B型肝炎病毒的釋出透過胞內運輸分選機制/ESCRT之調控

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研究生：	曾博群 Tseng, Po-Chun
論文名稱：	B型肝炎病毒的釋出透過胞內運輸分選機制/ESCRT之調控 Hepatitis B virus secretion via intracellular ESCRT machinery.
指導教授：	王慧菁 Wang, Lily Hui-Ching
口試委員:	王雯靜 Wang, Wen-Ching 桑自剛 Sang, Tzu-Kang
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	43
中文關鍵詞：	B型肝炎病毒、釋出、胞內運輸分選機制
外文關鍵詞：	ESCRT, ALIX, VTA1
相關次數：	點閱：2 下載：0
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B型肝病毒感染所引發的慢性B型肝炎是造成肝硬化和肝癌的主因，而慢性B型肝炎在接受抗病毒藥物治療後表面抗原(HBsAg)仍然存在，也是B型肝炎無法痊癒的原因。因此我們認為阻斷HBsAg的釋放可以使B型肝炎患者達到更好的預後以及痊癒。在本研究中，我們在肝癌細胞株 HuH-7中利用shRNA敲除了22個包含在胞內運輸分選機制(ESCRT)中的基因，想了解是否會影響HBsAg的釋出。在實驗中我們發現在22個基因當中只有敲除ALIX和VTA1會減少HBsAg和病毒e抗原(HBeAg)的胞外分泌，另一方面在細胞內的B型肝炎蛋白表現量並會不受到ALIX或VTA1的缺失而有所改變。我們進一步利用免疫螢光染色發現在被B肝病毒感染的肝細胞中大表面抗原 (LHBs) 和ALIX及VTA1有部分交疊的現象，除此之外在敲除ALIX和VTA1之後細胞內的LHBs有聚集且靠近細胞膜的情形。為了進一步確認實驗的準確性，我們利用RNA干擾 (siRNA)的方式敲除ALIX和VTA1，並發現只有VTA1對於HBsAg的分泌有抑制的情形。由於ESCRT參與在多囊體(MVB)形成，所以利用MVB抑制劑U18666A阻斷MVB的生成想知道是否能夠有效地阻止HBsAg/HBeAg的分泌。實驗結果顯示U18666A藉由累積HBsAg在細胞內來降低HBsAg和HBeAg的分泌。由於U18666A的揭示，我們想更進一步了解VTA1所調節的ATPase和跟U18666A同為抑制膽固醇生成的Statin藥物對於B肝病毒的釋出是否也能達到相同的抑制效果。出乎意料的是Statin在不影響細胞毒殺的情況下可降低HBsAg的分泌，但是對HBeAg則沒有作用。綜合上述實驗結果，我們發現ESCRT中的VTA1和膽固醇生成機制在HBsAg和HBeAg的分泌中都扮演著重要角色，而這些機制將可以提供慢性B肝患者一個新的治療策略。

Hepatitis B virus (HBV) infection is one major cause of liver cirrhosis and liver cancer. The present of abundant viral surface antigen (HBsAg) in bloodstream is a long lasting risk factor of patients who have drug resistance after treatment with antiviral drug. We therefore suggest that blocked of HBsAg secretion may help to cure HBV. In this study, we depleted 22 different endosomal sorting complex required for transport (ESCRT) components in HuH-7 cells and explored their essential roles in virion secretion. We found that extracellular secretion of HBsAg and HBeAg were significantly reduced in cells depleted with Alix or VTA1, but not other 20 ESCRT components. The knockdown efficiency of small hairpin RNA was verified by quantitative RT-PCR. Notably, knockdown of Alix and VTA1 did not affect intracellular expression of more viral antigens, such as HBsAg, large surface antigen/LHBS, and core antigen/HBcAg. We also found that intracellular LHBs partially co-localized with ALIX and VTA1. Depletion of ALIX or VTA1 changed intracellular distribution and accumulated in proximity to plasma membrane in hepatocytes. Finally, we confirmed depletion of ALIX and VTA1 by siRNA and found that only VTA1 reduce extracellular HBsAg level, but not ALIX. To test if inhibition of MVB may suppress HBsAg secretion, we treated cells with MVB inhibitor U18666A and found that extracellular HBsAg/HBeAg was reduced, but intracellular was increased. Finally, we tested specific compared selections for inhibition of extracellular HBsAg and HBeAg, including drugs that target to ATPase and cholesterol synthesis (the statin family). To our surprise, specific stain members displayed inhibitory roles of HBsAg secretion without affecting cell viability. In conclusion, we found that both ESCRT and cholesterol pathways are involved in the secretion of HBsAg and HBeAg. The underlying mechanism may serve novel therapeutic targets for treatment of chronic HBV infection.

