當細胞受到RNA病毒感染的時候，細胞質中的重要的病毒RNA接受器RIG-I，會偵測病毒RNA，例如登革病毒與流感病毒，之後活化下游訊息傳遞路徑，並且啟動抗病毒免疫。RIG-I分子的C端可以結合病毒的5端三磷酸RNA，打開RIG-I的封閉構形，使得N端的CARD片段釋放出來並且被泛素修飾接著進行寡聚合作用。接著此RIG-I複合物會活化下游位於粒線體的MAVS分子，誘導更下游第一型干擾素的產生，進而達到抗病毒的效果。TAPE (TBK1-Associated Protein in Endolysosomes)是我們實驗室感興趣的先天免疫調控分子，並且位於MAVS上游參與RLR的訊息傳遞路徑。我的論文主要工作是探討TAPE是如何調控RIG-I泛素化修飾以及寡聚合作用以及含有TAPE的endosomes在RLR的訊息傳遞中是否扮演關鍵的橋樑角色。不同於目前RIG-I活化後會位移到粒線體上與MAVS交互作用的想法，我們實驗室先前的證據指出RIG-I在受到RLR配體刺激之後會先位移到含有早期的endosome標示蛋白Rab5或是TAPE的endosome。在我的論文研究中更進一步探討RIG-I在RNA病毒感染之後是否會位移到endosome。共顎焦顯微鏡結果顯示RIG-I在仙台病毒感染之後會與TAPE位於相同位置。同樣地，RIG-I在仙台病毒感染之後會與Rab5早期的endosome標示蛋白重合。我接下來探討RIG-I是否會與其他的endosome標示蛋白有交互作用。共顎焦顯微鏡結果顯示polyI:C刺激後RIG-I會與晚期endosome標示蛋白Rab7部分重疊。此外，我的結果顯示調降Rab7表現會壓抑RIG-I和MDA5誘導的干擾素β活化。接下來我進一步探討TAPE是否調控RIG-I的泛素化修飾以及寡聚合作用。生化實驗分析結果指出TAPE基因剔除會降低RIG-I的K63連結多泛素鍊修飾。此外，光學結果顯示polyI:C刺激後TAPE會跟TRIM25重合。在光學定量實驗顯示TAPE基因剔除細胞會減少RIG-I蛋白複合物形成。這些結果證明TAPE調控RIG-I的泛素化修飾以及寡聚合作用。先前我們實驗室在老鼠胚胎纖維母細胞的結果中發現TAPE在RIG-I路徑活化第一型干擾素扮演著重要的角色。鑒於這些事實，我們更進一步探討在其他免疫細胞中TAPE對於RIG-I路徑活化的角色。體外實驗結果顯示傳統樹突細胞不論是在RLR配體刺激或RNA病毒的感染下，TAPE的缺失會降低干擾素β的產生。總結以上結果，我的論文工作證明在配體刺激或RNA病毒的感染下含有TAPE和Rab5的endosome在RIG-I訊息上會扮演一個平台的角色。

Cytosolic RNA sensor RIG-I plays a key role in detecting RNA virus infection, like dengue and influenza, to trigger antiviral immunity. The C-terminal domain of RIG-I functions to bind 5’ triphosphate viral RNA, and then the N-terminal tandem CARD domains of RIG-I will be released for K63-linked ubiquitination and the oligomerization of RIG-I. This RIG-I protein complex subsequently engages with a mitochondrial adaptor MAVS to induce downstream signaling to type I IFN-mediated antiviral defenses. Our previous study identified an innate immune regulator TAPE (TBK1-associated protein in endolysosomes) acting upstream of MAVS to regulate RIG-I signaling. Therefore, my current work aims to explore the molecular mechanism of how TAPE regulates RIG-I ubiquitination and oligomerization, and whether TAPE and its-associated endosomes act as a platform to bridge RIG-I signaling to MAVS. Distinct from the current idea that activated RIG-I is translocated to mitochondria for engaging with MAVS, our previous findings indicated that RIG-I was translocated to TAPE- and Rab5-containing endosomes after ligand stimulation. In my thesis, I attempted to further examine whether RIG-I is translocated to endosomes upon RNA virus infection. Confocal microscopy analyses showed that RIG-I was co-localized with TAPE after Sendai virus infection. Likewise, RIG-I was shown to be localized with an early endosomal marker Rab5 after Sendai virus infection. Next, I addressed if RIG-I interplays with other endosomal proteins during signaling. My microscopic analyses demonstrated that RIG-I was partially co-localized with a late endosomal marker Rab7 upon polyI:C stimulation. Moreover, my results also showed that knockdown of endolysosomal protein Rab7 impaired RIG-I CARD- and MDA5-mediated IFN-β induction. Next, I explored whether TAPE regulates the ubiquitination and oligomerization of RIG-I. Biochemical results revealed that TAPE knockout decreased K63-linked polyubiquitination of RIG-I. Our previous work has indicated that TAPE interacts with TRIM25 which is an E3 ubiquitin ligase of RIG-I. My thesis work further found that TAPE is co-localized with TRIM25 upon polyI:C stimulation. Microscopic quantitative analyses showed that TAPE deficiency decreased the formation of the RIG-I puncta in mammalian cells. These results suggest that TAPE regulates the ubiquitination and oligomerization of the RIG-I. Our previous work has demonstrated that TAPE was critical for RIG-I signaling to typeⅠIFN induction in primary mouse embryonic fibroblasts (MEFs). Given these facts, we further explored the functional role of TAPE in RIG-I signaling in conventional dendritic cells (cDCs). TAPE-deficient cDCs were impaired type I IFN induction in response to polyI:C and 5’pppRNA stimulation. In addition, western blot analysis also showed the impairment of IRF3 phosphorylation in TAPE-deficient cDCs upon stimulation. In conclusion, my thesis work suggests that TAPE- and Rab5-containing endosomes serve as a platform for RIG-I signaling during ligand stimulation and RNA virus infection. Ex vivo studies revealed that TAPE is essential for RIG-I signaling to IFN-β production upon RLR ligand stimulation.

