臺灣博碩士論文加值系統

日本腦炎病毒為一單股正向核糖核酸病毒，其基因體分別具有五端及三端非轉譯區域，非轉譯區域與病毒蛋白轉譯、病毒核糖核酸合成複製及病毒顆粒的包裝等有重要的調節作用，且目前認為具有參與這些步驟的細胞內宿主或者病毒蛋白質，會結合作用於非轉譯區。於是我們利用生物素標定非轉譯區域核糖核酸，進而再以卵白素磁珠結合被標定生物素的非轉譯區核糖核酸，連帶地將結合於非轉譯區域核糖核酸的宿主蛋白質沉澱出來，再經由質譜儀與蛋白質體學分析鑑定，我們發現宿主蛋白質Far upstream element (FUSE) binding protein 1 (FBP1)會結合於此非轉譯區域，於是我們利用改變細胞內FBP1表現量來探討FBP1對日本腦炎病毒的影響，當我們降低細胞內內生性FBP1表現量，發現會增加日本腦炎病毒的複製，而當我們增加FBP1表現量，則能抑制病毒的複製現象，由此得知FBP1對於日本腦炎病毒的複製扮演負調節者角色。FBP1主要存在於細胞核內，但當細胞受到日本腦炎病毒感染的早期，FBP1會由細胞核內移動至細胞核邊及細胞質中，且與日本腦炎病毒核糖核酸有部分結合的現象。進一步我們利用日本腦炎病毒報導基因複製子系統，實驗結果得知FBP1具有抑制日本腦炎病毒五端及三端非轉譯區域所控制的轉譯能力。因此，我們發現FBP1結合於日本腦炎病毒非轉譯區核糖核酸，且經由抑制日本腦炎病毒蛋白質表現，來對抗日本腦炎病毒的複製。
日本腦炎病毒感染時FBP1會出現在stress granule (SG)的位置，而目前已知一些病毒與SG路徑有交互作用，因此我們也研究SG在日本腦炎病毒感染時所扮演的角色，我們發現日本腦炎病毒感染會引發SGs的形成，而且不會抑制sodium arsenite所誘發的SGs。進一步發現可抑制RhoA活化的Y-27632藥物，以及可抑制RohA下游的ROCK1的另一藥物Toxin B，並不會抑制日本腦炎病毒和登革病毒所引發的SGs，結果顯示RhoA/ROCK1訊息傳導途徑可能不在這兩個病毒誘發SG形成時扮演重要的角色。此外我們發現monocyte chemotactic protein-induced protein 1 (MCPIP1)又稱為Zc3h12a，可有效地抑制日本腦炎病毒和登革病毒感染所引發的SGs形成。更進一步研究發現，各別單一表現日本腦炎病毒蛋白質core、prM-E、prM, E、NS1、NS2B、NS4A、和NS4B，但非NS2B3和NS5會有SGs形成現象。總結目前我們的結果，登革病毒和日本腦炎病毒的感染確實是會引發SGs的形成，但其是如何引發SGs形成，及SGs在登革病毒和日本腦炎病毒感染所扮演的角色，還尚待釐清。

The untranslated regions (UTRs) located at the 5and 3 ends of the Japanese encephalitis virus (JEV) genome, a positive-sense RNA, are involved in viral translation, the initiation of RNA synthesis, and the packaging of nascent virions. The cellular and viral proteins that participate in these processes are expected to interact with the UTRs. In this study, we used biotinylated RNA-protein pulldown and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses to identify that the far upstream element (FUSE) binding protein 1 (FBP1) binds with JEV 5 and 3 UTRs. The impact of FBP1 on JEV infection was determined in cells with altered FBP1 expression. JEV replication was enhanced by knockdown and reduced by the overexpression of FBP1, indicating a negative role for FBP1 in JEV infection. FBP1, a nuclear protein, was redistributed to the perinuclear region and appeared as cytoplasmic foci that partially colocalized with JEV RNA in the early stage of JEV infection. By using a JEV replicon reporter assay, FBP1 appeared to suppress JEV protein expression mediated by the 5 and 3UTRs. Thus, we suggest that FBP1 binds with the JEV UTR RNA and functions as a host anti-JEV defense molecule by repressing viral protein expression.
FBP1 was found to partially colocalize with stress granules (SGs) in JEV-infected cells and some viruses have been found to interact with the SG pathway. We thus study the role of SG in JEV infection. We found that JEV infection induced SGs formation and did not interfere with SG induction triggered by sodium arsenite stimulation. JEV- and Dengue virus serotype 2 (DEN-2)-induced SGs formation can not be blocked by specific inhibitors Y-27632 and Toxin B that inactive signaling of RhoA and its downstream kinase ROCK1, respectively, suggesting that RhoA/ROCK1 pathway is not involved in SGs induction during JEV and DEN-2 infection. In addition, monocyte chemotactic protein-induced protein 1 (MCPIP1), also known as Zc3h12a, could block the assembly of SGs in cells infected with JEV and DEN-2. Furthermore, several viral proteins such as core, prM, E, NS1, NS2B, NS4A, and NS4B, but not NS2B3 and NS5 are able to trigger SG formation . Overall, we demonstrate that SGs formation was induced in DEN-2- and JEV-infected cells. The mechanism and consequence of SGs formation during DEN-2 and JEV infection are of interest to be further studied.

