臺灣博碩士論文加值系統

數個登革病毒非結構(nonstructural, NS)蛋白除了參與病毒基因體RNA複製之外也參與病毒顆粒組裝，NS3蛋白是其中之一，但是機制未明。NS3蛋白由蛋白水解酶區與RNA解旋酶區組成的，蛋白水解酶區需與NS2B形成複合體才具有切割病毒多蛋白的活性。先前研究得知NS3蛋白RNA解旋酶區N369胺基酸一些置換突變可對病毒生活史造成不同的影響，例如：N369A與N369P突變株都無法在哺乳動物細胞中製造病毒顆粒，然而位在水解酶區的N158Y突變可專一補償N369A突變的缺陷。在本研究中我將分析在哺乳動物細胞中病毒RNA複製力、病毒多蛋白切割力和病毒蛋白穩定性受NS3蛋白N369A/P和N158Y+N369A突變的影響以期瞭解NS3蛋白在病毒顆粒組裝可能的角色。實驗結果顯示N369A突變或N158Y+N369A共同突變對各式活性都沒有顯著的影響，然而N369P突變會損害病毒複製力。由此排除病毒顆粒製造缺陷是源於N369A突變導致NS2B-NS3蛋白水解酶活性不彰所致。推測NS3 N369A突變可能影響病毒核殼的形成與病毒顆粒的裝配，而這需要進一步的研究。
登革病毒基因體RNA複製是由七種病毒非結構蛋白在被感染細胞內質網膜組成的複製複合體(Replication complex, RC)執行的。目前對複製複合體的架構及各非結構蛋白的功能所知有限。本論文第二部份擬深入探討NS4A和NS4B非結構膜蛋白間的交互作用。由先前病毒複製子的逆向遺傳研究得知NS4B膜蛋白第三穿膜區(TMD3)對複製活性有決定性的影響，而NS4A膜蛋白之I116M適應性突變可有效補救數個位在NS4B TMD3內的複製缺陷突變，這揭露了NS4A和NS4B膜蛋白間具有遺傳上的交互作用。NS4A和NS4B膜蛋白可分別形成雙體或多體亦可組成NS4A-NS4B複合體，但由於膜蛋白不易進行蛋白純化及生化特性分析。本論文擬使用雙分子螢光互補 (Bimolecular fluorescence complementary, BiFC)平台研究NS4B TMD3突變對於NS4B-NS4B和NS4A-NS4B蛋白之間交互作用的影響。我發現維持NS4B蛋白TMD3胺基酸官能基的分佈對蛋白間的交互作用是必需的，此外，NS4A I116M突變可補償NS4A-NS4B蛋白間的作用。NS4B TMD3內同一位置不同胺基酸置換也會對病毒蛋白間的交互作用及病毒複製造成不同的影響。目前的數據仍無法逐一釐清特定突變如何影響蛋白的交互作用，也未證實蛋白交互作用力與病毒複製能力的直接關係。未來，為深入瞭解NS4B TMD3在蛋白交互作用所扮演的角色，將使用BiFC平台繼續分析各式NS4B TMD3突變對NS4B-NS4B及NS4A-NS4B交互作用的影響，此外也將此分析平台運用在分析其他非結構蛋白(如NS1，NS3及NS5)與NS4B或NS4A的交互作用的研究中。

Most if not all dengue virus (DENV) nonstructural proteins are involved in viral RNA replication, whereas some nonstructural proteins including NS3 also have a role in infectious virion production. DENV NS3 protein contains an N-terminus protease domain and a C-terminus helicase domain, and NS3 protease needs to be co-factored with NS2B protein for viral polyprotein processing. Previous study in our laboratory showed that N369 substitutions in the NS3 helicase domain had various effects on DENV lifecycle events in the mammalian cell host; for example, both NS3 N369A/P mutations impaired virus production, whereas NS3 N158Y mutation in the protease domain specifically compensated for the N369A mutation in terms of virus production. To gain insight into the role of NS3 in infectious virus production, I studied the impact of N369A/P and N158Y+N369A mutations on viral RNA replication, viral polyprotein processing and viral protein stability. My findings show that N369P mutation impaired viral replication, whereas N369A/P alone and N369A+N158Y mutations did not alter the polyprotein processing function of NS3 and had no significant effect on the stability of viral structural and nonstructural proteins. These results exclude the possibility that inefficient cleavage by NS2B-NS3 protease is the main cause of NS3 N369 mutation-induced virus production deficiency. Whether NS3 N369 mutation affects nucleocapsid formation and subsequent virion assembly and egression warrants further investigation.
