4 research outputs found
Adenovirus vector-mediated assay system for hepatitis C virus replication
The efficient delivery of the hepatitis C virus (HCV) RNA subgenomic replicon into cells is useful for basic and pharmaceutical studies. The adenovirus (Ad) vector is a convenient and efficient tool for the transduction of foreign genes into cells in vitro and in vivo. However, an Ad vector expressing the HCV replicon has never been developed. In the present study, we developed Ad vector containing an RNA polymerase (pol) I-dependent expression cassette and a tetracycline-controllable RNA pol I-dependent expression system. We prepared a hybrid promoter from the tetracycline-responsive element and the RNA pol I promoter. Ad vector particles coding the hybrid promoter-driven HCV replicon could be amplified, and interferon, an inhibitor of HCV replication, reduced HCV replication in cells transduced with the Ad vector coding HCV replicon. This is the first report of the development of an Ad vector-mediated HCV replicon system
Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B
BACKGROUND: Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. PRINCIPAL FINDINGS: Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. CONCLUSIONS: We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology
Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines
<div><p>Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.</p></div
The roles of fatty acid synthase in hepatitis C virus replication
C型肝炎病毒是造成肝病的重要因素,引起脂肪肝、肝纖維化、肝硬化與肝癌的發生。由於缺乏C型肝炎病毒的研究模式,對其致病機轉不甚瞭解,導致病人受C型肝炎病毒的威脅日益增加。近年來隨著各種研究模式的建立,慢慢描繪出病毒生活史的輪廓。然而參與C型肝炎病毒複製複合體的細胞因子仍未完全清楚。為了瞭解C型肝炎病毒的複製機制,先前的研究利用C型肝炎病毒複製酶-NS5B蛋白質尋找與其結合的細胞蛋白質,結果發現NS5B蛋白質與脂肪酸合成酶產生交互作用。本研究進一步使用C型肝炎病毒複製細胞與病毒顆粒感染系統再次驗證NS5B蛋白質與脂肪酸合成酶間的結合,並鑑定NS5B的N端是與脂肪酸合成酶結合的重要區域,及探討脂肪酸合成酶與NS5B產生結合的區域。大量表現脂肪酸合成酶可增強C型肝炎病毒在病毒複製細胞的複製能力,以RNAi靜默脂肪酸合成酶表現能抑制C型肝炎病毒複製細胞與病毒顆粒感染細胞的病毒RNA與蛋白質表現。部分脂肪酸合成酶更參與了C型肝炎病毒複製複合體的形成,調控病毒複製的能力;NS5B活性試驗更顯示,純化的脂肪酸合成酶直接增強NS5B聚合酶活性。報導指出,苦瓜可抑制大鼠脂肪酸合成酶的表現。我們的研究顯示,苦瓜萃取物可降低脂肪酸合成酶在C型肝炎病毒複製細胞的表現,進而抑制C型肝炎病毒複製細胞及病毒顆粒感染細胞之病毒基因的表現。更進一步的研究顯示,純化自苦瓜的有效化合物抑制脂肪酸合成酶表現,是經由活化AMPK路徑所引起。綜觀本研究結果認為,脂肪酸合成酶是C型肝炎病毒複製時所需要的重要因子,可提供治療C型肝炎病毒一個新標的。Hepatitis C Virus (HCV) is a leading cause of liver disease, including steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. The prevalence of HCV is increased due to the lack of research model for uncovering the mechanisms of pathogenesis. In recent years, the possible processes of HCV life cycle were more understanding by establishment of various HCV culture systems. However, the cellular components of HCV replication complex are still not complete clear. To elucidate the replication mechanisms of HCV, we used NS5B, the RNA dependent RNA polymerase of HCV, as a bait to search its interacting proteins in Huh7 cells. Previously, we found that fatty acid synthase (FASN) binds to NS5B. In this study, the interaction of FASN and NS5B were further verified in HCV subgenome replicon cells and HCVcc infected cells. We defined that N terminal region is crucial for NS5B binding to FASN, whereas the NS5B binding fragments of FASN were also determined. Overexpression of FASN enhanced the replication activity in HCV replicon cells, whereas RNAi targeting on FASN expression inhibited the viral RNA and protein expression in HCV replicon cells and HCVcc viral infected cells. Moreover, part of FASN protein was recruited into HCV replication complex and modulated RNA replication activity. In addition, the NS5B RdRp activity was enhanced by purified FASN protein. Bitter melon, which was reported to inhibit FASN expression in rat model, suppressed the protein expression of FASN and further decreased HCV replication in HCV replicon cells as well as HCVcc viral infected cells. Furthermore, the effective compound purified from bitter melon inhibited FASN expression through AMPK activation. Taken together, these results suggested that FASN is a critical component for HCV replication and serves as a new target for HCV therapeutic strategy.中文摘要................................................i
英文摘要...............................................ii
目次..................................................iii
圖表次.................................................v
緒言...................................................1
第一章 C型肝炎病毒及參與病毒表現細胞因子文獻檢討...............2
第一節 C型肝炎病毒.......................................2
一、 C型肝炎病毒之介紹....................................2
二、 C型肝炎病毒之基因表現.................................2
三、 C型肝炎病毒之生命週期.................................4
四、 C型肝炎病毒之研究模式.................................4
第二節 細胞因子在C型肝炎病毒生成所扮演的角色..................6
一、 病毒因子參與C型肝炎病毒生活史之介紹......................6
