JC病毒样颗粒可直接转运进入细胞核

The JC virus-like particle transportes into nuclear

目的 探讨JC病毒 (JCV)病毒样颗粒 (VLP)是否可以直接转运进入细胞核。方法 应用JCV主要外壳蛋白VP1体外表达、重组VLP ,在其表面标记异硫氰基荧光素 (FITC) ,同时在其内部包裹荧光染料Cy3,感染培养的HeLa细胞和SVG细胞 ,荧光显微镜观察VLP入核转运。结果 HeLa和SVG细胞感染包裹Cy3的FITC VLP时 ,FITC与Cy3同时出现于细胞核内相同部位 ;而感染FITC VP1与Cy3混合物时 ,FITC虽可在细胞核内检测到 ,但Cy3信号几乎消失。包裹Cy3的VLP用SDS PAGE展开 ,荧光显像后行考马斯亮蓝 (CBB)染色 ,发现Cy3和VLP移行至不同部位 ,证明Cy3不能与VP1结合 ,提示VLP以完整的颗粒形式转运进入细胞核。应用包裹外源性DNA的VLP感染培养的HeLa和SVG细胞 ,发现包裹的DNA在细胞浆和细胞核内均可检测到 ,提示JCV入核过程与VLP相同。结论 VLP可以不经裂解直接转运进入细胞核 ,JCV入核转运可能与VLP相同。

Objective To determine whether the JC virus-like particles (VLP) can enter the nuclear as an intact particle. Methods The nuclear transport of VLP consisting of recombinant major capsid proteins VP1 were investigated. HeLa or SVG cells were incubated with fluorescein isothiocyanate(FITC)-labeled VLPs containing packaged Cy3 and nucleus import of VLPs were examined with confocal microscopy. Results When the cells were inoculated with FITC-labeled VLPs containing packaged Cy3, the FITC and Cy3 signals exhibited similar temporal and spatial patterns of accumulation in the nucleus of either HeLa or SVG cells. In contrast, when HeLa or SVG cells were inoculated with mixture of dissociated FITC-labeled VP1 and Cy3, the Cy3 signal was not detected within cells, even though FITC was detected in nucleus. VLPs containing packaged Cy3 were subjected to SDS-PAGE and the gel was analyzed by CBB staining for VP1 and with a fluorescence imager for Cy3 respectively, that Cy3 did not bind covalently to VP1 was confirmed as revealed by VP1 and Cy3 migrated to different positions. These findings indicated that the virion structure of VLP was preserved during transportion into the nucleus. When HeLa or SVG cells were incubated with VLPs containing packaged DNA, the DNA was detected using PCR within cytoplasm and nuclear, indicated that nuclear entrance of the VLP is not different from JCV. Conclusion The JCV VLP transported into nuclear in intact particle that is as same as VLP.