Dengue virus replication is mediated by an ER membrane-associated replication complex containing all seven viral nonstructural proteins. Viral nonstructural proteins engage in complicated protein interactions for replication complex formation, but the precise architecture/organization of the viral replication complex remains elusive. Previous mutagenesis analyses of the DENV selectable replicon showed that the TMD3 transmembrane segment of NS4B protein was critical for viral replication and that several NS4B TMD3 replication defective mutations could be rescued by NS4A I116M mutation, revealing a genetic interaction between NS4A and NS4B proteins. NS4A and NS4B are transmembrane proteins; each protein can form dimers or oligomers and the two proteins interact with each other, but their ER membrane-anchoring nature inhibits nonstructural protein purification for further biochemical characterization. In this thesis, a bimolecular fluorescence complementation (BiFC) assay was established to evaluate the impact of NS4B TMD3 mutation on NS4B-NS4B and NS4A-NS4B protein interactions. The results revealed that: (1) the distribution of NS4B TMD3 functional groups is critical for nonstructural protein interaction; (2) the intergenic NS4A I116M adaptive mutation has a compensatory effect on NS4A-NS4B interaction; and (3) different substitutions of the same residue of NS4B TMD3 may cause distinct effects on nonstructural protein interactions. Nevertheless, more studies are needed to reach a better understanding of the role of the NS4B TMD3 transmembrane segment in maintaining NS4B structure for proper nonstructural protein interactions and viral replication complex formation.

Contents
中文摘要 i
Abstract iii
Contents v
Figure contents vii
Chapter 1 – Introduction 1
1.1. Dengue virus (DENV) 1
1.2. DENV replication 2
1.3. NS3 in virus life cycle events 4
1.4. NS4B-NS4B and NS4A-NS4B interaction 5
Chapter 2 – Materials and Methods 9
2.1. Cell culture 9
2.2. Antibodies 9
2.3. Constructs 9
2.4. In vitro RNA synthesis 11
2.5. Cell electroporation with FL RNA 12
2.6. Cell transfection with plasmid DNA 12
2.7. Western blotting analysis 13
2.8. Immunofluorescence assay (IFA) 14
2.9. Visualization of bimolecular fluorescence complementation (BiFC)
and data analysis 14
Chapter 3 – Results 16
3.1. Studies of NS3 16
3.1.1. NS3 N369P mutation impairs viral RNA replication 16
3.1.2. NS3 N369A and N158Y+N369A mutations did not affect NS2B/3
protease activity 17 3.2. Studies of NS4B-NS4B and NS4A-NS4B interactions by BiFC 18
3.2.1. BiFC for NS4B TMD3 mutant study 18
3.2.2. Distribution of NS4B TMD3 functional groups is important for NS
protein interaction 20
3.2.3. Studies of NS4B P104K and P104E mutations 21
Chapter 4 – Discussion 22
4.1. Study of NS3 22
4.2. Studies of NS4B TMD3 mutation on NS4B-NS4B and NS4A-NS4B
interactions by BiFC 23
Chapter 5 – References 25
Chapter 6 – Figures 32
Chapter 7 – Appendix 50

Acosta EG, Castilla V and Damonte EB (2008) Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrin-mediated endocytosis. J. Gen. Virol. 89: 474-484.
Arias CF, Preugschat F and Strauss JH (1993) Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. Virology 193: 888-899.
Bartelma G and Padmanabhan R (2002) Expression, purification, and characterization of the RNA 5'-triphosphatase activity of dengue virus type 2 nonstructural protein 3. Virology 299: 122-132.
Bartenschlager R and Miller S (2008) Molecular aspects of Dengue virus replication. Future Microbiol 2: 155-165
Bazan JF and Fletterick RJ (1989) Detection of a trypsin-like serine protease domain in flaviviruses and pestviruses. Virology 171: 637-639.
Benarroch D, Selisko B, Locatelli GA, Maga G, Romette JL and Canard B (2004) The RNA helicase, nucleotide 5’-triphosphatase and RNA 5’-triphosphatase activities of dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motid in the helicase catalytic core. Virology 328: 208-218.
