探討細胞因子參與C型肝炎病毒進入宿主細胞之機制

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Title:	探討細胞因子參與C型肝炎病毒進入宿主細胞之機制 Investigation of cellular factors involved in hepatitis C virus entry
Authors:	盧孟弘;Meng-Hong Lu
Contributors:	健康照護學院醫學檢驗生物技術學系碩士班
Keywords:	病毒;感染;細胞因子;HCV;entry;cellular factor
Date:	2010
Issue Date:	2010-09-29 12:10:29 (UTC+8)
Abstract:	C型肝炎病毒是目前世界上造成肝臟疾病的重要致病源。近年來的研究顯示C型肝炎病毒透過其病毒套膜上的醣蛋白E1與E2形成的複合物與細胞膜表面的受器進行交互作用，並利用clathrin-mediated endocytosis方式感染宿主細胞。Disabled-2 (Dab2) 是一種cargo-specific的adaptor蛋白，它同時具有cargo-selection及lattice polymerization的功能幫助clathrin蛋白在細胞膜組裝成型。為探討Dab2蛋白是否參與在C型肝炎病毒感染的過程，首先利用RNA 干擾技術將Huh 7.5細胞內生性的Dab2蛋白表現量降低，隨後感染HCV偽病毒 (HCVpv)，結果顯示，在Dab2表現量降低的穩定表達細胞株中，HCV偽病毒感染率顯著的下降。接著，我們將細胞內的Dab2蛋白表現量回復之後，發現HCV偽病毒感染率也跟著回升，但是將clathrin的另一個adaptor蛋白，ARH過量表現卻沒有使HCV偽病毒的感染率回升。免疫螢光染色的實驗結果顯示，C型肝炎病毒進行clathrin-mediated endocytosis時，HCV Core蛋白與細胞的Dab2蛋白有co-localization的現象。為了更進一步瞭解Dab2如何調控C型肝炎病毒進入細胞的機制，我們構築Dab2不同功能性區域刪除或突變的蛋白。結果顯示HCV偽病毒在Dab2蛋白的PTB domain刪除時，感染能力下降，而Dab2蛋白的Myosin VI結合位的突變蛋白則影響不大。這些研究結果顯示Dab2蛋白在C型肝炎病毒進入細胞過程具有重要的角色。 Hepatitis C virus (HCV) is a major pathogen causing liver diseases. It has been known that HCV interacts with cell surface receptors through the viral protein complexes composed of the viral envelop glycoproteins E1 and E2. Recent studies revealed that HCV enters host cells through clathrin-mediated endocytosis pathway. Disabled-2 (Dab2) is a cargo-specific adaptor protein, which modulate clathrin-coat assembly at plasma membrane by synchronizing cargo selection and lattice polymerization events. To determine whether Dab2 involved in HCV entry, RNA interference technology was performed to deplete endogenous Dab2 expression in Huh7.5 cells followed by infection with HCV pseudotyped virus (HCVpv). Our data revealed that HCVpv infection was decreased in Dab2 knockdown cells. The reduced infectivity could be compensated by overexpression of Dab2 but not another clathrin adaptor molecule autosomal recessive hypercholesterolemia (ARH) in Dab2 knockdown stable cells. Moreover, Dab2 colocalized with HCV core protein in clathrin coated vesicle during HCV cell culture viral particle (HCVcc) infection. To advance investigate the functional domain of Dab2 involved in HCV entry, the deleted or mutated Dab2 was constructed. The data demonstrated that the PTB domain but not myosin VI binding motif of Dab2 was essential for HCV entry. Taken together, our results suggest that Dab2 plays an important role in HCV entry into host cells.
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