Importin α/β与importin β双重途径介导鹿眼蛱蝶浓核病毒结构蛋白VP1入核转运

Dual pathways of importin α/β and importin β mediate the nuclear translocation of the structural protein VP1 of Junonia coenia densovirus

摘要:

目的 分析鹿眼蛱蝶浓核病毒(Junonia coenia densovirus, JcDV)结构蛋白VP1在棉铃虫(Helicoverpa armigera)表皮细胞系HaEpi内的入核转运动态及转运途径，为明确JcDV的装配与增殖过程提供理论依据。方法 构建与绿色荧光蛋白(green fluorescent protein, GFP)融合表达的VP1野生型、突变体质粒，转染至HaEpi细胞内，分析VP1的亚细胞定位动态特征，鉴定核定位信号(nuclear localization signal, NLS)及关键氨基酸残基；克隆HaEpi细胞表达的入核转运受体基因，构建与红色荧光蛋白DsRed2融合表达的质粒，分析其亚细胞定位；采用共定位、免疫共沉淀(co-immunoprecipitation, Co-IP)技术分析VP1与入核转运受体的相互作用。结果 转染后6 h，VP1定位于细胞质内，之后直至48 h逐渐转运至细胞核内；VP1的NLS位于325-EGTKRKADTPVEEGPSKKGAH-345，其中K328、R329、K341是影响核定位的关键氨基酸残基。入核转运受体Haimpα1、Haimpα4、Haimpα7定位于细胞核内，而Haimpβ1主要位于细胞核膜周围。共定位与Co-IP结果表明，VP1与Haimpα1、Haimpα4、Haimpβ1存在相互作用，但与Haimpα7无相互作用。结论 JcDV结构蛋白VP1可通过importin α/β和importin β双重途径转运入核。

Abstract:

Objective We explored the nuclear translocation dynamics and pathways of the structural protein VP1 of Junonia coenia densovirus (JcDV) in the epidermal cell line HaEpi derived from the larvae of Helicoverpa armigera, aiming to provide theoretical support for clarifying the assembly and proliferation processes of JcDV.Methods The wild-type and mutant plasmids of VP1 protein fused with green fluorescent protein (GFP) were constructed and transfected into HaEpi cells. Subsequently, the subcellular localization dynamics of the VP1 protein were analyzed, and the nuclear localization signal (NLS) and key amino acid residues of the protein were identified. The importin genes expressed by HaEpi cells were cloned. Subsequently, the plasmids of importins fused with DsRed2 were constructed to analyze their subcellular localization. The co-localization and co-immunoprecipitation (Co-IP) assays were employed to analyze the interactions between VP1 protein and importins.Results The VP1 protein was located in the cytoplasm at 6 h post transfection, and then gradually translocated to the nucleus until 48 h. The NLS of VP1 protein was located at 325-EGTKRKADTPVEEGPSKKGAH-345, among which K328, R329, K341 were the key amino acid residues affecting the nuclear localization. The importins Haimpα1, Haimpα4, and Haimpα7 were located in the nucleus, while Haimpβ1 was mainly located around the nuclear membrane. The co-localization and Co-IP results indicated that the VP1 protein interacted with Haimpα1, Haimpα4, and Haimpβ1 but not with Haimpα7.Conclusion The structural protein VP1 of JcDV can be translocated into the nucleus through the dual pathways of importin α/β and importin β.