Bera AK, Kuhn RJ and Smith JL (2007) Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease. J. Biol. Chem. 282: 12883-12892.
Bhatt,S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, Drak, JM, Brownstein JS, Hoen AG, Sankoh O et al. (2013) The global distribution and burden of dengue. Nature 496: 504-507.
Byk LA and Gamarnik A (2016) Properties and functions of the dengue virus capsid protein. Ann. Rev. Virol. 3: 263-281.
Chambers TJ, Nestorowicz A, Amberg SM and Rice CM (1993) Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J. Virol. 67: 6797-6807.
Chatel-Chaix L, Cortese M, Romero-Brey I, Bender S, Neufeldt CJJ, Fischl W, Scaturro P, Schieber N, Schwab Y, Fischer B, Scaturro P, Schieber N, Schwab Y, Fischer B et al., (2016) Dengue virus perturbs mitochondrial morphodynamics to dampen innate immune responses. Cell Host Microbe 20: 342-356.
Chatel-Chaix L and Bartenschlager R (2014) Dengue virus-and hepatitis C virus-induced replication and assembly compartments: the enemy inside—caught in the web. J. Virol. 88: 5907-5911.
Chiang PY and Wu HN (2016) The role of surface basic amino acids of dengue virus NS3 helicase in viral RNA replication and enzyme activities. FEBS Lett. 590: 2307-2320.
Chu PW and Westaway EG (1985) Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. Virology 140: 68-79.
Dong H, Chang DC, Hua MHC, Lim SP, Chionh YH, Hia F, Lee YH, Kukkaro P, Lok S-M and Dedon PC (2012) 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase. PLoS Pathog 8: e1002642.
Falgout B, Pethel M, Zhang Y and Lai C (1991) Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J. Virol. 65: 2467-2475.
Guzman MG and Harris E (2015) Dengue. The Lancet 385: 453-465.
Hanley KA, Manlucu LR, Gilmore LE, Blaney JE, Hanson CT, Murphy BR and Whitehead SS (2003) A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4. Virology 312: 222-232.
Kakumani PK, Ponia SS, Sood V, Chinnappan M, Banerjea AC, Medigeshi GR, Malhotra P, Mukherjee SK and Bhatnagar RK (2013) Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor. J. Virol. 87: 8870-8883.
Kapoor M, Zhang L, Ramachandra M, Kusukawa J, Ebner KE and Padmanabhan R (1995) Association between NS3 and NS5 Proteins of Dengue Virus Type 2 in the Putative RNA Replicase Is Linked to Differential Phosphorylation of NS5. J. Biol. Chem. 270: 19100-19106.
Kerppola, TK (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat. Proto. 1: 1278-1286.
Klema VJ, Padmanabhan R and Choi KH (2015) Flaviviral replication complex: Coordination between RNA synthesis and 5′-RNA capping. Viruses 7: 4640-4656.
Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones CT, Mukhopadhyay S, Chipman PR and Strauss EG (2002) Structure of dengue virus: implications for flavivirus organization, maturation,and fusion. Cell 108: 717-725.
Lee CM, Xie X, Zou J, Li S-H, Lee MYQ, Dong H, Qin C-F, Kang C, Shi P-Y (2015) Determinants of dengue virus NS4A protein oligomerization. J. Virol. 89: 6171–6183.
Leung JY, Pijlman GP, Kondratieva N, Hyde J, Mackenzie JM and Khromykh AA (2008) Role of Nonstructural protein NS2A in flavivirus assembly. J. Virol. 82: 4731-4741.
Li Y, Kim YM, Zou J, Wang QY, Gayen S, Wong YL, Lee le T, Xie X, Huang Q, Lescar J, Shi PY and Kang C (2015) Secondary structure and membrane topology of dengue virus NS4B N-terminal 125 amino acids. Biochim Biophys Acta. 1848: 3150-3157.
Lim SP, Wang Q-Y, Noble CG, Chen Y-L, Dong H, Zou B, Yokokawa F, Nilar S, Smith P and Beer D (2013) Ten years of dengue drug discovery: progress and prospects. Antiviral Res.100: 500-519.
Lin C, Amberg SM, Chambers TJ and Rice CM (1993) Cleavage at a novel site in the NS4A region by the yellow fever virus NS2B-3 proteinase is a prerequisite for processing at the downstream 4A/4B signalase site. J. Virol. 67: 2327-2335.
