脂质转运蛋白与VAMP相关蛋白相互作用维持丙型肝炎病毒复制体脂质组分的分子机制

Like all positive-sense RNA viruses, HCV induces cytoplasmic membrane alterations in infected cells, which have been termed replication organelles (ROs) and are believed to be sites of HCV RNA synthesis. HCV ROs have specialized lipid composition, and are enriched in cholesterol and phosphatidylinositol 4-phosphate (PI4P). An important gap in our knowledge is that despite recent progress in defining cellular pathways necessary for virus RO formation, we do not know all of the factors that shape their lipid composition. Lipid transfer proteins (LTPs) are proteins with a lipid-binding domain that have the ability to transport specific lipids from one liposome to another. Most lipid transfer proteins tether membranes from different organelles to form membrane contact sites (MCS) where they mediate nonvesicular lipid transfer. In general, LTPs can interact with VAMP associated proteins at the ER and at the same time, interact with negatively charged phospholipids from the other organelles. .We and others have identified and characterized a cellular phosphoinositide signaling pathway necessary for RO formation of HCV as well as several other positive-sense RNA viruses from different families. HCV infection is accompanied by PI4K activation and increased levels of PI4P in viral RO membranes. PI4P can then recruit OSBP, a LTP, which transfers cholesterol to HCV ROs by exchanging PI4P for cholesterol. However, ultrastructural analysis has revealed that the majority of HCV ROs are not in physical continuity with a cellular membrane compartment, raising the question of how phosphatidylinositol (PI) is supplied to the ROs. In mammalian cells, nonvesicular PI transfer is mediated by several different phosphatidylinositol transfer proteins (PITPs), which function through interacting with VAMP associated proteins at the ER. These PITPs are then able to transfer PI from the ER to other organelles. Here we propose that a specific PITP is required to resupply HCV ROs with PI, which can then be converted to PI4P that is necessary to drive HCV infection. Therefore, in this study we will first identify the specific PITP that is essential for HCV viral infection and then study the mechanism of how it mediates PI transfer to the HCV RO. .In addition, it has been shown that the ER resident protein VAMP associated proteins- VAPA and VAPB are necessary for efficient HCV replication, and interact with the HCV NS5A and NS5B nonstructural proteins. However, the mechanistic details of how VAPA and VAPB support HCV replication are not well understood. Here we propose that VAPs function by recruiting downstream LTP effectors to support HCV replication organelle formation. In this study, we will first study the specific roles of VAPs in HCV infection and we will further conduct Mass Spectrometry to identify possible LTPs other than OSBP and the PITP described above that interact with VAPs. We will further study the roles of these newly identified VAP interacting proteins in HCV replication and RO formation. .Successful completion of these aims will yield a comprehensive overview of the cellular lipid transfer proteins that support formation of the HCV replication organelle as well as their specific roles in RO formation and function.

丙型肝炎病毒重塑宿主内质网膜作为复制场所（复制体）。由于多数病毒复制体并未与内质网膜连接，目前尚不清楚病毒在持续感染过程中如何维持其特殊脂质组分。申请人前期研究表明，脂质转运蛋白OSBP介导了胆固醇至病毒复制体的转运，同时将PI4P转运至内质网，但病毒如何维持复制体PI4P含量则尚不清楚。哺乳动物细胞内磷脂酰肌醇转运蛋白负责磷脂酰肌醇的非膜泡转运。鉴于此，本研究将首先筛选出对丙型肝炎病毒复制起作用的磷脂酰肌醇转运蛋白，并进一步分析其如何与VAMP相关蛋白相互作用并维持复制体PI4P含量。进而研究VAMP相关蛋白在丙型肝炎病毒复制中发挥的作用，阐明各结构域功能，并通过质谱鉴定出所有与VAMP相关蛋白相互作用的脂质转运蛋白，通过表型筛选及功能分析，最终阐明各脂质转运蛋白如何相互作用调节并维持丙型肝炎病毒复制体脂质组分。这对于潜在药物靶点的发现及阐明细胞内膜结构及脂质变化都具有重要意义。

正链RNA病毒重塑宿主内膜系统作为自己的复制场所，同时在病毒复制过程中会广泛利用宿主因子来满足其快速、大量扩增的需求。病毒复制场所的形成过程及病毒如何转运所需原料是RNA病毒研究的一个重点。膜接触位点是细胞内不同细胞器之间的一种特殊物理结构，在信号转导及双向小分子转运过程中发挥重要功能。其中内质网与不同细胞器之间的MCS由内质网锚定蛋白VAPs(VAMP associated proteins)介导。本研究的主要内容是阐明在内质网与病毒复制场所之间，VAP分子是如何招募其他分子形成MCS，及各种脂质转运分子如何在MCS处发挥脂质转运功能，从而保证病毒持续复制。.研究发现，在HCV复制过程中，细胞内的两种VAP同源分子-VAPA和VAPB都发挥了重要作用，而且二者在功能上具有一定的可替代性。对VAP分子结构域进行功能分析发现VAP分子必须定位于内质网才能发挥其辅助病毒复制的功能。关键位点突变实验则提示VAP分子通过招募富含FFAT motif的蛋白来发挥功能。通过免疫共沉淀-质谱鉴定的方法我们发现了一种全新的脂质转运蛋白（Nir2）可以与VAP分子相互作用形成内质网与病毒复制体之间的MCS。Nir2功能分析则发现Nir2可以向病毒复制体转运PI，而后者会被病毒复制体内的PI4K激酶磷酸化生成PI4P，维持病毒复制。而且Nir2在转运PI的同时会反向转运一分子的磷脂酰氨酸（PA）至内质网，完成其双向脂质交换的功能。此研究结果解释了空间上相对独立的病毒复制场所如何维持其高水平PI4P 以促进病毒复制，同时与我们之前关于OSBP功能的研究共同构成了磷酸肌醇在内质网与病毒复制场所之间循环流动的环路模型。.此外，我们还对磷脂酰肌醇通路在新冠病毒、肠道病毒感染中发挥的作用进行了初步分析。基于上述研究共发表SCI论文6篇，申请专利1项，参加国内外线下学术会议4次，培养了2名博士生和2名硕士生，完成了预期目标。