Abstract..............................................................................................................................................................i
中文摘要.............................................................................................................................................................ii
Contents...........................................................................................................................................................iv
1. Introduction....................................................................................................................................................1
1-1 Hepatitis B virus...........................................................................................................................................1
1-2 Acute and chronic hepatitis B......................................................................................................................1
1-3 HBV surface antigen is an important biomarker of disease progression....................................................2
1-4 Endosomal Sorting Require for Transport (ESCRT)....................................................................................3
1-5 Potential mechanism of virion secretion    ..................................................................................................5
2. Specific aim....................................................................................................................................................6
3. Material and method.......................................................................................................................................7
4. Results...........................................................................................................................................................10
4-1 ESCRT is involved in the secretion pathway of HBsAg/HBeAg....................................................................10
4-2 LHBs partially co-localize with ALIX and VTA1............................................................................................10
4-3 Depletion of ALIX or VTA1 changed intrahepatic distribution of LHBs........................................................10
4-4 Depletion of ALIX and VTA1 reduced HBsAg and HBeAg secretio...............................................................11
4-5 Intracellular abundance of HBsAg was not increased upon the depletion of VTA1.....................................11
4-6 Depletion of ALIX and VTA1 using mixed pool of lentivirus.........................................................................12
4-7 Depletion ALIX or VTA1 by siRNA changed intracellular LHBs distribution.................................................12
4-8 Confirm HBsAg/HBeAg secretion inhibition by siRNA.................................................................................12
4-9 ATPase inhibitors reduced cell viability.    ..................................................................................................13
4-10 U18666A blocks extracellular HBsAg/HBeAg secretion by accumulating inner cells...............................14
4-11 Cholesterol upstream inhibitor family statin decreased HBsAg secretion    .............................................14
4-12 Drugs that increased intrahepatic LHBs and blocked the secretion of HBsAg and HBeAg......................14
5. Discussion    ..................................................................................................................................................16
6. Reference    ...................................................................................................................................................18
7. Figure    .........................................................................................................................................................23
Figure 1. Knockdown of ESCRT components VTA1 and ALIX is associated with HBsAg/HBeAg secretion.......23
Figure 2. HuH-7 cells were infected with Ad-HBV for 3 days by immunofluorescence....................................24
Figure 3. Intrahepatic distribution of LHBs in the presence and absence of VTA1 and ALIX............................26
Figure 4. Knockdown of ALIX or VTA1 inhibited HBsAg/HBeAg secretion.........................................................27
Figure 5. Knockdown of ALIX or VTA1 induced a different pattern of intracellular protein................................28
Figure 6. Mixture of shRNA blocked HBsAg secretion significantly...................................................................29
Figure 7. Intracellular LHBs accumulation was increased upon the depletion of ALIX and VTA1 by siRNA........30
Figure 8. Inhibition of HBsAg secretion occurred only deplete VTA1 but not ALIX by siRNA.............................31
Figure 9. ATPase inhibitors could not reduce extracellular HBsAg/HBeAg secretion.........................................32
Figure 10. MVB Inhibitor U18666A suppressed extracellular HBsAg and HBeAg...............................................33
Figure 11. Cholesterol upstream inhibitor family statin decreased HBsAg secretion..........................................34
Figure 12. Drug that increased intrahepatic LHBs blocked the secretion of HBsAg and HBeAg........................35
Figure 13. Schematic model of Hepatitis B virus surface antigen secretion pathway.........................................36
8. Tables..............................................................................................................................................................37











                                

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Usami, Y., Popov, S., Popova, E., Inoue, M., Weissenhorn, W., and H, G.G. (2009). The ESCRT pathway and HIV-1 budding. Biochem Soc Trans 37, 181-184.