1. D. Goubau, S. Deddouche, C. Reis e Sousa, Cytosolic sensing of viruses. Immunity 38, 855-869 (2013).
2. Y. K. Chan, M. U. Gack, RIG-I-like receptor regulation in virus infection and immunity. Current opinion in virology 12, 7-14 (2015).
3. M. Yoneyama et al., The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 5, 730-737 (2004).
4. H. Kato et al., Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J Exp Med 205, 1601-1610 (2008).
5. T. Kawai, S. Akira, The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nature immunology 11, 373-384 (2010).
6. R. Zust et al., Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat Immunol 12, 137-143 (2011).
7. O. Takeuchi, S. Akira, Pattern recognition receptors and inflammation. Cell 140, 805-820 (2010).
8. V. Hornung et al., 5'-Triphosphate RNA is the ligand for RIG-I. Science 314, 994-997 (2006).
9. A. M. Bruns, C. M. Horvath, Activation of RIG-I-like receptor signal transduction. Critical reviews in biochemistry and molecular biology 47, 194-206 (2012).
10. S. Rothenfusser et al., The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I. J Immunol 175, 5260-5268 (2005).
11. A. Komuro, C. M. Horvath, RNA- and virus-independent inhibition of antiviral signaling by RNA helicase LGP2. J Virol 80, 12332-12342 (2006).
12. K. S. Childs, R. E. Randall, S. Goodbourn, LGP2 plays a critical role in sensitizing mda-5 to activation by double-stranded RNA. PLoS One 8, e64202 (2013).
13. A. M. Bruns, G. P. Leser, R. A. Lamb, C. M. Horvath, The innate immune sensor LGP2 activates antiviral signaling by regulating MDA5-RNA interaction and filament assembly. Mol Cell 55, 771-781 (2014).
14. R. B. Seth, L. Sun, C. K. Ea, Z. J. Chen, Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122, 669-682 (2005).
15. T. Kawai et al., IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol 6, 981-988 (2005).
16. L. G. Xu et al., VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell 19, 727-740 (2005).
17. E. Meylan et al., Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167-1172 (2005).
18. J. A. Gorman et al., The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity. Nat Immunol 18, 744-752 (2017).
19. G. I. Rice et al., Gain-of-function mutations in IFIH1 cause a spectrum of human disease phenotypes associated with upregulated type I interferon signaling. Nat Genet 46, 503-509 (2014).
20. F. Rutsch et al., A specific IFIH1 gain-of-function mutation causes Singleton-Merten syndrome. Am J Hum Genet 96, 275-282 (2015).
21. D. J. Smyth et al., A genome-wide association study of nonsynonymous SNPs identifies a type 1 diabetes locus in the interferon-induced helicase (IFIH1) region. Nat Genet 38, 617-619 (2006).
22. M. U. Gack et al., TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916-920 (2007).
23. F. Civril et al., The RIG-I ATPase domain structure reveals insights into ATP-dependent antiviral signalling. EMBO Rep 12, 1127-1134 (2011).
24. F. Ferrage et al., Structure and dynamics of the second CARD of human RIG-I provide mechanistic insights into regulation of RIG-I activation. Structure 20, 2048-2061 (2012).
25. E. Kowalinski et al., Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell 147, 423-435 (2011).
26. M. Okamoto, T. Kouwaki, Y. Fukushima, H. Oshiumi, Regulation of RIG-I Activation by K63-Linked Polyubiquitination. Front Immunol 8, 1942 (2017).
