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研究生： 陳欣瑜
Chen, Hsin-Yu
論文名稱： 探討週期蛋白依賴性激酶參與 B 型肝炎病毒 cccDNA 合成之機轉
Investigating the effects of cyclin-dependent kinases in hepatitis B virus cccDNA synthesis
指導教授： 黃溫雅
Huang, Wenya
學位類別： 碩士
Master
系所名稱： 醫學院 - 醫學檢驗生物技術學系
Department of Medical Laboratory Science and Biotechnology
論文出版年： 2025
畢業學年度： 113
語文別： 英文
論文頁數： 86
中文關鍵詞： B型肝炎病毒 、共價閉合環狀DNA 、鬆弛環狀DNA 、細胞週期 、細胞週期蛋白依賴性激酶1 、Flap核酸內切酶1
外文關鍵詞： Hepatitis B virus, covalently closed circular DNA, relaxed circular DNA, cell cycle, cyclin-dependent kinase 1, flap endonuclease 1
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    • 摘要 I Abstract III Acknowledgments V Table of Contents VII List of Figures X List of Appendices XI I. Introduction 1 1.1. Hepatitis B virus 1 1.1.1. Epidemiology of hepatitis B virus 2 1.1.2. Clinical manifestations and disease burden 2 1.1.3. Structure and serological markers of HBV 3 1.1.4. Life cycle of hepatitis B virus 3 1.1.5. Mechanism of HBV cccDNA synthesis 5 1.1.6. Current therapeutic approaches 5 1.1.7. Treatment challenges and limitations 6 1.2. Interactions between the cell cycle and viruses 7 1.2.1. Cell cycle and cyclin-dependent kinases 7 1.2.2. Regulation of viral replication by the cell cycle 8 1.2.3. Viral manipulation of cell cycle progression 8 1.2.4. Cell cycle regulation by HBV 9 1.3. Detection methods for HBV cccDNA 10 1.4. Previous study in the lab 11 II. Specific aims 12 III. Materials and Methods 14 3.1. Cell culture 14 3.2. MTS assay 15 3.3. Treatments with CDK inhibitors and nocodazole 15 3.4. HBV cccDNA extraction 16 3.5. T5 and T7 exonuclease treatments 16 3.6. HBV cccDNA real-time quantitative PCR 17 3.7. Production of HBV virions 17 3.8. HbcAg immunofluorescence assay 18 3.9. HBV infection 18 3.10. Western blotting assay 19 3.11. Cell cycle analysis 20 3.12. Plasmid construction 20 3.13. Stable cell construction 21 3.14. Site-directed mutagenesis 21 IV. Results 22 4.1 CDK1 plays a crucial role in the progression of HBV cccDNA synthesis 22 4.2 CDK1 is involved in cccDNA synthesis in an infectious cell model 23 4.3 G2/M arrest induced by HBV replication fails to enhance cccDNA synthesis 24 4.4 CDK1 regulates FEN1 phosphorylation to stimulate cccDNA synthesis 25 4.5 Establishment of cccDNA reporter assay 27 4.6 Inhibition of CDK2 inhibitor AUZ454 promotes cccDNA synthesis but decreases HBeAg production 30 4.7 CDK4/6 inhibition by Ribociclib suppresses cccDNA formation and HBeAg production 30 4.8 CDK9 plays a crucial role in the progression of HBV cccDNA synthesis 31 V. Discussion 32 VI. References 38

    Allweiss, L., Testoni, B., Yu, M., Lucifora, J., Ko, C., Qu, B., Lütgehetmann, M., Guo, H., Urban, S., Fletcher, S. P., Protzer, U., Levrero, M., Zoulim, F. & Dandri, M. (2023). Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure. Gut, 72(5), 972-983.
    Bagga, S. & Bouchard, M. J. (2014). Cell cycle regulation during viral infection. Methods Mol Biol, 1170, 165-227.
    Bao, C. Y., Hung, H. C., Chen, Y. W., Fan, C. Y., Huang, C. J. & Huang, W. (2020). Requirement of cyclin-dependent kinase function for hepatitis B virus cccDNA synthesis as measured by digital PCR. Ann Hepatol, 19(3), 280-286.
    Bashir, T., Horlein, R., Rommelaere, J. & Willwand, K. (2000). Cyclin A activates the DNA polymerase delta -dependent elongation machinery in vitro: A parvovirus DNA replication model. Proc Natl Acad Sci U S A, 97(10), 5522-5527.