Vanlandschoot, P., Van Houtte, F., Roobrouck, A., Farhoudi, A., and Leroux-Roels, G. (2002). Hepatitis B virus surface antigen suppresses the activation of monocytes through interaction with a serum protein and a monocyte-specific receptor. J Gen Virol 83, 1281-1289.
von Schwedler, U.K., Stuchell, M., Muller, B., Ward, D.M., Chung, H.Y., Morita, E., Wang, H.E., Davis, T., He, G.P., Cimbora, D.M., et al. (2003). The protein network of HIV budding. Cell 114, 701-713.
Votteler, J., and Sundquist, W.I. (2013). Virus budding and the ESCRT pathway. Cell Host Microbe 14, 232-241.
Wang, H.C., Huang, W., Lai, M.D., and Su, I.J. (2006). Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Sci 97, 683-688.
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WHO (2017). Global hepatitis report, 2017.
Xiao, J., Xia, H., Zhou, J., Azmi, I.F., Davies, B.A., Katzmann, D.J., and Xu, Z. (2008). Structural basis of Vta1 function in the multivesicular body sorting pathway. Dev Cell 14, 37-49.
Xu, H.Z., Liu, Y.P., Guleng, B., and Ren, J.L. (2014). Hepatitis B Virus-Related Hepatocellular Carcinoma: Pathogenic Mechanisms and Novel Therapeutic Interventions. Gastrointest Tumors 1, 135-145.
Xu, Z., Jensen, G., and Yen, T.S. (1997). Activation of hepatitis B virus S promoter by the viral large surface protein via induction of stress in the endoplasmic reticulum. J Virol 71, 7387-7392.
Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., Huang, Y., Qi, Y., Peng, B., Wang, H., et al. (2012). Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1, e00049.
Chen, Y., Wei, H., Sun, R., and Tian, Z. (2005). Impaired function of hepatic natural killer cells from murine chronic HBsAg carriers. Int Immunopharmacol 5, 1839-1852.
Chou, S.F., Tsai, M.L., Huang, J.Y., Chang, Y.S., and Shih, C. (2015). The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion. PLoS Pathog 11, e1005123.
Freed, E.O. (2015). HIV-1 assembly, release and maturation. Nat Rev Microbiol 13, 484-496.
Garrus, J.E., von Schwedler, U.K., Pornillos, O.W., Morham, S.G., Zavitz, K.H., Wang, H.E., Wettstein, D.A., Stray, K.M., Cote, M., Rich, R.L., et al. (2001). Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107, 55-65.
Hetz, C. (2012). The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13, 89-102.
Hildt, E., Saher, G., Bruss, V., and Hofschneider, P.H. (1996). The hepatitis B virus large surface protein (LHBs) is a transcriptional activator. Virology 225, 235-239.
Hurley, J.H. (2010). The ESCRT complexes. Crit Rev Biochem Mol Biol 45, 463-487.
Hurley, J.H., and Hanson, P.I. (2010). Membrane budding and scission by the ESCRT machinery: it's all in the neck. Nat Rev Mol Cell Biol 11, 556-566.
Jiang, B., Himmelsbach, K., Ren, H., Boller, K., and Hildt, E. (2015). Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies. J Virol 90, 3330-3341.
Lambert, C., Doring, T., and Prange, R. (2007). Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and gamma 2-adaptin. Journal of virology 81, 9050-9060.
McCullough, J., Fisher, R.D., Whitby, F.G., Sundquist, W.I., and Hill, C.P. (2008). ALIX-CHMP4 interactions in the human ESCRT pathway. Proceedings of the National Academy of Sciences of the United States of America 105, 7687-7691.
Morita, E., Sandrin, V., McCullough, J., Katsuyama, A., Baci Hamilton, I., and Sundquist, W.I. (2011). ESCRT-III protein requirements for HIV-1 budding. Cell Host Microbe 9, 235-242.