Lindenbach BD, Murray CL, Thiel HJ and Rice CM. Flviviridae. In Knipe, DM and Howley, P editors. Fields Virology. Philadelphia: Lippincott Williams & Wilins; 2013. p. 712-746.
Lindenbach BD and Rice CM (1999) Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. J. Virol. 73: 4611–4621.
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 3.1, Hierarchical Structure of Proteins.
Luo D, Xu T, Hunke C, Grüber G, Vasudevan SG and Lescar J (2008a) Crystal structure of the NS3 protease-helicase from dengue virus. J. Virol. 82: 173-183.
Luo D, Xu T, Watson RP, Scherer-Becker D, Sampath A, Jahnke W, Yeong SS, Wang CH, Lim SP, Strongin A, Vasudevan SG and Lescar J (2008b) Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein. EMBO J. 27: 3209–3219.
Mackenzie JM, Khromykh AA, Jones MK and Westaway EG (1998) Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology 245: 203-215.
Matusan AE, Pryor MJ, Davidson AD and Wright PJ (2001) Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. J. Virol. 75: 9633-9643.
McLean JE, Wudzinska A, Datan E, Quaglino D and Zakeri Z. (2011) Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication. J. Biol. Chem. 286: 22147-22159.
Miller S, Kastner S, Krijnse-Locker J, Bühler S and Bartenschlager R (2007) The non-structural protein 4A of dengue virus is an integral membrane protein inducing membrane alterations in a 2K-regulated manner. J. Biol. Chem. 282: 8873-8882.
Miller S, Sparacio S and Bartenschlager R (2006) Subcellular localization and membrane topology of the dengue virus type 2 non-structural protein 4B. J. Biol. Chem. 281: 8854-8863.
Morell M, Espargaro A, Aviles FX and Ventura S (2008) Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry. Nat. Protoc. 3, 22–33.
Muller DA and Young PR (2013) The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker. Antiviral Res. 98:192-208.
Muñoz-Jordán JL, Sánchez-Burgos GG, Laurent-Rolle M and García-Sastre A (2003) Inhibition of interferon signaling by dengue virus. Proc. Natl. Acad. Sci. USA 100: 14333-14338.
Muñoz-Jordán JL, Laurent-Rolle M, Ashour J, Martínez-Sobrido L, Ashok M, Lipkin WI and García-Sastre A (2005) Inhibition of alpha/beta interferon signaling by the NS4B protein of flaviviruses. J. Virol. 79: 8004-8013.
Murray CL, Jones CT and Rice CM (2008) Architects of assembly: roles of Flaviviridae nonstructural proteins in virion morphogenesis. Nat. Rev. Micro. 6: 699-708.
Naik NG and Wu HN (2015) Mutation of putative N-glycosylation sites on dengue virus NS4B decreases RNA replication. J. Virol. 89: 6746-6760.
Nestorowicz A, Chambers TJ and Rice CM (1994) Mutagenesis of the yellow fever virus NS2A/2B cleavage site: effects on proteolytic processing, viral replication, and evidence for alternative processing of the NS2A protein. Virology 199: 114-123.
Niyomrattanakit P, Winoyanuwattikun P, Chanprapaph S, Angsuthanasombat C, Panyim S and Katzenmeier G (2004) Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation. J. Virol. 78: 13708-13716.
Patkar CG and Kuhn RJ (2008) Yellow fever virus NS3 plays an essential role in virus assembly independent of its known enzymatic functions. J. Virol. 82: 3342-3352.
Rastogi M, Sharma N and Singh SK (2016) Flavivirus NS1: a multifaceted enigmatic viral protein. Virol. J. 13: 131.
Ray D, Shah A, Tilgner M, Guo Y, Zhao Y, Dong H, Deas TS, Zhou Y, Li H and Shi P-Y (2006) West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5. J. Virol. 80: 8362-8370.
Sansom MS and Weinstein H (2000) Hinges, swivels and switches: the role of prolines in signalling via transmembrane α-helices. Trends in Pharm. Sci. 21: 445-451.
Sahili AE and Lescar L (2017) Dengue virus non-structural protein 5. Viruses: 9: 91-110.
Scaturro P, Cortese M, Chatel-Chaix L and Bartenschlager R (2015) Dengue virus non-structural protein 1 modulates infectious particle production via Interaction with the structural proteins. PloS Pathog. 11: e1005277.
