Function of Lipid Droplets in Viral Entry and Membrane Fusion

描述（由申请人提供）：我们研究项目的广泛长期目标是通过表征病毒感染所必需的细胞辅因子，特别是正链RNA病毒，如丙型肝炎病毒（HCV）和登革病毒（DENV），来提高对病毒-宿主细胞相互作用的认识。这些病毒感染了全世界数百万人，对人类健康构成重大威胁。了解这些病毒如何与宿主细胞相互作用对于诊断、抗病毒药物和预防性疫苗的开发至关重要。在初步研究中，我们已经发现了脂滴的关键作用， DENV和HCV中的相关蛋白在膜融合步骤进入宿主细胞。虽然已经报道了脂滴在病毒组装中的作用，但这些细胞器在病毒进入和膜融合中的功能以前尚未提出。在这项研究中，我们将研究脂滴及其相关蛋白CIDEB的作用机制，以促进各种RNA病毒的进入。具体目标如下：（1）确定脂滴及其募集早期内体在病毒进入中的作用。我们的工作假设，这一目标是，LD和招聘的病毒含有早期内涵体的LD完成病毒进入。我们将使用药理学和遗传操作来减少DENV和HCV靶细胞中的LD，然后确定这些病毒的感染是否在特定步骤（如内吞作用、膜融合或衣壳释放）受到抑制。(2)确定CIDEB在正链RNA病毒感染周期中的功能。我们已经证明了CIDEB敲低（KD）与RNAi减少DENV/HCV感染，并假设CIDEB可以作为一个新的宿主靶点治疗感染的正链RNA病毒。我们将使用CIDEB敲除（KO）模型，使用TAL效应核酸酶（TALEN）技术生成，以确定CIDEB在各种RNA病毒的生命周期中的重要性。拟议研究的结果不仅将为脂滴和CIDEB的病毒和宿主相关功能提供重要见解，还可能揭示针对医学重要RNA病毒的抗病毒干预的新治疗靶点。

DESCRIPTION (provided by applicant): The broad, long-term goal of our research program is to advance knowledge of virus-host cell interactions by characterizing cellular cofactors essential for viral infections, particularly positive-strand RNA viruses such as hepatitis C virus (HCV) and dengue virus (DENV). These viruses infect millions of people worldwide and pose major threats to human health. Understanding how these viruses interact with the host cell is of critical importance to the development of diagnostics, antiviral drugs, and a prophylactic vaccine. In preliminary studies, we have uncovered a critical role of lipid droplets and one of its associated proteins in DENV and HCV entry into host cells at the membrane fusion step. Although a role of the lipid droplets in viral assembly has been reported, a function of these organelles in viral entry and membrane fusion has not been proposed before. In this study, we will investigate the mechanism of action for lipid droplets and its associated protein, CIDEB, to facilitate the entry of diverse RNA viruses. The following specific aims are proposed: (1) to define the roles of lipid droplets and their recruitment of early endosomes in virus entry. Our working hypothesis of this aim is that LDs and the recruitment of virus-containing early endosomes to LDs are required for completing viral entry. We will use pharmacological and genetic manipulations to reduce LDs in the target cells of DENV and HCV and then determine if infection by these viruses is inhibited at specific steps such as endocytosis, membrane fusion, or capsid release. (2) to determine the function of CIDEB during the infection cycle of positive-strand RNA viruses. We have demonstrated the CIDEB knockdown (KD) with RNAi reduced DENV/HCV infection and hypothesize that CIDEB can serve as a new host target for treating infections by positive-stranded RNA viruses. We will use CIDEB knockout (KO) models, generated using TAL effector nuclease (TALEN) technology, to determine the importance of CIDEB in the life cycles of various RNA viruses. The results of the proposed studies will not only provide significant insights into the virus- and host-related functions of lipid droplets and CIDEB but also may reveal new therapeutic targets for antiviral intervention directed at medically important RNA viruses.