27. M. U. Gack et al., Roles of RIG-I N-terminal tandem CARD and splice variant in TRIM25-mediated antiviral signal transduction. Proc Natl Acad Sci U S A 105, 16743-16748 (2008).
28. W. Zeng et al., Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315-330 (2010).
29. Y. K. Chan, M. U. Gack, Viral evasion of intracellular DNA and RNA sensing. Nat Rev Microbiol 14, 360-373 (2016).
30. C. Chiang, M. U. Gack, Post-translational Control of Intracellular Pathogen Sensing Pathways. Trends Immunol 38, 39-52 (2017).
31. J. G. Sanchez et al., Mechanism of TRIM25 Catalytic Activation in the Antiviral RIG-I Pathway. Cell Rep 16, 1315-1325 (2016).
32. H. Oshiumi et al., The ubiquitin ligase Riplet is essential for RIG-I-dependent innate immune responses to RNA virus infection. Cell Host Microbe 8, 496-509 (2010).
33. C. Cadena et al., Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity. Cell 177, 1187-1200 e1116 (2019).
34. Y. Shi et al., Ube2D3 and Ube2N are essential for RIG-I-mediated MAVS aggregation in antiviral innate immunity. Nat Commun 8, 15138 (2017).
35. J. A. Potter, R. E. Randall, G. L. Taylor, Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain. BMC structural biology 8, 11 (2008).
36. F. Hou et al., MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell 146, 448-461 (2011).
37. K. Onoguchi et al., Virus-infection or 5'ppp-RNA activates antiviral signal through redistribution of IPS-1 mediated by MFN1. PLoS Pathog 6, e1001012 (2010).
38. C. Odendall et al., Diverse intracellular pathogens activate type III interferon expression from peroxisomes. Nature immunology 15, 717-726 (2014).
39. E. Dixit et al., Peroxisomes are signaling platforms for antiviral innate immunity. Cell 141, 668-681 (2010).
40. S. M. Horner, H. M. Liu, H. S. Park, J. Briley, M. Gale, Jr., Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus. Proc Natl Acad Sci U S A 108, 14590-14595 (2011).
41. H. M. Liu et al., The mitochondrial targeting chaperone 14-3-3epsilon regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity. Cell Host Microbe 11, 528-537 (2012).
42. H. Wu, Higher-order assemblies in a new paradigm of signal transduction. Cell 153, 287-292 (2013).
43. J. C. Kagan, V. G. Magupalli, H. Wu, SMOCs: supramolecular organizing centres that control innate immunity. Nat Rev Immunol 14, 821-826 (2014).
44. S. C. Lin, Y. C. Lo, H. Wu, Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 465, 885-890 (2010).
45. J. M. Blander, A long-awaited merger of the pathways mediating host defence and programmed cell death. Nature reviews. Immunology 14, 601-618 (2014).
46. A. Lu et al., Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156, 1193-1206 (2014).
47. A. Peisley, B. Wu, H. Yao, T. Walz, S. Hur, RIG-I forms signaling-competent filaments in an ATP-dependent, ubiquitin-independent manner. Mol Cell 51, 573-583 (2013).
48. A. Peisley, B. Wu, H. Xu, Z. J. Chen, S. Hur, Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I. Nature 509, 110-114 (2014).
49. A. Peisley et al., Cooperative assembly and dynamic disassembly of MDA5 filaments for viral dsRNA recognition. Proc Natl Acad Sci U S A 108, 21010-21015 (2011).
50. B. Wu et al., Structural basis for dsRNA recognition, filament formation, and antiviral signal activation by MDA5. Cell 152, 276-289 (2013).
51. B. Wu et al., Molecular imprinting as a signal-activation mechanism of the viral RNA sensor RIG-I. Mol Cell 55, 511-523 (2014).
52. S. Liu et al., MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades. Elife 2, e00785 (2013).
53. J. P. Lin, Y. K. Fan, H. M. Liu, The 14-3-3eta chaperone protein promotes antiviral innate immunity via facilitating MDA5 oligomerization and intracellular redistribution. PLoS Pathog 15, e1007582 (2019).
54. L. Basel-Vanagaite et al., The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation. Journal of medical genetics 43, 203-210 (2006).
55. A. Rogaeva, K. Galaraga, P. R. Albert, The Freud-1/CC2D1A family: transcriptional regulators implicated in mental retardation. Journal of neuroscience research 85, 2833-2838 (2007).
56. A. Nakamura, M. Naito, T. Tsuruo, N. Fujita, Freud-1/Aki1, a novel PDK1-interacting protein, functions as a scaffold to activate the PDK1/Akt pathway in epidermal growth factor signaling. Molecular and cellular biology 28, 5996-6009 (2008).
57. C. H. Chang et al., TBK1-associated protein in endolysosomes (TAPE) is an innate immune regulator modulating the TLR3 and TLR4 signaling pathways. The Journal of biological chemistry 286, 7043-7051 (2011).
