Qu Qiumin, Sawa Hirofumi, Suzuki Tadaki, Semba Shingo, Henmi Chizuka, Okada Yuki, Tsuda Masumi, Tanaka Shinya, Atwood Walter J, Nagashima Kazuo
Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, and CREST, JST, Sapporo 060-8638, Japan.
J Biol Chem. 2004 Jun 25;279(26):27735-42. doi: 10.1074/jbc.M310827200. Epub 2004 Apr 6.
JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. Although transport of virions to the nucleus is an important step in JCV infection, the mechanism of this process has remained unclear. The outer shell of the JCV virion comprises the major capsid protein VP1, which possesses a putative nuclear localization signal (NLS), and virus-like particles (VLPs) consisting of recombinant VP1 exhibit a virion-like structure and physiological functions (cellular attachment and intracytoplasmic trafficking) similar to those of JCV virions. We have now investigated the mechanism of nuclear transport of JCV with the use of VLPs. Wild-type VLPs (wtVLPs) entered the nucleus of most HeLa or SVG cells. The virion structure of VLPs was preserved during transport to the nucleus as revealed by confocal microscopy of cells inoculated with fluorescein isothiocyanate-labeled wtVLPs containing packaged Cy3. The nuclear transport of wtVLPs in digitonin-permeabilized cells was dependent on the addition of importins alpha and beta and was prevented by wheat germ agglutinin or by antibodies to the nuclear pore complex. The nuclear entry of VLPs composed of VP1 with a mutated NLS was greatly inhibited, compared with that of wtVLPs, in both intact and permeabilized cells. Unlike wtVLPs, the mutant VLPs did not bind to importins alpha or beta. Limited proteolysis analysis revealed that the NLS of VP1 was exposed on the surface of wtVLPs. These results suggest that JCV VLPs bind to cellular importins via the NLS of VP1 and are transported into the nucleus through the nuclear pore complex.
JC病毒（JCV）属于双链DNA病毒的多瘤病毒科，可导致人类进行性多灶性白质脑病。尽管病毒粒子向细胞核的转运是JCV感染的重要步骤，但这一过程的机制仍不清楚。JCV病毒粒子的外壳由主要衣壳蛋白VP1组成，VP1具有一个假定的核定位信号（NLS），由重组VP1组成的病毒样颗粒（VLP）具有与JCV病毒粒子相似的病毒样结构和生理功能（细胞附着和胞浆内运输）。我们现在利用VLP研究了JCV的核转运机制。野生型VLP（wtVLP）进入了大多数HeLa或SVG细胞的细胞核。用含有包裹的Cy3的异硫氰酸荧光素标记的wtVLP接种细胞后进行共聚焦显微镜观察，结果显示VLP的病毒粒子结构在转运至细胞核的过程中得以保留。在洋地黄皂苷通透处理的细胞中，wtVLP的核转运依赖于导入蛋白α和β的添加，并受到麦胚凝集素或针对核孔复合体的抗体的抑制。与wtVLP相比，由具有突变NLS的VP1组成的VLP在完整细胞和通透处理的细胞中的核进入均受到极大抑制。与wtVLP不同，突变型VLP不与导入蛋白α或β结合。有限蛋白酶解分析表明，VP1的NLS暴露在wtVLP的表面。这些结果表明，JCV VLP通过VP1的NLS与细胞导入蛋白结合，并通过核孔复合体转运至细胞核。
