Abstract
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.
Highlights
JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans
These observations indicate that Wild-type VLPs (wtVLPs) are able to enter the nucleus of both permissive and nonpermissive cells, and, together with the results of previous studies [4, 19], they suggest that the neurotropism of JCV is attributable to intranuclear mechanisms such as DNA replication, transcription, or virus assembly
Inoculation of HeLa or SVG cells with fluorescein isothiocyanate (FITC)-labeled wtVLPs containing packaged Cy3 resulted in the appearance of both FITC and Cy3 fluorescence signals in nuclei, whereas Cy3 fluorescence was not detected in cells inoculated with a mixture of dissociated FITC-labeled wtVLPs and Cy3
Summary
Limited proteolysis analysis revealed that the NLS of VP1 was exposed on the surface of wtVLPs. 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. We have investigated the mechanism of JCV nuclear entry with VLPs that exhibit both morphological characteristics and physiological functions, including hemagglutination activity, cell attachment, and cellular trafficking, similar to those of JCV virions (18 –20). Limited proteolysis revealed that the NH2-terminal region of VP1 that includes the NLS was exposed on the outer surface of VLPs. In addition, limited proteolysis revealed that the NH2-terminal region of VP1 that includes the NLS was exposed on the outer surface of VLPs These findings suggest that the entry of JCV VLPs into the nucleus of host cells is mediated by interaction of the NLS of VP1 with importins and the NPC
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