Smit JM, Moesker B, Rodenhuis-Zybert I and Wilschut J (2011) Flavivirus cell entry and membrane fusion. Viruses 3: 160-171.
Stern O, Hung Y-F, Valdau O, Yaffe Y, Harris E, Hoffmann,S. Willbold D and Sklan EH (2013) An N-terminal amphipathic helix in dengue virus nonstructural protein 4A mediates oligomerization and is essential for replication. J. Virol. 87: 4080-4085.
Tay MYF, Saw WG, Zhao Y, Chan KWK, Singh D, Chong Y, Forwood JK, Ooi EE, Grüber G, Lescar J and Luo D (2015) The C-terminal 50 Amino Acid Residues of Dengue NS3 Protein Are Important for NS3-NS5 Interaction and Viral Replication. J. Biol. Chem. 290: 2379-2394.
Teoh PG, Huang ZS, Pong WL, Chen PJ and Wu HN (2014) Maintenance of dimer conformation by the dengue virus core protein a4-a4' helix pair is critical for nucleocapsid formation and virus production. J. Virol. 88: 7998-8015.
van Cleef KWR, Overheul GJ, Thomassen MC, Kaptein SJF, Davidson AD, Jacobs M, Neyts J, van Kuppeveld JM and van Rij RP (2013) Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication. Antiviral Res. 99: 165-171.
Wang CC, Huang ZS and Wu HN (2009) Analysis of the nucleotide triphosphatase, RNA triphosphatase, and unwinding activities of the helicase domain of dengue virus NS3 protein. FEBS Lett. 583: 691-696.
Wang Q-Y, Dong H, Zou B, Karuna R, Wan KF, Zou J, Susila A, Yip A, Shan C and Yeo KL (2015) Discovery of dengue virus NS4B inhibitors. J. Virol. 89: 8233-8244.
Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, Fuller SD, Antony C, Krijnse-Locker J and Bartenschlager R (2009) Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell host & microbe 5: 365-375.
Westaway EG, Mackenzie JM, Kenney MT, Jones, MK and Khromykh AA (1997). Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. J. Virol. 71: 6650-6661.
Wu RH, Tasi MH, Tsai KN, Tian JN, Wu JS, Wu SY, Chern JH, Chen CH and Yueh A (2017) Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments. J. Virol. 91: e01836-16.
Xie X, Wang Q-Y, Xu HY, Qing M, Kramer L, Yuan Z and Shi P-Y (2011) Inhibition of dengue virus by targeting viral NS4B protein. J. Virol. 85: 11183-11195.
Xie X, Zou J, Puttikhunt C, Yuan Z, Shi P-Y (2015) Two distinct sets of NS2A molecules are responsible for dengue virus RNA synthesis and virion assembly. J. Virol. 89: 1298–1313.
Youn S, Li T, McCune BT, Edeling MA, Fremont DH, Cristea IM and Diamond MS (2012) Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus. J. Virol. 86: 7360-7371.
Yu L, Takeda K and Markoff L (2013) Protein-protein interactions among West Nile non-structural proteins and transmembrane complex formation in mammalian cells. Virology. 446: 365–377.
Zhang Y, Zhang W, Ogata S, Clements D, Strauss JH, Baker TS, Kuhn RJ and Rossmann MG (2004) Conformational changes of the flavivirus E glycoprotein. Structure 12:, 1607-1618.
Zmurko J, Neyts J and Dallmeier K (2015) Flaviviral NS4b, chameleon and jack‐in‐the‐box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention. Rev. Med. Virol. 25: 205-223.
Zou J, Wang QY, Xie X, Lu S, Yau YH, Yuan Z, Shochat SG, Kang C, Lescar J and Shi P-Y (2015a) Mapping the interactions between the NS4B and NS3 proteins of dengue virus. J. Virol. 89: 3471-3483.
Zou J, Xie X, Chandrasekaran R, Reynaud A, Yap L., Wang Q-Y, Dong H, Kang C, Yuan Z and Lescar J (2014) Dimerization of flavivirus NS4B protein. J. Virol. 88: 3379-3391.
Zou J, Xie X, Wang Q-Y, Dong H, Lee MY, Kang C, Yuan Z and Shi P-Y (2015b) Characterization of dengue virus NS4A and NS4B protein interaction. J. Virol. 89: 3455-3470.