    Beck, J. & Nassal, M. (2007). Hepatitis B virus replication. World J Gastroenterol, 13(1), 48-64.
    Bello, K. E., Mat Jusoh, T. N. A., Irekeola, A. A., Abu, N., Mohd Amin, N. A. Z., Mustaffa, N. & Shueb, R. H. (2023). A Recent Prevalence of Hepatitis B Virus (HBV) Genotypes and Subtypes in Asia: A Systematic Review and Meta-Analysis. Healthcare (Basel), 11(7).
    Bressy, C., Droby, G. N., Maldonado, B. D., Steuerwald, N. & Grdzelishvili, V. Z. (2019). Cell Cycle Arrest in G(2)/M Phase Enhances Replication of Interferon-Sensitive Cytoplasmic RNA Viruses via Inhibition of Antiviral Gene Expression. J Virol, 93(4).
    Cai, D., Wang, X., Yan, R., Mao, R., Liu, Y., Ji, C., Cuconati, A. & Guo, H. (2016). Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators. Antiviral Res, 132, 26-37.
    Cheng, P., Li, Y., Yang, L., Wen, Y., Shi, W., Mao, Y., Chen, P., Lv, H., Tang, Q. & Wei, Y. (2009). Hepatitis B virus X protein (HBx) induces G2/M arrest and apoptosis through sustained activation of cyclin B1-CDK1 kinase. Oncol Rep, 22(5), 1101-1107.
    Conners, E. E., Panagiotakopoulos, L., Hofmeister, M. G., Spradling, P. R., Hagan, L. M., Harris, A. M., Rogers-Brown, J. S., Wester, C. & Nelson, N. P. (2023). Screening and Testing for Hepatitis B Virus Infection: CDC Recommendations - United States, 2023. MMWR Recomm Rep, 72(1), 1-25.
    Davy, C. & Doorbar, J. (2007). G2/M cell cycle arrest in the life cycle of viruses. Virology, 368(2), 219-226.
    de Almeida Pondé, R. A. (2022). Detection of the serological markers hepatitis B virus surface antigen (HBsAg) and hepatitis B core IgM antibody (anti-HBcIgM) in the diagnosis of acute hepatitis B virus infection after recent exposure. Microbiol Immunol, 66(1), 1-9.
    Dusheiko, G. (1997). Side effects of alpha interferon in chronic hepatitis C. Hepatology, 26(3 Suppl 1), 112s-121s.
    Eller, C., Heydmann, L., Colpitts, C. C., El Saghire, H., Piccioni, F., Jühling, F., Majzoub, K., Pons, C., Bach, C., Lucifora, J., Lupberger, J., Nassal, M., Cowley, G. S., Fujiwara, N., Hsieh, S. Y., Hoshida, Y., Felli, E., Pessaux, P., Sureau, C., Schuster, C., Root, D. E., Verrier, E. R. & Baumert, T. F. (2020). A genome-wide gain-of-function screen identifies CDKN2C as a HBV host factor. Nat Commun, 11(1), 2707.
    Fan, Y., Sanyal, S. & Bruzzone, R. (2018). Breaking Bad: How Viruses Subvert the Cell Cycle. Front Cell Infect Microbiol, 8, 396.
    Feuer, R., Mena, I., Pagarigan, R., Slifka, M. K. & Whitton, J. L. (2002). Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro. J Virol, 76(9), 4430-4440.
    Flemington, E. K. (2001). Herpesvirus lytic replication and the cell cycle: arresting new developments. J Virol, 75(10), 4475-4481.
    Frey, M. R., Saxon, M. L., Zhao, X., Rollins, A., Evans, S. S. & Black, J. D. (1997). Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells. J Biol Chem, 272(14), 9424-9435.
    Gao, Y., Zhang, Z., Huang, X., You, M., Du, C., Li, N., Hao, Y., Wang, K., Ding, X., Yang, F., Cheng, S. Q., Luo, J., Chen, R. & Yang, P. (2025). HBV-associated hepatocellular carcinomas inhibit antitumor CD8(+) T cell via the long noncoding RNA HDAC2-AS2. Nat Commun, 16(1), 2055.
    Gerlich, W. H. (2013). Medical virology of hepatitis B: how it began and where we are now. Virol J, 10, 239.
    Henneke, G., Koundrioukoff, S. & Hübscher, U. (2003). Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation. Oncogene, 22(28), 4301-4313.