58. P. Burkhard, J. Stetefeld, S. V. Strelkov, Coiled coils: a highly versatile protein folding motif. Trends in cell biology 11, 82-88 (2001).
59. M. A. Lemmon, Membrane recognition by phospholipid-binding domains. Nature reviews. Molecular cell biology 9, 99-111 (2008).
60. X. M. Ou et al., Freud-1: A neuronal calcium-regulated repressor of the 5-HT1A receptor gene. The Journal of neuroscience : the official journal of the Society for Neuroscience 23, 7415-7425 (2003).
61. A. Rogaeva, P. R. Albert, The mental retardation gene CC2D1A/Freud-1 encodes a long isoform that binds conserved DNA elements to repress gene transcription. The European journal of neuroscience 26, 965-974 (2007).
62. Y. Usami et al., Regulation of CHMP4/ESCRT-III function in human immunodeficiency virus type 1 budding by CC2D1A. Journal of virology 86, 3746-3756 (2012).
63. R. Jaekel, T. Klein, The Drosophila Notch inhibitor and tumor suppressor gene lethal (2) giant discs encodes a conserved regulator of endosomal trafficking. Developmental cell 11, 655-669 (2006).
64. N. Drusenheimer et al., The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling. PLoS Genet 11, e1005749 (2015).
65. M. Zhao, X. D. Li, Z. Chen, CC2D1A, a DM14 and C2 domain protein, activates NF-kappaB through the canonical pathway. J Biol Chem 285, 24372-24380 (2010).
66. A. Matsuda et al., Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways. Oncogene 22, 3307-3318 (2003).
67. M. C. Manzini et al., CC2D1A regulates human intellectual and social function as well as NF-kappaB signaling homeostasis. Cell reports 8, 647-655 (2014).
68. C. Y. Yang, T. H. Yu, W. L. Wen, P. Ling, K. S. Hsu, Conditional Deletion of CC2D1A Reduces Hippocampal Synaptic Plasticity and Impairs Cognitive Function through Rac1 Hyperactivation. J Neurosci 39, 4959-4975 (2019).
69. K. R. Chen et al., TBK1-associated protein in endolysosomes (TAPE)/CC2D1A is a key regulator linking RIG-I-like receptors to antiviral immunity. J Biol Chem 287, 32216-32221 (2012).
70. M. Zhao, J. Raingo, Z. J. Chen, E. T. Kavalali, Cc2d1a, a C2 domain containing protein linked to nonsyndromic mental retardation, controls functional maturation of central synapses. Journal of neurophysiology 105, 1506-1515 (2011).
71. H. Hu, S. C. Sun, Ubiquitin signaling in immune responses. Cell research 26, 457-483 (2016).
72. L.-C. Wang, (2017).
73. K. R. Chen, National Cheng Kung University, (2016).
74. 羅尹秋, 國立成功大學, (2012).
75. C. D. Hodge, L. Spyracopoulos, J. N. Glover, Ubc13: the Lys63 ubiquitin chain building machine. Oncotarget 7, 64471-64504 (2016).
76. X. Jiang et al., Ubiquitin-induced oligomerization of the RNA sensors RIG-I and MDA5 activates antiviral innate immune response. Immunity 36, 959-973 (2012).
77. H. Kato et al., Cell type-specific involvement of RIG-I in antiviral response. Immunity 23, 19-28 (2005).
78. H. Kato et al., Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441, 101-105 (2006).
79. M. T. Sanchez-Aparicio, J. Ayllon, A. Leo-Macias, T. Wolff, A. Garcia-Sastre, Subcellular Localizations of RIG-I, TRIM25, and MAVS Complexes. J Virol 91, (2017).
80. N. Nakamura et al., Endosomes are specialized platforms for bacterial sensing and NOD2 signalling. Nature 509, 240-244 (2014).
81. A. T. Irving et al., The immune receptor NOD1 and kinase RIP2 interact with bacterial peptidoglycan on early endosomes to promote autophagy and inflammatory signaling. Cell Host Microbe 15, 623-635 (2014).
82. H. Yanai et al., HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 462, 99-103 (2009).
83. A. Mukherjee et al., Retinoic acid-induced gene-1 (RIG-I) associates with the actin cytoskeleton via caspase activation and recruitment domain-dependent interactions. J Biol Chem 284, 6486-6494 (2009).
84. T. Ohman, J. Rintahaka, N. Kalkkinen, S. Matikainen, T. A. Nyman, Actin and RIG-I/MAVS signaling components translocate to mitochondria upon influenza A virus infection of human primary macrophages. J Immunol 182, 5682-5692 (2009).
85. H. Lin et al., The long noncoding RNA Lnczc3h7a promotes a TRIM25-mediated RIG-I antiviral innate immune response. Nature Immunology 20, 812-823 (2019).