C型肝炎病毒(HCV)與愛滋病病毒(HIV)都屬於RNA病毒，由於缺乏治療藥物以及預防性疫苗，因此對於公共衛生安全具有嚴重的威脅性。本研究的目標是想要發展一個可以攜帶HCV次基因複製體(subgenomic replicon)的重組flavi/retroviral嵌合載體，用來表現RNAi或是細胞激素(cytokine)以探討研發治療性抗病毒疫苗或預防性疫苗的可能性。我們利用反轉錄病毒設計了一個新穎的假核組裝系統(pseudocore assembly system，PCAS)，利用HIV-1次基因質體(僅含gag-pol讀碼框架)，將HIV結構蛋白Gag中的核鞘蛋白(nucleocapsid，NC)或是鞘蛋白及核鞘蛋白(capsid-nucleocapsid，CA-NC)置換成HCV核心蛋白(core)的序列，產生一個沒有感染性的HIV/HCV重組病毒的結構蛋白顆粒體(virus-like particles，VLP)，以便初步探討HCV core在此重組病毒顆粒體上的組裝及RNA包裝的功用。我們將HIV NC或CA-NC之基因序列置換成不同區段的HCV core基因序列(分別為1-79及1-191a.a.相對應的基因序列)建構出許多嵌合質體，並且其重組基因序列也經由定序而確認。有報導指出將HIV NC置換成白胺酸區域(leucine zipper domain)可以支持HIV-1進行組裝，因此我們認為HCV core所提供的同型類白胺酸區域(leucine-like homotypic region)或許也可驅使HIV進行組裝。我們將這些嵌合質體轉染至293T細胞，利用西方墨點法以及同位密度蔗糖梯度(isotypic sucrose gradient)離心來分析細胞內或細胞外釋放出的嵌合病毒蛋白質以及可能形成的類病毒顆粒(virus-like particles，VLPs)。此外由於HIV蛋白酶(HIV protease)的活性會影響HIV類病毒顆粒組裝的穩定性而減低病毒顆粒形成，我們也發現額外帶有protease缺失的重組病毒載體，可有效的形成VLPs，進一步證實了這個假核系統用以研究HCV core組裝機制的可行性與疫苗發展的潛力。我們目前正用重組病毒載體來分析是否有任何的遺傳物質包裹在此嵌合病毒蛋白形成的VLPs中，並預測此嵌合病毒之結構蛋白僅對HCV genome有專一親和性的功能。未來我們也將探討VLPs在組裝時，是否能將可表達綠色螢光(GFP)或細胞激素的HCV RNA次基因複製體包裝至在VLPs中。這個研究最終的目標是要利用此假核系統去定義在HIV以及HCV core當中有那段序列是病毒組裝的功能性區域，以用來縮小嵌合病毒載體的大小或增加HCV core在RNA包裝上的專一性，以方便未來HIV/HCV疫苗的研發。

HCV and HIV are RNA viruses and both pose serious threats to the public health which demands therapeutic drugs or preventive vaccines urgently. Our goal is to develop a recombinant flavi/retroviral vector that is capable of delivering HCV subgenomic replicons expressing RNAi or cytokines for antiviral research and vaccine development. A novel retroviral (HIV) pseudocore assembly system (PCAS) was designed to generate HIV/HCV genetic constructs using subgenomic HIV proviral backbone (gag-pol frame) for the preliminary investigation of the role of HCV core in replacing HIV nucleocapsid (NC) or capsid-nucleocapsid (CA-NC) for virus-like particle (VLP) assembly and RNA packaging in vivo. Several HIV/HCV genetic constructs via swapping the HIV-1 NC or CA-NC regions with HCV core in the HIV gag-pol frame were constructed and their sequences were confirmed by automatic sequencing. Since exchanging HIV-NC with leucine zipper domain supports HIV-1 assembly, it is most likely that the core provided by HCV with its homotypic leucine zipper-like domains will drive particle assembly. We transfected these chimera viral vectors into 293T cells and investigated the intracellular and extracellular protein production of these chimeric VLPs using Western blot and sucrose fractionation analysis. Because the HIV protease activity is known to affect Gag protein processing and assembly, deletion of the protease functional domain from the recombinant HIV/HCV constructs was found to efficiently facilitate particle formation. Our results clearly demonstrated the feasibility of using PAS system to study HCV core assembly and RNA packaging as well as to develop recombinant HIV/HCV vaccine. We are currently analyzing if any kind of genetic materials is carried into the VLPs. We will also investigate whether trans-provided HCV subgenomic RNA expressing GFP or cytokines can be actively packaged into the viral particles for subsequent replication and expression in HCV infected cells. Our ultimate goal is to define the functional motifs in HCV core for viral assembly and packaging using the PAS system and to develop HIV-HCV chimeric VLPs for vaccine research.

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