The roles of nuclear localization signal in nuclear import of JC virus-like particles

Objective: To study the roles of nuclear localization signal(NLS) in nuclear import of JC virus-like particles (VLP). Methods: The three amino acids: Lys⁵ , Arg⁶, and Lys⁷, of JCV wild-type VP1 (wtVP1) within amino terminal were replaced by Ala, Gly, and Ala, respectively, through site-directed mutagenesis for construction of the mutant of VP1 NLS(△NLS-VP1) . The DNA fragment encoding either wtVP1 or △NLS-VP1 was subcloned into the prokaryotic expression vector pET-15b, and was introduced into competent E. coli by transformation. The VLP composed of VP1 was prepared after expression of recombinant protein VP1 was induced by IPTG. SVG cells were inoculated with FTTC-labeled VLP and nuclear import of VLP was examined with a laser scanning confocal microscope. Results: The wtVLP was transported into nucleus of SVG cells after inoculation, but △NLS-VLP did not, even though these particles was able to enter the cytoplasm. In vitro transport assay using digitonin-permeabilized SVG cells, wtVLP were able to enter the nucleus of SVG cells at present of cytosolic extract or both importin α and β, but the △NLS-VLP did not. After transfection, the wtVP1 mainly expressed in nucleus, but NLS-VP1 most expressed in cytoplasm. When the cells were inoculated with VLP containing packaged DNA, the DNA packaged with wtVLP was detected in nucleus, but the DNA packaged with △NLS-VLP was not detected in nucleus. In vitro overlay assay, wtVLP bound with both importin α and β, but △NLS-VLP bound with neither importin α nor importin β. Conclusion: VP1 NLS is necessary for nuclear import of VLP, and the transport of VLP into nuclear is mediated by the interaction between VP1 NLS and importins.