    Hojatizadeh, M., Amiri, M. M., Mobini, M., Hassanzadeh Makoui, M., Ghaedi, M., Ghotloo, S., Peyghami, K., Jeddi-Tehrani, M., Golsaz-Shirazi, F. & Shokri, F. (2023). Cross-Reactivity of HBe Antigen-Specific Polyclonal Antibody with HBc Antigen. Viral Immunol, 36(6), 378-388.
    Hu, J. & Liu, K. (2017). Complete and Incomplete Hepatitis B Virus Particles: Formation, Function, and Application. Viruses, 9(3).
    Jeng, W. J., Papatheodoridis, G. V. & Lok, A. S. F. (2023). Hepatitis B. Lancet, 401(10381), 1039-1052.
    Jindal, A., Kumar, M. & Sarin, S. K. (2013). Management of acute hepatitis B and reactivation of hepatitis B. Liver Int, 33 Suppl 1, 164-175.
    Königer, C., Wingert, I., Marsmann, M., Rösler, C., Beck, J. & Nassal, M. (2014). Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses. Proc Natl Acad Sci U S A, 111(40), E4244-4253.
    Kim, S. S., Cheong, J. Y. & Cho, S. W. (2011). Current Nucleos(t)ide Analogue Therapy for Chronic Hepatitis B. Gut Liver, 5(3), 278-287.
    Kitamura, K., Que, L., Shimadu, M., Koura, M., Ishihara, Y., Wakae, K., Nakamura, T., Watashi, K., Wakita, T. & Muramatsu, M. (2018). Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus. PLoS Pathog, 14(6), e1007124.
    Li, X., Zhao, J., Yuan, Q. & Xia, N. (2017). Detection of HBV Covalently Closed Circular DNA. Viruses, 9(6).
    Lin, C. L. & Kao, J. H. (2011). Recent advances in the treatment of chronic hepatitis B. Expert Opin Pharmacother, 12(13), 2025-2040.
    Liu, H., Xi, J. & Hu, J. (2021). Regulation of Hepatitis B Virus Replication by Cyclin Docking Motifs in Core Protein. J Virol, 95(12).
    Lok, A. S. F. (2024). Toward a Functional Cure for Hepatitis B. Gut Liver, 18(4), 593-601.
    Long, Q., Yan, R., Hu, J., Cai, D., Mitra, B., Kim, E. S., Marchetti, A., Zhang, H., Wang, S., Liu, Y., Huang, A. & Guo, H. (2017). The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation. PLoS Pathog, 13(12), e1006784.
    MacLachlan, J. H. & Cowie, B. C. (2015). Hepatitis B virus epidemiology. Cold Spring Harb Perspect Med, 5(5), a021410.
    Malumbres, M. (2014). Cyclin-dependent kinases. Genome Biol, 15(6), 122.
    Malumbres, M. & Barbacid, M. (2005). Mammalian cyclin-dependent kinases. Trends Biochem Sci, 30(11), 630-641.
    Marchetti, A. L. & Guo, H. (2020). New Insights on Molecular Mechanism of Hepatitis B Virus Covalently Closed Circular DNA Formation. Cells, 9(11).
    Minosse, C., Coen, S., Visco Comandini, U., Lionetti, R., Montalbano, M., Cerilli, S., Vincenti, D., Baiocchini, A., Capobianchi, M. R. & Menzo, S. (2016). Simple and Reliable Method to Quantify the Hepatitis B Viral Load and Replicative Capacity in Liver Tissue and Blood Leukocytes. Hepat Mon, 16(10), e28751.
    Mohd-Ismail, N. K., Lim, Z., Gunaratne, J. & Tan, Y. J. (2019). Mapping the Interactions of HBV cccDNA with Host Factors. Int J Mol Sci, 20(17).
    Nassal, M. (2015). HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut, 64(12), 1972-1984.
    Norbury, C. & Nurse, P. (1992). Animal cell cycles and their control. Annu Rev Biochem, 61, 441-470.
    Olaru, I. D., Beliz Meier, M., Mirzayev, F., Prodanovic, N., Kitchen, P. J., Schumacher, S. G. & Denkinger, C. M. (2023). Global prevalence of hepatitis B or hepatitis C infection among patients with tuberculosis disease: systematic review and meta-analysis. EClinicalMedicine, 58, 101938.
