臺灣博碩士論文加值系統

登革感染症是藉由登革病毒感染的蚊子所傳播，感染症好發於熱帶與亞熱帶地區。登革感染症還會導致高致死率之重症疾病（如：登革出血熱、登革休克症候群），目前仍然缺乏有效的抗登革病毒之藥物或治療方法。登革病毒感染起始於和特定的受體結合，經由內吞作用(endocytosis)進入細胞中。先前的研究發現登革病毒感染宿主細胞後會誘導「自噬作用」(autophagy)，而自噬作用活化可以增強病毒的複製與子代病毒生產。我們實驗室之前的研究也發現，在登革病毒感染後15分鐘即能夠誘導自噬作用，且病毒與自噬小體(autophagosome)有colocalization現象。然而，登革病毒如何在感染初期即能誘導自噬作用，其詳細機制仍缺乏相關研究。根據研究證實，T-cell immunoglobulin and mucin domain 1 (TIM-1) 和TAM (Tyro3, Axl, Mer) 受體也是增強登革病毒感染宿主細胞的特定受體，此兩種受體可以辨認並結合凋亡細胞或病毒膜上的磷脂絲氨酸(phosphatidylserine, PS)。有研究顯示，TIM-1受體參與的胞吞作用(phagocytosis)以及Axl受體在肝臟發炎機制中，皆有誘導自噬作用的現象。但是這兩種受體在登革病毒感染所誘導之自噬作用過程中，其詳細機制仍未有相關研究。因此，本篇研究主要探討在登革病毒感染之初期，登革誘導之自噬作用是否透過TIM-1和Axl受體傳入活化自噬作用訊息，我分別使用螢光顯微鏡技術和病毒斑分析法來確認自噬作用活化和登革病毒子代生產。實驗結果顯示，TIM-1受體在抗體中和下，分別在登革病毒感染後15、30、60分鐘，自噬作用的活化情形有下降趨勢。而且，在病毒感染後48小時，其病毒子代生產也隨之減少。除了使用抗體中和，更進一步使用siRNA降低細胞的TIM-1受體表現量 (TIM-1 knockdown)。在TIM-1 knockdown細胞株的實驗結果中，發現登革病毒誘導之自噬作用，其活化程度更顯著地降低，且感染病毒48小時後，病毒子代的生產也隨之減少。由結果可知登革誘導之自噬作用可能藉由TIM-1受體傳入活化訊息。即使針對TIM-1抗體中和是否會阻斷病毒進入細胞，還未有足夠的實驗結果。但是本篇研究仍提供另一種觀點，探討在登革病毒感染初期，病毒與宿主細胞上特定受體反應、以及登革病毒誘導之自噬作用之間的關係。

Dengue disease is transmitted by dengue virus-infected mosquito and mostly occurs in tropical and subtropical area. Dengue disease could develop into severe diseases (dengue hemorrhage fever and dengue shock syndrome) that have high mortality rate and there are still not effective treatment or anti-dengue drugs. Dengue virus (DENV) infection begins from the binding of DENV particle with specific receptors and internalizes into host cells through receptor-mediated endocytosis. In addition, DENV infection induces autophagy that enhances viral replication and production. Our previous studies revealed that DENV-induced autophagy and the colocalization of virus particles with autophagosomes at 15 mins post-infection. However, the mechanism of the DENV-induced autophagy at the early phase of infection is still not elucidated. T-cell immunoglobulin and mucin domain 1 (TIM-1) and TAM (Tyro3, Axl, and Mer) receptors are putative DENV receptors through binding with phosphatidylserine (PS) on the DENV membrane and are required for enhancing DENV entry and infectivity. Moreover, TIM-1 and Axl were correlated with autophagy induction in phagocytosis and hepatic inflammation signaling, respectively. To investigate whether DENV-induced autophagy is mediated by either TIM-1 or Axl receptors, I used fluorescence imaging and plaque assay to verify the autophagy activation and virus production. Neutralization of TIM-1 by TIM-1 antibody reduced DENV-induced autophagy at 15, 30, and 60 mins post DENV-infection and decreased the viral production at 48 hrs post DENV-infection. In addition to TIM-1 neutralization, TIM-1 expression was further lowered by siRNA knockdown. The DENV-induced autophagy was significantly declined at 15, 30, and 60 mins post DENV-infection and the viral production decrease at 48 hrs post DENV-infection on TIM-1 knockdown. The results showed that the activation of DENV-induced autophagy could be mediated by TIM-1 receptor. Even though my experiments did not show the effect of TIM-1 neutralization on DENV entry clearly, this study has revealed another view of the interaction between the cellular receptors such as TIM-1 and DENV-induced autophagy at the early phase of DENV infection.