二、 與C型肝炎病毒RNA聚合酶NS5B結合的細胞因子................6
第三節 C型肝炎病毒之治療...................................6
第四節 研究動機..........................................7
第二章 脂肪酸合成酶在C型肝炎病毒複製中所扮演的角色..............8
第一節 摘要.............................................8
第二節 前言.............................................8
一、 脂肪酸合成酶之基因與蛋白質結構.........................8
二、 調控脂肪酸合成酶表現之機制.............................9
三、 脂肪酸合成酶之功能...................................10
四、 脂肪酸合成酶與病毒間關係之研究.........................10
第三節 實驗材料與方法.....................................12
一、 實驗材料...........................................12
二、 質體建構...........................................14
三、 細胞培養與藥物處理...................................15
四、 細胞轉染...........................................16
五、 shRNA lentiviral 生產..............................16
六、 C型肝炎病毒生產 (cell culture produced HCV virus, HCVcc)................................................16
七、 西方墨點法..........................................18
八、 複製複合體(crude replication complex, CRC)之研究.....19
九、 蔗糖濃度梯度試驗.....................................20
十、 免疫沉澱法..........................................20
十一、 免疫螢光染色.......................................21
十二、 即時偵測聚合酶反應 (real-time PCR)..................21
十三、 螢光酶活性試驗(Luciferase assay)...................22
十四、 NS5B RNA聚合酶活性試驗.............................23
十五、 統計方法..........................................24
第四節 結果.............................................25
一、 脂肪酸合成酶與NS5B在病毒複製系統中的交互作用..............25
二、 脂肪酸合成酶表現與C型肝炎病毒複製成正相關.................25
三、 C型肝炎病毒的表現需要脂肪酸合成酶的參與..................26
四、 脂肪酸合成酶參與C型肝炎病毒複製複合體的形成...............27
五、 脂肪酸合成酶與NS5B具有交互作用的區域....................28
六、 NS5B與脂肪酸合成酶具有交互作用的區域....................29
七、 脂肪酸合成酶能促進NS5B RNA聚合酶活性...................29
第五節 討論.............................................30
第三章 苦瓜萃取物抑制C型肝炎病毒之機制.......................32
第一節 摘要.............................................32
第二節 前言.............................................32
一、 天然藥物與C型肝炎病毒相關研究..........................32
二、 脂肪酸合成酶抑制劑與C型肝炎病毒的關係....................33
三、 苦瓜與細胞代謝的關係..................................33
第三節 實驗材料與方法.....................................35
一、 實驗材料............................................35
二、 藥物處理............................................35
三、 苦瓜萃取物成分對HCVcc病毒的影響........................35
四、 三酸甘油酯的定量.....................................36
五、 統計方法............................................36
第四節 結果..............................................37
一、 苦瓜萃取物成分對C型肝炎病毒複製的影響.....................37
二、 苦瓜萃取物成分對C型肝炎病毒感染的影響.....................37
三、 苦瓜萃取物成分對AMPK的影響.............................37
四、 苦瓜萃取物成分對脂質代謝之影響...........................38
第五節 討論..............................................39
第四章 總結...............................................42
第一節 脂肪酸合成酶結合NS5B並參與C型肝炎病毒複製................42
第二節 苦瓜萃取物經由誘導AMPK路徑抑制C型肝炎病毒的表現...........42
第三節 結論...............................................42
第五章 圖表...............................................44
第六章 參考文獻............................................67
第七章 附錄...............................................80
圖表次
圖 1. NS5B與脂肪酸合成酶在HCV replicon與HCVcc感染系統中產生交互作用。.....................................................44
圖 2. 脂肪酸合成酶與HCV的複製成正相關。.......................46
圖 3. 抑制脂肪酸合成酶的表現可降低HCV replicon細胞中HCV RNA的生成。.....................................................47
圖 4. 棕櫚酸(C16:0脂肪酸)對HCV複製的影響。....................48
圖 5. 脂肪酸合成酶對HCVcc病毒感染的影響。......................49
圖 6. 脂肪酸合成酶在細胞detergent resistant membrane的相對位置。.....................................................51
圖 7. 抑制脂肪酸合成酶的表現導致HCV replication complex內的病毒蛋白質減少。..................................................52
圖 8. 抑制脂肪酸合成酶表現導致HCV replication complex內HCV RNA的表現降低。...................................................53
圖 9. 與NS5B產生交互作用的FASN domain能增加HCV的複製能力。......................................................54
圖 10. NS5B的N端與脂肪酸合成酶產生交互作用。....................56
圖 11. FASN能增加NS5B RdRp活性。............................58
圖 12. 苦瓜萃取物對C型肝炎病毒複製的影響。......................60
圖 13. 苦瓜萃取物成分在Huh7/Rep-Feo細胞抑制HCV的表現。..........61
圖 14. 苦瓜萃取物成分對HCVcc病毒感染細胞能力的影響。.............62
圖 15. 苦瓜萃取物成分對於活化AMPK與抑制FASN表現,進而抑制HCV的表現。......................................................63
圖 16. 苦瓜萃取物成分抑制C型肝炎病毒表現與PPAR alpha路徑無關。....65
圖 17. 苦瓜萃取物成分對三酸甘油酯的影響。.......................66
附表1. 質體建構之引子序列....................................80
附表2. FASN knockdown之序列...............................82
附表3. 即時定量聚合酶反應之引子序列............................83
附圖 1. C型肝炎病毒基因體架構。...............................84
附圖 2. 脂肪酸合成催化反應。..................................85
附圖 3. 脂肪酸合成酶之立體結構。..............................86
附圖 4. NS5B活性試驗原理示意圖。..............................8