人SR-BI基因多态性调控的脂质代谢对HCV入侵和感染的影响

Human scavenger receptor class B type I (SR-BI) not only plays an important role in the lipoprotein metablism but also mediates primary attachment of HCV particles to heoatocytes. HCV life cycle has a close correlation with host lipid metabolism and makes use of the envelop protein obtained from host cells to escape immune clearance and establish persistent infection. In recent years, the influence of host genome single nucleotide polymorphism (SNPs) on virus-host interaction has received much concern. For HCV, most of the researches have focused on the gene IL28B and HCV prognosis. Currently, there are few reports on virus entry and host genome SNPs. SR-BI is the receptor for HDL and LDL mediating lipid metabolism, it was reported that some SR-BI SNPS led to high blood HDL level in population. Since HDL enhances the infectivity of HCV, SR-BI polymorphism might result in different susceptibility to HCV infection. Our preliminary experiments have confirmed that some reported SR-BI SNPs influenced HCV infection in vitro, yet the process is still obscure in natural HCV infections. This project aims to map SR-BI SNPs related with susceptibility to HCV entry and infectivity, confirm the relationship between SR-BI SNPs and HCV life cycle in cell models with the methods of site mutangenesis, RNA interference, flow cytometry, HCVcc、HCVpp and confocal microscopy, and then discuss the possible mechanisms involved. Finally, we try to illustrate the correlation between HCV progression and SR-BI polymorphism through HCV patients follow-up. This project will also be supplement to the knowledge on HCV entry and virus-host interaction, and provide basic research data for HCV prevention and treatment.

B类I型清道夫受体（SR-BI）是体内脂质代谢重要的调节因子，也是丙型肝炎病毒（HCV）入侵肝细胞的首位受体。HCV为单正链RNA包膜病毒，其生命周期与脂质代谢密切相关。近年，全基因组单核苷酸多态性位点（SNPs）与HCV感染的关系备受关注，主要集中在IL28B基因型影响HCV疗效，受体SNPs研究仅有一篇claudin-1的报道。SR-BI作为高密度脂蛋白（HDL）的受体，人群中存在基因变异，突变者体内HDL升高，而HDL可以显著增加HCV感染性。本课题拟在已报道SR-BI SNPs影响胆固醇代谢及前期实验基础上，寻找HCV相关SR-BI SNPs，采用点突变、RNAi、流式细胞术、HCVcc、HCVpp和激光共聚焦等，细胞模型检测SR-BI SNPs对HCV生命周期的影响并探讨其作用机制，并通过病人随访观察SR-BI SNPs与疾病进程间的关系，以期为HCV感染的防治提供基础研究数据。

B类I型清道夫受体（SR-BI）是体内脂质代谢重要的调节因子，也是丙型肝炎病毒（HCV）入侵肝细胞的首位受体。HCV为单正链RNA包膜病毒，其生命周期与脂质代谢密切相关。据报道，SR-BI单核苷酸多态性位点（SNP）与人群HDL水平相关。为研究SR-BI SNP与HCV关系，首先筛选获得了SR-BI表达抑制的Huh7-siSR-BI和Huh7.5.1- siSR-BI稳定细胞系，利用定点突变技术构建了SR-BI S112F、T175A和P297S等突变体。将SR-BI突变体和野生型质粒转染Huh7.5.1-siSR-BI细胞，加入HCVpp感染，5天后检测荧光素酶活性，显示SR-BI S112F明显降低HCV的入侵，T175A则增强HCV的入侵。利用HCVcc模型、免疫荧光和qRT-PCR检测，SR-BI S112F降低HCVcc感染性，而T175A可以增强HCVcc感染性。进而利用免疫荧光、流式细胞仪、荧光定量PCR和Western Blot，结果表明SR-BI SNP不影响SR-BI膜蛋白的分布，SR-BI S112F蛋白表达较其他组降低，S112F与E2蛋白的结合能力比野生型下降，而T175A与E2蛋白的结合能力较野生型上升。SR-BI S112F、T175A和P297S摄取细胞外Dil-HDL的能力降低。对SR-BI S112F进一步分析，发现SR-BI第112位氨基酸由丝氨酸突变为苯丙氨酸后，降低了蛋白的亲水性，使得SR-BI蛋白表达降低，进而降低了HCVcc的感染性。由于SR-BI在HCV感染中的重要性，研究了靶向SR-BI的microRNA对HCV感染的影响。将miR-185-5p mimics和inhibitor在Huh7细胞中验证其有效性，然后再以HCVcc感染转染了miR-185-5p mimic和inhibitor的Huh7细胞。结果表明，与对照相比，miR-185-5p mimics可以抑制HCVcc的感染性。继而利用双荧光报告系统和Dil-HDL摄入实验，证明miR-185-5p可与SR-BI的3’-UTR 第69~74nt区结合，抑制SR-BI的表达，进而影响了Huh7细胞对HCVcc的敏感性。本项目的开展，为探讨SR-BI在HCV复制中的作用以及基于SR-BI的miRNA在HCV治疗方面的前景奠定了基础。