the hepatitis delta (delta) viral genome.[erratum appears in Nature 1987 Jul 30-Aug 5;328(6129):456]. Nature 323, 508-514.
Wang, T. C. & Chao, M. (2005). RNA recombination of hepatitis delta virus in natural mixed-genotype infection and transfected cultured cells. J Virol 79, 2221-2229.
Watanabe, H., Nagayama, K., Enomoto, N., Chinzei, R., Yamashiro, T., Izumi, N., Yatsuhashi, H., Nakano, T., Robertson, B. H., Nakasone, H., Sakugawa, H. & Watanabe, M. (2003). Chronic hepatitis delta virus infection with genotype IIb variant is correlated with progressive liver disease. J Gen Virol 84, 3275-3289.
Wilson, K. S. & von Hippel, P. H. (1995). Transcription termination at intrinsic terminators: the role of the RNA hairpin. Proc Natl Acad Sci U S A 92, 8793-8797.
Wu, H. N. & Lai, M. M. (1989). Reversible cleavage and ligation of hepatitis delta virus RNA.[erratum appears in Science 1989 Mar 17;243(4897):1420]. Science 243, 652-654.
Wu, J. C., Chen, C. M., Sheen, I. J., Lee, S. D., Tzeng, H. M. & Choo, K. B. (1995). Evidence of transmission of hepatitis D virus to spouses from sequence analysis of the viral genome. Hepatology 22, 1656-1660.
Wu, J. C., Chiang, T. Y. & Sheen, I. J. (1998). Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis. J Gen Virol 79 ( Pt 5), 1105-1113.
Xia, Y. P., Yeh, C. T., Ou, J. H. & Lai, M. M. (1992). Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex. J Virol 66, 914-921.
Zhang, Y. Y., Tsega, E. & Hansson, B. G. (1996). Phylogenetic analysis of hepatitis D viruses indicating a new genotype I subgroup among African isolates. J Clin Microbiol 34, 3023-3030.