Reignat, S., Webster, G.J., Brown, D., Ogg, G.S., King, A., Seneviratne, S.L., Dusheiko, G., Williams, R., Maini, M.K., and Bertoletti, A. (2002). Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J Exp Med 195, 1089-1101.
Roh, S., and Kim, K. (2003). Overcoming tolerance in hepatitis B virus transgenic mice: a possible involvement of regulatory T cells. Microbiol Immunol 47, 453-460.
Ron, D., and Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8, 519-529.
Rost, M., Mann, S., Lambert, C., Doring, T., Thome, N., and Prange, R. (2006). Gamma-adaptin, a novel ubiquitin-interacting adaptor, and Nedd4 ubiquitin ligase control hepatitis B virus maturation. J Biol Chem 281, 29297-29308.
Stieler, J.T., and Prange, R. (2014). Involvement of ESCRT-II in hepatitis B virus morphogenesis. PLoS One 9, e91279.
Strack, B., Calistri, A., Craig, S., Popova, E., and Gottlinger, H.G. (2003). AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114, 689-699.
Tamai, K., Shiina, M., Tanaka, N., Nakano, T., Yamamoto, A., Kondo, Y., Kakazu, E., Inoue, J., Fukushima, K., Sano, K., et al. (2012). Regulation of hepatitis C virus secretion by the Hrs-dependent exosomal pathway. Virology 422, 377-385.
Tralhao, J.G., Roudier, J., Morosan, S., Giannini, C., Tu, H., Goulenok, C., Carnot, F., Zavala, F., Joulin, V., Kremsdorf, D., et al. (2002). Paracrine in vivo inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation: an alternative mechanism of HBx-related pathogenesis. Proc Natl Acad Sci U S A 99, 6991-6996.
Usami, Y., Popov, S., Popova, E., Inoue, M., Weissenhorn, W., and H, G.G. (2009). The ESCRT pathway and HIV-1 budding. Biochem Soc Trans 37, 181-184.
Vanlandschoot, P., Van Houtte, F., Roobrouck, A., Farhoudi, A., and Leroux-Roels, G. (2002). Hepatitis B virus surface antigen suppresses the activation of monocytes through interaction with a serum protein and a monocyte-specific receptor. J Gen Virol 83, 1281-1289.
von Schwedler, U.K., Stuchell, M., Muller, B., Ward, D.M., Chung, H.Y., Morita, E., Wang, H.E., Davis, T., He, G.P., Cimbora, D.M., et al. (2003). The protein network of HIV budding. Cell 114, 701-713.
Votteler, J., and Sundquist, W.I. (2013). Virus budding and the ESCRT pathway. Cell Host Microbe 14, 232-241.
Wang, H.C., Huang, W., Lai, M.D., and Su, I.J. (2006). Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. Cancer Sci 97, 683-688.
Watanabe, T., Sorensen, E.M., Naito, A., Schott, M., Kim, S., and Ahlquist, P. (2007). Involvement of host cellular multivesicular body functions in hepatitis B virus budding. Proc Natl Acad Sci U S A 104, 10205-10210.
WHO (2017). Global hepatitis report, 2017.
Xiao, J., Xia, H., Zhou, J., Azmi, I.F., Davies, B.A., Katzmann, D.J., and Xu, Z. (2008). Structural basis of Vta1 function in the multivesicular body sorting pathway. Dev Cell 14, 37-49.
Xu, H.Z., Liu, Y.P., Guleng, B., and Ren, J.L. (2014). Hepatitis B Virus-Related Hepatocellular Carcinoma: Pathogenic Mechanisms and Novel Therapeutic Interventions. Gastrointest Tumors 1, 135-145.
Xu, Z., Jensen, G., and Yen, T.S. (1997). Activation of hepatitis B virus S promoter by the viral large surface protein via induction of stress in the endoplasmic reticulum. J Virol 71, 7387-7392.
Yan, H., Zhong, G., Xu, G., He, W., Jing, Z., Gao, Z., Huang, Y., Qi, Y., Peng, B., Wang, H., et al. (2012). Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1, e00049.

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