家蚕细胞质型多角体病毒受体鉴定及入侵细胞的分子机理研究

In our previous studies, sequencing of Bombyx mori cypovirus (BmCPV) genome, predicting of BmCPV receptor genes through the bioinformatics analysis of B. mori immunoglobulin superfamily (IgSF) genes, cloning of B. mori SRC kinase gene and establishing platform for generating transgenic silkworm have be accomplished. In this study, we are going to screen and identify BmCPV receptor and characterize functional BmCPV entry so as to understand the molecular mechanism of BmCPV cell entry. Based on the former studies, we will capture BmCPV receptor candidate genes with the interaction between BmCPV structural protein VP3 and proteins located on the midgut cell surface, and validate with yeast two-hybrid system. The receptor candidate genes will be further determined by the changes of BmCPV infectivity to silkworm after inhibiting or deleting the expression and ectopic expression of the candidate genes. Endocytosis pathway of BmCPV will be studied by using specific inhibitors for internalization. The roles of β1 integrin and SRC kinase in the course of BmCPV cell entry are characterized according to the changes of BmCPV infectivity to silkworm and observation of BmCPV cell entry with electron microscope after both of suppressing the expression of β1 integrin and SRC kinase genes through RNAi and inhibiting the activity of SRC kinase with specific inhibitor. The study can not only disclose the BmCPV receptor and characterize the basic molecular mechanisms of BmCPV cell entry, but also theoretically provide the relevant targets, which will help improve the resistance of silkworm to BmCPV by the knockout or suppression expression of the related genes of silkworm to virus cell entry, and promote research and development of therapeutic drug to cytoplasmic polyhedrosis of silkworm.

为了筛选和鉴定家蚕质型多角体病毒(BmCPV)的受体，探讨BmCPV有效入侵的分子机制，我们在已完成BmCPV基因组测序、通过生物信息学分析家蚕免疫球蛋白超家族(IgSF)预测BmCPV受体、SRC激酶基因克隆和家蚕转基因平台建立的基础上，拟通过BmCPV与中肠细胞表面蛋白互作和酵母双杂交捕获、验证BmCPV受体候选基因；通过抑制或缺失候选基因表达以及异位表达候选基因,研究家蚕对BmCPV的感染性和组织特异性，鉴定受体；通过特异性抑制剂研究细胞内吞BmCPV方式；通过RNAi抑制β1整联蛋白、SRC激酶的表达，抑制剂抑制SRC激酶活性，根据家蚕对BmCPV的感染性及BmCPV入侵细胞过程观察，探讨β1整联蛋白和SRC激酶在BmCPV入侵细胞中的功能。本项目不仅能明确BmCPV受体和入侵的基本分子机制，并可为敲除或抑制病毒入侵相关的家蚕基因，提高家蚕对BmCPV抗性以及研发防治药物提供靶标。

家蚕质型多角体病毒（Bombyx mori cypovirus, BmCPV）是引起养蚕歉收的重要病原，至今不明BmCPV的受体以及BmCPV的感染机制。本研究采用Co-IP、VOPBA、pull-down等技术筛选了与BmCPV互作的中肠组织细胞膜蛋白，通过质谱对相关蛋白进行了鉴定。结合信息学分析，通过敲降、抗体封闭和共定位等方法对衔接蛋白-1 (AP)，网格蛋白 (CLAT)，整合素β (ITGB)，酪氨酸蛋白激酶 (SRC)，激活蛋白激酶受体 (RACK)，唐氏综合征细胞黏附分子-1（Dscam1），Nogo受体1 (NgR1)，紧密接合蛋白ZO-1(BmZO）和阿片结合蛋白 (OBP) 等23个候选受体蛋白和1个共受体进行鉴定，证明BmCPV的受体由ITGB、RACK、CLAT、AP、OBP等构成；细胞表面的神经节甘脂GM2是BmCPV的共受体。OBP和ITGB在中肠组织特异性高水平表达可能与BmCPV中肠感染组织特异性有关。敲降受体基因表达和/或抗体封闭受体蛋白、内吞抑制剂（丹（磺）酰戊二胺、氯丙嗪）抑制BmCPV侵染、电镜观察结果表明，BmCPV利用ITGB通过CLAT介导的内吞进入细胞。广谱性的酪氨酸蛋白激酶抑制剂金雀异黄素和SRC家族的特异性抑制剂PP2可通过可促进内化的BmCPV异常定位至溶酶体，BmCPV的异常定位可被溶酶体抑制剂leupeptin部分拯救，表明SRC对BmCPV的有效感染有重要影响。丹（磺）酰戊二胺、氯丙嗪、金雀异黄素、PP2添食可明显抑制BmCPV对家蚕的感染性。抑制Dscam1、NgR1、BmZO可显著抑制BmCPV的感染性，表明BmCPV入侵细胞存在冗余途径。相关研究明确了BmCPV受体和入侵的基本分子机制，为敲除或抑制病毒入侵相关的家蚕基因，提高家蚕对BmCPV抗性以及研发防治药物提供靶标。此外，建立了基于体外转录病毒RNA的BmCPV反向遗传学系统，为研究BmCPV基因功能、研发重组CPV杀虫剂提供了方法；发现BmCPV基因组编码功能性小分子多肽，打破了BmCPV基因组片段为单顺反子的认识。