脂筏在禽呼肠孤病毒感染中的作用机制研究

Avian reovirus (ARV) is an important pathogen in domestic fowls. Lipid rafts, specific microdomains on cell membrane that enriched cholesterol and sphingolipids, are known to be involved in the replication cycle of various types of virus. However, the relationship between lipid rafts and ARV infection remains to be defined. Our preliminary results showed that lipid rafts disruption induced by cholesterol depletion significantly inhibited ARV replication in Vero cells. On the basis of the above finding, here we propose this new project to further elucidate the role of lipid rafts in ARV infection and its molecular mechanism. After lipid rafts disruption, the quantitative analysis of bound virions, genome loads and protein expression, syncytium formation, titration of progeny viruses will be characterized, to evaluate the involvement of lipid rafts in ARV attachment, entry, assembly and release progress. Sub-cellular localization assay, isolation of lipid rafts as detergent-resistant membranes (DRMs) and relevant bio-mass spectrometry analysis, will be utilized to study the interaction between lipid rafts and the structural and non- structural proteins of ARV. To explore the mechanism of lipid rafts in ARV-mediated membrane fusion, the overexpression vector of viral non-structural protein p10, and mutants of the cysteines associated with cholesterol binding motif in the ectodomain will be constructed. Membrane fusion activity, sub-cellular localization and DRMs analysis will be carried out to evaluate the interaction between cystine loop of p10 and lipid rafts in cell-cell fusion. This new project will utilize lipid rafts as the point of penetration to study the virus-host interactions during ARV infection. It will pave the way and provide new insights into the pathogenesis and control of ARV.

禽呼肠孤病毒（avian reovirus，ARV）是家禽的重要病原之一。脂筏是细胞膜上富含鞘脂类和胆固醇的微结构域，在多种病毒的感染过程中发挥重要作用。目前，脂筏与ARV感染的相关性尚不明确。在前期研究中，申请者发现用胆固醇去除剂破坏细胞膜的脂筏，对ARV的复制有显著影响。为进一步明确脂筏在ARV感染中的作用及相关机制，本项目将从病毒吸附量、病毒蛋白的表达水平、合胞体数量、子代病毒滴度等方面，系统分析脂筏对ARV吸附、侵入、组装和释放过程的影响；通过亚细胞定位、脂筏分离结合生物质谱鉴定分析，研究ARV结构蛋白及非结构蛋白在病毒感染各阶段与脂筏的互作关系；构建ARV p10蛋白的表达载体，并突变p10蛋白的胆固醇结合相关氨基酸位点，探讨脂筏在ARV介导膜融合中的作用机制。该项目以脂筏为切入点，从分子水平分析ARV与宿主的相互作用，为阐明ARV的致病机理乃至防控ARV提供新依据。

禽呼肠孤病毒（avian reovirus，ARV）是家禽的重要病原之一。脂筏是细胞膜上富含鞘脂类和胆固醇的微结构域，在多种病毒的感染过程中有重要作用。目前，脂筏与ARV感染的相关性尚不明确。本项目使用药物破坏细胞膜脂筏，结合外源性胆固醇补充实验，从病毒吸附量、ARV病毒蛋白的转录水平及表达水平、合胞体数量、子代病毒滴度等方面，系统分析脂筏破坏对ARV吸附、侵入、组装和释放过程的影响。证明了脂筏中的细胞胆固醇是ARV侵入和侵入细胞后复制的关键决定因素，脂筏参与了ARV的入侵及随后的病毒复制过程，但细胞膜脂筏的破坏不影响ARV在靶细胞上的吸附。结合ARV部分编码蛋白与脂筏的亚细胞定位分析，明确了脂筏在ARV感染周期中的重要性。通过透射电镜技术观察、内吞途径抑制实验，探讨ARV的入胞途径，发现ARV通过小窝（一类特殊类型的脂筏）依赖型内吞途径入胞。通过脂筏组分分离鉴定、分析了ARV编码的σC、σB、σNS、μNS、p10、p17蛋白在ARV感染过程中与脂筏标志蛋白的共分离情况。结合IFA和激光共聚焦技术、免疫共沉淀技术，siRNA实验，发现ARV结构蛋白σB蛋白与宿主小窝蛋白（Caveolin-1）在ARV内吞过程中存在互作关系。针对ARV侵入后阶段，运用蛋白质谱分析技术，筛选了可能与σNS、μNS相互作用的脂筏相关蛋白。此外，构建了p10蛋白表达载体，并突变p10蛋白的胆固醇结合相关氨基酸位点，发现p10蛋白N-端胞外域的胱氨酸环在p10脂筏定位和诱导细胞融合中具有关键作用。综上，本研究以脂筏为切入点，探究脂筏在ARV感染周期中的作用及相关机制，分析宿主蛋白与ARV感染的关系，为阐明ARV的致病机理乃至防控ARV提供新依据。