KPNB1和Ran蛋白共同介导新城疫病毒基质蛋白的入核转运

【目的】鉴定与新城疫病毒(Newcastle disease virus,NDV)基质蛋白(matrix protein,M)入核相关的细胞蛋白,以阐明NDV M蛋白细胞核定位的分子机制。【方法】从鸡胚成纤维细胞中分别克隆核转运受体蛋白KPNA1–KPNA6和KPNB1基因,将其构建到真核表达载体,并与表达NDV M蛋白的重组真核表达载体分别共转染HEK-293T细胞,通过免疫共沉淀方法鉴定与NDV M蛋白相互作用的核转运受体蛋白。另外,将M蛋白与Ran蛋白突变体或与M蛋白互作的核转运受体蛋白缺失体分别共表达,通过荧光共定位确定M蛋白入核转运相关的细胞蛋白。【结果】构建的重组真核表达载体在HEK-293T细胞中能够正确表达;通过间接免疫荧光观察发现,重组蛋白中除Myc-KPNA2蛋白定位在细胞质外,其它核转运受体蛋白均与M蛋白表现出相同的细胞核定位。免疫共沉淀试验结果表明,M蛋白与KPNA1蛋白和KPNB1蛋白均存在相互作用。进一步通过荧光共定位观察发现,M蛋白与KPNA1蛋白缺失体(DN-KPNA1)共表达不改变M蛋白的细胞核定位,而与KPNB1蛋白缺失体(DN-KPNB1)共表达后导致M蛋白变为细胞质定位,说明M蛋白入核转运需要KPNB1蛋白的参与。另外,将M蛋白与Ran蛋白突变体Ran-Q69L共表达,荧光观察发现M蛋白同样由细胞核定位变为细胞质定位,说明M蛋白入核转运还需要Ran蛋白的辅助。【结论】KPNB1和Ran蛋白共同介导NDV M蛋白的入核转运,其过程是KPNB1蛋白首先和M蛋白发生相互作用并形成复合物,然后通过Ran蛋白的辅助作用完成入核转运。

The nuclear import of Newcastle disease virus matrix protein depends on KPNB1 and Ran protein

Abstract: [Objective] The aim of this study was to identify the transport proteins that mediates the nuclear import of Newcastle disease virus (NDV) matrix (M) protein. [Methods] Chicken KPNA1 to KPNA6 gene and KPNB1 gene were cloned from DF-1 cells and then inserted into eukaryotic expression vectors. The constructed recombinant plasmids with a combination of grouping were transfected into HEK-293T cells to identify the transport proteins interacting with NDV M protein by co-immunoprecipitation (Co-IP) assay. Moreover, fluorescent co-localization assay was used to verify the transport proteins by co-expressing M and Ran protein mutant or M and its interactive protein deletant. [Results] The recombinant proteins could normally express in plasmid-transfected HEK-293T cells. Indirect immunofluorescence detection showed that the recombinant proteins except for Myc-KPNA2 displayed the same nuclear localization as NDV M protein. The results of Co-IP revealed that M protein could interact with KPNA1 and KPNB1. Further fluorescent co-localization indicated that co-expression of M and DN-KPNA1 did not change the nuclear localization of M, whereas co-expression of M and DN-KPNB1 or M and Ran-Q69L disrupted the nuclear localization of M, demonstrating that the nuclear import of M protein was dependent on KPNB1 and Ran protein. [Conclusion] KPNB1 and Ran protein jointly mediated the nuclear import of NDV M protein, showing that KPNB1 protein interacted with NDV M protein to form binary complex and then entered into the nucleus with the assistance of Ran protein.