中文摘要 i
Abstract ii
Contents iii
Figures contents v
Table of Abbreviations vi
Introduction 1
1.Dengue virus (DENV) 1
2.DENV infectious life cycle 2
3.Autophagy in DENV infection 4
4.The roles of TIM-1 and Axl receptors in DENV infection 5
Hypothesis and Specific Aims 8
Materials and Methods 9
1.Cell culture 9
2.DENV amplification 10
3.Plaque assay 10
4.Immunofluorescence staining and flow cytometry analysis 11
5.Fluorescence image captured by confocal microscope 11
6.Quantification of DENV-induced autophagy activation by GFP-LC3 puncta numbers 12
7.DENV production 13
8.TIM-1 siRNA knockdown 14
9.DENV were labeled with fluorescence dye 14
Results 16
1.TIM-1 receptor expressed in Huh7.5, Huh7.5-GFP-LC3, and A549 cell lines. Axl receptor only expressed in A549 cell line. 16
2.TIM-1 receptor neutralization reduced DENV-induced autophagy 16
3.The effect of TIM-1 neutralization led to decrease of DENV production 17
4.TIM-1 knockdown efficiency was sustained from 48h to 96h siRNA post-transfection 18
5.TIM-1 knockdown effectively reduced DENV-induced autophagy 19
6.The DENV production was lowered in TIM-1-knockdown cells 19
Discussion 21
Conclusion 24
References 25

Figures contents
Figures contents
Figure 1. The expression of TIM-1 receptors........30
Figure 2. The expression of Axl receptors..........31
Figure 3. The distribution of TIM-1 and Axl by confocal microscope.......33
Figure 4. The primary screening of mono- and polyclonal TIM-1 antibodies for
DENV-induced autophagy ..........................34
Figure 5. DENV-induced autophagy in TIM-1 neutralization.........35
Figure 6. DENV production in TIM-1 neutralization ...........36
Figure 7. The verification of DENV entrance in TIM-1 neutralization.....38
Figure 8. The knockdown efficiency of TIM-1 siRNA in Huh7.5 cells.......40
Figure 9. The knockdown efficiency of TIM-1 siRNA in Huh7.5-GFP-LC3 cells.....42
Figure 10. DENV-induced autophagy in TIM-1 knockdown .........................43
Figure 11. DENV production in TIM-1 knockdown ...............................44

