臺灣博碩士論文加值系統

登革病毒非結構蛋白3 (NS3) 由蛋白水解酶區與解螺旋酶區所組成。NS3蛋白水解酶需與NS2B形成複合體以切割病毒多聚蛋白，NS3解螺旋酶則可促使RNA結構改變，這兩個酵素活性對於病毒複製是必須的。然而目前對於登革病毒NS3蛋白，在病毒組裝和釋出過程中所扮演的角色所知甚微。先前藉著在哺乳動物BHK-21細胞中表達登革病毒全基因體RNA的研究發現一些位於NS3蛋白「解螺旋酶區」N369胺基酸的置換突變株在未顯著影響病毒RNA複製能力的情況下，竟然無法製造出具有感染力的病毒，這也進一步指出NS3蛋白應參與在病毒的製程裡，而這個鮮被報導的現象，即是本研究探討NS3的最初動機。意外的是，一個位在NS3「蛋白水解酵素區」的N158Y適應性突變可成功地讓特定N369突變株製造具有感染力的病毒。有鑑於此，我聚焦在NS2B/NS3的蛋白水解功能，探討NS3 N369突變能否透過NS2B/NS3複合體影響病毒多聚蛋白鏈的切割 (含核殼Core結構蛋白的熟成以及除NS1蛋白外所有非結構蛋白的切割) 而喪失製造病毒的能力。一些實驗結果顯示NS3 N369不同的突變並不會影響NS2B/NS3在切割非結構蛋白的活性，因此暗示問題可能發生在核殼蛋白無法熟成或效率不彰。在本研究中我利用可持續表達NS3 N369突變蛋白之登革次基因體複製子(subgenomic replicon)的細胞株大量表達登革病毒結構蛋白，以檢視病毒結構蛋白的切割和核殼蛋白的熟成過程。螢光免疫染色和西方墨點分析的結果顯示，無論是能夠或不能夠製造出具有感染力病毒的NS3 N369突變蛋白或是伴隨著NS3 N158Y適應性突變的NS3 N369突變蛋白，催化核殼蛋白熟成的能力與野生型NS3蛋白相比並無顯著差異。總結，NS3 N369置換突變所引發之製造具感染力病毒能力的缺失，並非肇因於NS3蛋白水解酶與解螺旋酶的功能喪失。未來將需要更多的研究來解答此因果關係，而我們也將探討病毒組裝和釋出的過程是否受到NS3 N369置換突變的影響。

Dengue virus (DENV) non-structural protein 3 (NS3) is comprised of protease and helicase domains. NS3 protease is co-factored with NS2B to process viral polyprotein, whereas NS3 helicase facilitates RNA structural re-arrangements. Both protease and helicase activities of DENV NS3 are indispensable for viral replication, whereas it is not clear if DENV NS3 has a role in virus assembly and egress. Previous studies of DENV full-length (FL) infectious RNA in mammalian BHK-21 cells revealed that several NS3 “helicase” domain N369 mutants are replication-competent but fail to produce infectious virus, suggesting that NS3 participates in virus production. Of note, an adaptive mutation of the NS3 “protease” domain (N158Y) has been shown to rescue some N369 mutants, enabling them to produce infectious virus. This prompted me to investigate further the role of NS3 in infectious virus production. Here, I aimed to investigate the mechanism of how the NS3 N369 mutation impairs virus production, with a focus on the NS2B/NS3 protease function. Several experiments have shown that different NS3 N369 mutations do not affect the non-structural protein processing activity of NS2B/NS3, which suggests impairment or inefficiency of core protein maturation of NS3 N369 mutants. In this study, I overexpressed DENV structural proteins in stable cell lines harboring an NS3 N369 mutant DENV subgenomic replicon to examine viral structural protein processing and core protein maturation. Immunofluorescence and immunoblotting assays showed that the core protein maturation capabilities of different NS3 N369 mutants, as well as those with the NS3 N158Y adaptive mutation, were not significantly different from that of the wild type NS3. Thus, NS3 N369 mutation-induced virus production defects may not be caused by impairment of NS3 protease or helicase functioning. Overall then, the cause-and-effect mechanism behind the defective phenotype remains unanswered and needs more investigation. It would be informative to examine if the process of virus assembly and egress is affected under an NS3 N369 mutant background.

CONTENTS
Contents I
致謝 II
中文摘要 III
ABSTRACT IV
LIST of TABLES, FIGURES and APPENDIXES V
ABBREVIATIONS VI
1. INTRODUCTION 1
1.1 Dengue virus 1
1.2 DENV life cycle 1
1.3 DENV genome structure and viral protein function 1
1.4 Virus production defect of NS3 N369 mutants and rescue activity of NS3 N158Y 3
1.5 NS2B/NS3-mediated structural protein cleavage of C-CI 3
1.6 Specific aims 4
2. MATERIALS AND METHODS 5
2.1 Viral genes and cells 5
2.2 NS3 N369 SGR stable cell lines 5
2.3 Mini-expression cassettes of structural proteins 5
2.4 In vitro RNA transcription and RNA electroporation 6
2.5 Immunofluorescence assay 6
2.6 Immunoblotting analysis 7
2.7 Antibodies 7
3. RESULTS 9
3.1 Characterization of NS3 mutant SGR stable cell lines 9
3.2 Mini-expression cassettes for viral structural protein expression 9
3.3 V5-C-HA is not a substrate of NS2B/NS3 and accumulates in the nucleolus 10
3.4 V5-C-CI-HA is not completely ER-anchored and is only partially cleaved by
NS2B/NS3 11
3.5 V5-C-CI-prM-HA demonstrates structural protein cleavage 12
3.6 All NS3 mutants have sufficient structural protein cleavage ability 12
3.7 Translation of mini-RNA does not alter protein expression patterns of SGR cell lines 13
4. DISCUSSION 14
5. REFERENCES 15
6. TABLE 18
7. FIGURES 19
8. APPENDIX 26

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