宿主因子OATP1A2在禽戊型肝炎病毒感染过程中的作用及分子机制

Avian Hepatitis E virus (HEV) infection may cause the big liver and spleen disease and the decrease of egg production in the broiler and breeder chickens. The diseases cause seriously economic loss and destroy the healthy progress in poultry industry in China. However, because of the absence of high effective cell culture system and pathogenesis for the virus, it seriously hinders the prevention and pathogenesis of the virus. Previously, we identified the binding between the organic anion-transporting polypeptide 1A2 (OATP1A2) and avian HEV capsid protein using the GST-Pull down and SHOTGUN assays. Then in the project, based on the binding, we will identify the interaction between OATP1A2 and avian HEV capsid protein in vivo and vitro using the co-immunoprecipitation (Co-IP), virus overlay protein blot assay (VOPBA) and cellular co-localization assays. Subsequently, the function of OATP1A2 protein in the virus entering into the host cells will be determined using RNA interfering, antibody blocking and the target expressed in un-susceptible cell assays. Finally, the key amino acids of the interaction between OATP1A2 and avian HEV capsid proteins will be determined using truncated protein expression, mutated key amino acids, stimutated in virus infection clone and the target protein edited with CRISP/Cas9 assays. In the end, the OATP1A2 protein as the receptor for avian HEV will be determined. The high effective cell culture system will be established. The mechanism of interaction between two target proteins will be illustrated. The results of the project will pave the way for the research of avian HEV pathogenesis and designing of antivirus medicine and vaccine.

禽戊型肝炎病毒（Hepatitis E virus, HEV）感染导致鸡大肝大脾病和产蛋率下降，严重危害我国养禽业健康发展。然而，病毒高效培养体系的缺乏以及病毒与宿主互作机制研究的不深入严重制约了对该病的针对性防控。本项目将以我们前期筛选的与禽HEV衣壳蛋白特异结合的宿主膜蛋白OATP1A2为研究基础，利用Co-IP，VOPBA和细胞共定位等技术进一步确认两者的互作；然后通过RNA干扰，抗体封闭和靶蛋白在非易感细胞系表达重建等实验分析OATP1A2蛋白在病毒感染细胞过程中的作用，同时利用BiFC系统，定点突变以及CRISPR/Cas9基因编辑等技术揭示两者互作的分子基础及其在病毒感染中的作用。最终，明确OATP1A2蛋白的表达丰度与病毒感染关系，并以此建立病毒高效培养体系；同时阐明该蛋白在禽HEV感染宿主细胞过程中的作用及分子机制。为禽HEV致病机制研究以及抗病毒药物和疫苗研发奠定基础。

禽戊型肝炎病毒（Hepatitis E virus, HEV）感染导致鸡大肝大脾病和产蛋率下降，严重危害我国养禽业健康发展。然而，病毒高效培养体系的缺乏以及病毒与宿主互作机制研究的不深入严重制约了对该病的针对性防控。本项目以我们前期筛选的与禽HEV衣壳蛋白特异结合的宿主膜蛋白OATP1A2为研究基础，首先利用Co-IP，激光共聚焦细胞共定位等技术进一步确认禽HEV衣壳蛋白与OATP1A2的互作；利用荧光定量RT-PCR检测宿主细胞OATP1A2的表达量与禽HEV感染呈正相关，而在体内该宿主因子的表达量与病毒的组织嗜性也呈正相关；重构了OATP1A2过表达细胞系LMH，通过RNA干扰，OATP1A2在非易感细胞系中的表达证明了OATP1A2表达量的改变，影响禽HEV衣壳蛋白对宿主细胞的吸附以及病毒对细胞的感染，过表达促进病毒感染，敲低抑制病毒感染；同时利用OATP1A2的抑制剂进一步证明了上述影响趋势；利用截短表达，GST-Pull down和ELISA证明OATP1A2的胞外区与禽HEV衣壳蛋白的aa 313-549区域发生直接相互作用，然后通过蛋白阻断和抗体封闭实验，证实两者的直接互作参与禽HEV感染宿主细胞，初步解析了禽HEV感染宿主细胞的分子机制。本项目的研究证明了宿主因子OATP1A2是禽HEV感染宿主细胞的辅助因子，但并不是其唯一的受体，并通过过表达OATP1A2细胞系的构建，将该病毒体外感染细胞效率提高了20%。项目阐明了OATP1A2在禽HEV感染宿主细胞过程中的作用及分子机制,为禽HEV致病机制研究以及抗病毒药物和疫苗研发奠定基础。