Nuclear Entry Mechanism of the Human Polyomavirus JC Virus-like Particle

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 ␣ and ␤ 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 ␣ or ␤. 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.Progressive multifocal leukoencephalopathy is a fatal demyelinating disease of the central nervous system and is caused by JC virus (JCV) 1 (1). Although previously rare, this condition is now commonly seen in patients of different age groups as a result of the increasingly widespread use of immunosuppressive chemotherapy and the prevalence of acquired immune deficiency syndrome (2). JCV belongs to the polyomavirus family, which also includes simian virus 40 (SV40), murine polyomavirus, and BK virus, and is a nonenveloped, icosahedral DNA virus. The circular double-stranded DNA genome comprises 5130 bp and can be functionally divided into three regions: an early coding region, a late coding region, and a noncoding regulatory region (3). The regulatory region, which contains the promoter-enhancer for early and late gene transcription as well as the origin of DNA replication, is located between the early and late coding regions. The early gene encodes the viral regulatory protein, T antigen, whereas the late genes encode the structural capsid proteins VP1, VP2, and VP3 as well as agnoprotein.The early events of JCV infection include attachment of the virion to the host cell surface via a receptor that contains sialic acid (4, 5). The cytoplasmic transport of JCV in eukaryotic cells is dependent on a complex network of three types of cytoskeletal elements: microtubules, microfilaments,...

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DNA-tumor virus entry—From plasma membrane to the nucleus

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