CONTENTS Pages
中文摘要 1
Abstract 3
CHAPTER 1: INTRODUCTION 6
CHAPTER 2: MATERIALS AND METHODS
Cell lines, virus, and chemicals 11
In vitro transcription 11
Isolation of proteins that bound with biotinylated JEV UTR-Luc by pull-down assay 12
Tryptic digestion and mass spectrometry 13
Plasmid constructs 14
Lentiviral vectors preparation 17
Immunoprecipitation and RT–PCR 17
Western immunoblotting 18
Immunofluorescence assay (IFA) 19
Confocal imaging 19
CHAPTER 3: RESULTS
Identification of FBP1 interacting with JEV UTRs 22
FBP1 is a negative regulator against JEV and DEN-2 replication in human cells 24
Overexpression of FBP1 suppresses JEV replication 27
Subcellular localization of FBP1 and viral RNA in JEV-infected cells 27
FBP1 colocalizes with SG markers under stress condition 28
FBP1 is a negative regulator for JEV and DEN-2 UTR-mediated protein expression 29
JEV infection induces and does not interfere with SG assembly 31
RhoA/ROCK1-dependent pathway may not be involved in JEV-induced SG formation 31
Zc3h12a represses SG formation triggered by JEV and DEN-2 infection 32
The ability of JEV viral proteins to trigger SG formation 33
CHAPTER 4: DISCUSSION 35
CHAPTER 5: REFERENCES 42
TABLES
Table 1: LC-MS/MS result of the identified peptides matching the amino acid sequence of FBP1 49
Table 2: The result of JEV viral proteins as a SGs formation inducer 50
FIGURES
Fig. 1: Identification of host factors interacting with JEV UTRs 52
Fig. 2: Specific interaction of FBP1 with JEV 5 and 3 UTR 54
Fig. 3: Reduction of FBP1 expression enhanced JEV protein expression and viral production 56
Fig. 4: The cells with FBP1 knockdown showed increased JEV viral RNA replication 58
Fig. 5: DEN-2 protein expression and viral production were increased in cells with reduced FBP1 expression 59
Fig. 6: Rescue of FBP1 expression in HeLa-shFBP1 cells repressed JEV production 61
Fig. 7: Overexpression of FBP1 reduced JEV protein expression and viral production 63
Fig. 8: The JEV viral RNA replication levels were decreased in FBP1 overexpressed cells 65
Fig. 9: Cellular localization of FBP1 and dsRNA 66
Fig. 10: Stress caused FBP1 to accumulate in SGs 67
Fig. 11: FBP1 negatively regulated JEV UTR-dependent protein expression measured by a JEV reporter replicon 68
Fig. 12: FBP1 negatively regulated DEN-2 UTR-dependent protein expression measured by a DEN-2 reporter replicon 70
Fig. 13: JEV infection did not block SGs formation in response to arsentie treatment 72
Fig. 14: JEV and DEN-2 infection induced SGs formation 74
Fig. 15: The effect of ROCK1-specific inhibitor, Y-27632 on JEV and DEN-2 induced SGs formation 75
Fig. 16: The effect of a RhoA inhibitor, Toxin B, on JEV-induced SGs formation 77
Fig. 17: Zc3h12a expression blocked the ability of cells to form SGs in response to JEV or DEN-2 infection 79
Fig. 18: Screening of JEV viral proteins as a SGs formation inducer 82
Fig. 19: Screening of JEV viral proteins as a SGs formation inducer 85

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