原型泡沫病毒Bel1蛋白核定位信号精细定位及相互作用核输入蛋白初探

Identification of Prototype Foamy Virus Bel1 Nuclear Localization Signal and Its Corresponding Importins

Bel1是原型泡沫病毒(Prototype foamy virus,PFV)的反式激活因子,在病毒的复制周期中发挥关键作用。研究表明,Bel1含有核定位信号(Nuclear localization signal,NLS),但其精确氨基酸组成尚不明确,介导其入核的核输入蛋白亦未见报道。本研究利用插入Bel1截短片段的EGFP-GST融合表达体系,通过绿色荧光观察其亚细胞定位,首次精确确定Bel1NLS序列为215PRQKRPR221;定点突变明确了K218、R219和R221为Bel1核定位的必需残基,证明Bel1NLS属单分型核定位信号;GST-Pulldown实验显示这段序列可与α核输入蛋白(Importinα/Karyopherin alpha,official symbol:KPNA)KPNA1、KPNA6和KPNA7相互作用,即Bel1可能藉此转运入核。

Bel1,a transactivator of prototype foamy virus（PFV）,plays pivotal roles in the replication of PFV.Previous studies have shown that Bel1bears a nuclear localization signal（NLS）,but its amino acid sequence remains unclear and the corresponding importins have not been identified.In this report,we inserted various fragments of Bel1into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy.We found that the 215PRQKRPR221 fragment could direct nuclear localization,which accords with the consensus sequence K（K/R）X（K/R）of monopartite NLS.Point mutation experiments revealed that K218,R219,and R221 are essential for the nuclear localization of Bel1.The results of the GST-pulldown showed that the Bel1fragment with residues 215-223,which bears the NLS,interacts with KPNA1,KPNA6,and KPNA7.This result suggests that KPNA1,KPNA6,and KPNA7maybe involved in Bel1nuclear translocation.