    Panda, M., Kalita, E., Rao, A. & Prajapati, V. K. (2023). Mechanism of cell cycle regulation and cell proliferation during human viral infection. Adv Protein Chem Struct Biol, 135, 497-525.
    Perrillo, R. P. (2005). Current treatment of chronic hepatitis B: benefits and limitations. Semin Liver Dis, 25 Suppl 1, 20-28.
    Poli, A., Mongiorgi, S., Cocco, L. & Follo, M. Y. (2014). Protein kinase C involvement in cell cycle modulation. Biochem Soc Trans, 42(5), 1471-1476.
    Qi, Y., Gao, Z., Xu, G., Peng, B., Liu, C., Yan, H., Yao, Q., Sun, G., Liu, Y., Tang, D., Song, Z., He, W., Sun, Y., Guo, J. T. & Li, W. (2016). DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus. PLoS Pathog, 12(10), e1005893.
    Rabe, B., Vlachou, A., Panté, N., Helenius, A. & Kann, M. (2003). Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci U S A, 100(17), 9849-9854.
    Schreiner, S. & Nassal, M. (2017). A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation-and Beyond? Viruses, 9(5).
    Schweitzer, A., Horn, J., Mikolajczyk, R. T., Krause, G. & Ott, J. J. (2015). Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet, 386(10003), 1546-1555.
    Summers, J. & Mason, W. S. (1982). Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell, 29(2), 403-415.
    Tanaka, T., Okuyama-Dobashi, K., Murakami, S., Chen, W., Okamoto, T., Ueda, K., Hosoya, T., Matsuura, Y., Ryo, A., Tanaka, Y., Hagiwara, M. & Moriishi, K. (2016). Inhibitory effect of CDK9 inhibitor FIT-039 on hepatitis B virus propagation. Antiviral Res, 133, 156-164.
    Tang, L., Sheraz, M., McGrane, M., Chang, J. & Guo, J. T. (2019). DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA. PLoS Pathog, 15(4), e1007742.
    Tong, S. & Revill, P. (2016). Overview of hepatitis B viral replication and genetic variability. J Hepatol, 64(1 Suppl), S4-s16.
    Tsukuda, S. & Watashi, K. (2020). Hepatitis B virus biology and life cycle. Antiviral Res, 182, 104925.
    Verrier, E. R., Colpitts, C. C., Bach, C., Heydmann, L., Weiss, A., Renaud, M., Durand, S. C., Habersetzer, F., Durantel, D., Abou-Jaoudé, G., López Ledesma, M. M., Felmlee, D. J., Soumillon, M., Croonenborghs, T., Pochet, N., Nassal, M., Schuster, C., Brino, L., Sureau, C., Zeisel, M. B. & Baumert, T. F. (2016). A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses. Hepatology, 63(1), 35-48.
    Wang, T., Zhao, R., Wu, Y., Kong, D., Zhang, L., Wu, D., Li, C., Zhang, C., Yu, Z. & Jin, X. (2011). Hepatitis B virus induces G1 phase arrest by regulating cell cycle genes in HepG2.2.15 cells. Virol J, 8, 231.
    Wei, L. & Ploss, A. (2021). Mechanism of Hepatitis B Virus cccDNA Formation. Viruses, 13(8).
    Wilkins, T., Sams, R. & Carpenter, M. (2019). Hepatitis B: Screening, Prevention, Diagnosis, and Treatment. Am Fam Physician, 99(5), 314-323.
    Xia, Y., Cheng, X., Li, Y., Valdez, K., Chen, W. & Liang, T. J. (2018). Hepatitis B Virus Deregulates the Cell Cycle To Promote Viral Replication and a Premalignant Phenotype. J Virol, 92(19).
    Xu, C., Guo, H., Pan, X. B., Mao, R., Yu, W., Xu, X., Wei, L., Chang, J., Block, T. M. & Guo, J. T. (2010). Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus. J Virol, 84(18), 9332-9340.
    Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., Huang, Y., Qi, Y., Peng, B., Wang, H., Fu, L., Song, M., Chen, P., Gao, W., Ren, B., Sun, Y., Cai, T., Feng, X., Sui, J. & Li, W. (2012). Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife, 1, e00049.
    Zhao, Q., Liu, H., Tang, L., Wang, F., Tolufashe, G., Chang, J. & Guo, J. T. (2024). Mechanism of interferon alpha therapy for chronic hepatitis B and potential approaches to improve its therapeutic efficacy. Antiviral Res, 221, 105782.

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