1.WHO. WHO report on Global Surveillance of Epidemic-prone Infectious Disease - Dengue and Dengue Hemorrhagic Fever. 2014; Available from: http://www.who.int/csr/resources/publications/dengue/CSR_ISR_2000_1/en/.
2.Bhatt, S., et al., The global distribution and burden of dengue. Nature, 2013. 496(7446): p. 504-7.
3.CDC, T. 全國登革熱本土病例及境外移入病例趨勢圖. 2018; Available from: https://nidss.cdc.gov.tw/ch/SingleDisease.aspx?dc=1&dt=4&disease=061&position=1.
4.Gubler DJ, C.G., Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg Infect Dis, 1995. 1: p. 3.
5.Gubler, D.J., Dengue and dengue hemorrrhagic fever. CLINICAL MICROBIOLOGY REVIEWS, 1998. 11: p. 17.
6.Chambers TJ, H.C., Galler R, Rice CM, Flavivirus genome organization, expression, and replication. Annu Rev Microbial, 1990. 44: p. 40.
7.Perera, R. and R.J. Kuhn, Structural proteomics of dengue virus. Curr Opin Microbiol, 2008. 11(4): p. 369-77.
8.Ying Zhang, J.C., Paul R.Chipman, Wei Zhang, Sergei V.Pletnev, Dagmar Sedlak, Timothy S.Baker, James H.Strauss, Richard J.Kuhn and Michael G.Rossmann, <2003_Structures of immature flavivirus particles.pdf>. The EMBO Journal, 2003. 22(11): p. 9.
9.Zhang, W., et al., Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol, 2003. 10(11): p. 907-12.
10.Idress S., A.U., A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan Genet Vaccines Ther, 2012. 10(6): p. 10.
11.Freire, J.M., et al., Intracellular nucleic acid delivery by the supercharged dengue virus capsid protein. PLoS One, 2013. 8(12): p. e81450.
12.Ewers, H. and M. Schelhaas, Analysis of virus entry and cellular membrane dynamics by single particle tracking. Methods Enzymol, 2012. 506: p. 63-80.
13.Cruz-Oliveira, C., et al., Receptors and routes of dengue virus entry into the host cells. FEMS Microbiol Rev, 2015. 39(2): p. 155-70.
14.van der Schaar, H.M., et al., Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells. PLoS Pathog, 2008. 4(12): p. e1000244.
15.Rodenhuis-Zybert, I.A., J. Wilschut, and J.M. Smit, Dengue virus life cycle: viral and host factors modulating infectivity. Cell Mol Life Sci, 2010. 67(16): p. 2773-86.
16.Suomalainen, M. and U.F. Greber, Uncoating of non-enveloped viruses. Curr Opin Virol, 2013. 3(1): p. 27-33.
17.Grove, J. and M. Marsh, The cell biology of receptor-mediated virus entry. J Cell Biol, 2011. 195(7): p. 1071-82.
18.Perera-Lecoin, M., et al., Flavivirus entry receptors: an update. Viruses, 2013. 6(1): p. 69-88.
19.Mercer, J. and U.F. Greber, Virus interactions with endocytic pathways in macrophages and dendritic cells. Trends Microbiol, 2013. 21(8): p. 380-8.
20.Heaton, N.S. and G. Randall, Dengue virus and autophagy. Viruses, 2011. 3(8): p. 1332-41.
21.Patricia Boya, F.R., Patrice Codogno, Emerging regulation and functions of autophagy.pdf. Nature Cell Biology, 2013. 15(7).
22.Zeng, X. and T.J. Kinsella, Impact of Autophagy on Chemotherapy and Radiotherapy Mediated Tumor Cytotoxicity: "To Live or not to Live". Front Oncol, 2011. 1: p. 30.
23.Rubinsztein, D.C., C.F. Bento, and V. Deretic, Therapeutic targeting of autophagy in neurodegenerative and infectious diseases. J Exp Med, 2015. 212(7): p. 979-90.
24.Pyo, J.O., J. Nah, and Y.K. Jung, Molecules and their functions in autophagy. Exp Mol Med, 2012. 44(2): p. 73-80.
25.Chiramel, A.I., N.R. Brady, and R. Bartenschlager, Divergent roles of autophagy in virus infection. Cells, 2013. 2(1): p. 83-104.
26.Parzych, K.R. and D.J. Klionsky, An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal, 2014. 20(3): p. 460-73.
27.Lee, Y.R., et al., Autophagic machinery activated by dengue virus enhances virus replication. Virology, 2008. 374(2): p. 240-8.
28.Heaton, N.S. and G. Randall, Dengue virus-induced autophagy regulates lipid metabolism. Cell Host Microbe, 2010. 8(5): p. 422-32.
29.Chu, L.W., et al., Single-virus tracking approach to reveal the interaction of Dengue virus with autophagy during the early stage of infection. J Biomed Opt, 2014. 19(1): p. 011018.
30.Jemielity, S., et al., TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine. PLoS Pathog, 2013. 9(3): p. e1003232.
31.Gordon J. Freeman, J.M.C., Dale T. Umetsu, Rosemarie H., Dekruyff, R. H., TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunological Reviews, 2010. 235: p. 18.
32.Albacker, L.A., et al., TIM-4, a receptor for phosphatidylserine, controls adaptive immunity by regulating the removal of antigen-specific T cells. J Immunol, 2010. 185(11): p. 6839-49.
33.Kim, H.Y., et al., T-cell immunoglobulin and mucin domain 1 deficiency eliminates airway hyperreactivity triggered by the recognition of airway cell death. J Allergy Clin Immunol, 2013. 132(2): p. 414-25 e6.
34.Takaharu Ichimura, E.J.P.v.A., Benjamin D. Humphreys, Lakshman Gunaratnam, Jeremy S. Duffield, and Joseph V. Bonventre, Kidney injury molecule–1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells. The Journal of Clinical Investigation, 2008. 118: p. 11.
35.Munz, C., The different autophagic roads by which phagosomes travel to lysosomes. EMBO J, 2015. 34(19): p. 2391-2.
36.Meertens, L., et al., The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry. Cell Host Microbe, 2012. 12(4): p. 544-57.
37.Moller-Tank, S. and W. Maury, Phosphatidylserine receptors: enhancers of enveloped virus entry and infection. Virology, 2014. 468-470: p. 565-80.
38.Rothlin, C.V., et al., TAM receptors are pleiotropic inhibitors of the innate immune response. Cell, 2007. 131(6): p. 1124-36.
39.Bhattacharyya, S., et al., Enveloped viruses disable innate immune responses in dendritic cells by direct activation of TAM receptors. Cell Host Microbe, 2013. 14(2): p. 136-47.
40.Han, J., et al., Autophagy induced by AXL receptor tyrosine kinase alleviates acute liver injury via inhibition of NLRP3 inflammasome activation in mice. Autophagy, 2016. 12(12): p. 2326-2343.
41.Dejarnac, O., et al., TIM-1 Ubiquitination Mediates Dengue Virus Entry. Cell Rep, 2018. 23(6): p. 1779-1793.
42.Boulant, S., M. Stanifer, and P.Y. Lozach, Dynamics of virus-receptor interactions in virus binding, signaling, and endocytosis. Viruses, 2015. 7(6): p. 2794-815.
43.Greber, U.F., Signaling in viral entry. Cell Mol Life Sci, 2002. 59(4): p. 19.
44.Mercer, J., M. Schelhaas, and A. Helenius, Virus entry by endocytosis. Annu Rev Biochem, 2010. 79: p. 803-33.