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Originally published In Press as doi:10.1074/jbc.M310827200 on April 6, 2004

J. Biol. Chem., Vol. 279, Issue 26, 27735-27742, June 25, 2004
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Nuclear Entry Mechanism of the Human Polyomavirus JC Virus-like Particle

ROLE OF IMPORTINS AND THE NUCLEAR PORE COMPLEX*

Qiumin Qu{ddagger}, Hirofumi Sawa{ddagger}§, Tadaki Suzuki{ddagger}, Shingo Semba{ddagger}, Chizuka Henmi{ddagger}, Yuki Okada{ddagger}, Masumi Tsuda{ddagger}, Shinya Tanaka{ddagger}, Walter J. Atwood||, and Kazuo Nagashima{ddagger}

From the {ddagger}Laboratory of Molecular and Cellular Pathology and §21st Century COE Program for Zoonosis Control, Hokkaido University School of Medicine, and CREST, JST, Sapporo 060-8638, Japan and the ||Department of Molecular Microbiology and Immunology, Brown University, Providence, Rhode Island 02912

Received for publication, October 1, 2003 , and in revised form, April 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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, and intermediate filaments (6). After reaching the nucleus, the viral DNA undergoes replication and is transcribed into RNA, which is followed by the production of viral proteins and virion maturation. Successful JCV infection thus depends on the import of the virion into the nucleus, but the mechanism of this import has remained unknown.

Entry of macromolecules into the nucleus is an active process and is controlled by the interactions of transport factors (importins) both with their respective macromolecular cargoes and with the nuclear pore complex (NPC) (7). The nuclear entry of proteins that contain a classical nuclear localization signal (NLS) is mediated by importin {alpha} and {beta} heterodimers (8). Importin {alpha} recognizes and binds directly to the NLS, whereas importin {beta}, which interacts directly with a protein component of the nuclear pore (nucleoporin), binds the trimeric complex of the NPC and mediates its translocation into the nucleus (9).

The major capsid proteins (VP1) of SV40 and murine polyomavirus contain an NLS (10, 11). SV40 virions enter the nucleus through the NPC in a manner that is sensitive to wheat germ agglutinin (WGA) or to a monoclonal antibody specific for nucleoporin (12, 13). However, mutant SV40 virions that are devoid of an NLS have not been generated, leaving unresolved the question of whether such mutant virions would be able to enter the nucleus.

The NH2-terminal 12 amino acids of JCV VP1 (MAPTKRKGERKD) include a stretch of basic residues that is similar to the NLS of murine polyomavirus VP1 or SV40 VP1 and is a putative NLS of JCV VP1 (14, 15). This NLS of JCV VP1 was shown to be inefficient in mediating nuclear transport (16). However, JCV virus-like particles (VLPs) consisting exclusively of recombinant VP1 purified from either Escherichia coli or a baculovirus-insect cell expression system and assembled by the formation of disulfide bonds (17) were found to be transported to the nucleus of host cells (18, 19). We have now 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). VLPs comprised of wild-type VP1 (wtVLPs), but not those consisting of VP1 with a mutated NLS ({Delta}NLS-VLPs), were imported into the nucleus of both HeLa and SVG cells. An in vitro transport assay revealed that wtVLPs entered the nucleus in the presence of a cytosolic fraction or of importins {alpha} and {beta}, and the nuclear entry of wtVLPs was prevented by WGA or antibodies to the NPC. 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—HeLa human cervical carcinoma cells (JCRB 9004) were obtained from the Health Science Research Resources Bank (Osaka, Japan). SVG human fetal glial cells were as described (5). Both cell lines were cultured under 5% CO2 at 37 °C in Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, penicillin, and streptomycin (Sigma).

Plasmid Construction, Cell Transfection, and Immunofluorescence Analysis—The wtVP1 gene of JCV was subcloned from pBR-Mad1 (21) into the prokaryotic expression vector pET-15b (Novagen, Madison, WI). For construction of {Delta}NLS-VP1, three amino acids, Lys5, Arg6, and Lys7, of wtVP1 were replaced with Ala, Gly, and Ala, respectively, by site-directed mutagenesis. The DNA fragment encoding {Delta}NLS-VP1 was also subcloned into pET-15b. To examine localization of recombinant wtVP1 and {Delta}NLS-VP1 in SVG cells, we subcloned the corresponding DNA fragments into the eukaryotic expression vector pCXN2Flag (22), which generates FLAG epitope-tagged recombinant proteins. The inserted DNA fragments of all plasmids were confirmed by sequencing.

Twenty-four hours after transfection of SVG cells with the use of Optifect (Invitrogen, Carlsbad, CA), cells were lysed in RIPA buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulfonyl fluoride), and expression of the recombinant proteins was confirmed by immunoblot analysis with a horseradish peroxidase-conjugated mouse monoclonal antibody to FLAG (M2, Sigma). For immunocytofluorescence analysis, the cells were fixed with 3% paraformaldehyde for 15 min at room temperature and then exposed to 70% methanol for 5 min at -20 °C. The fixed cells were incubated with the M2 antibody to FLAG (1:500 dilution), and immune complexes were detected with Alexa 488-conjugated goat antibodies to mouse immunoglobulin G (Molecular Probes, Eugene, OR). The cells were then examined with a laser-scanning confocal microscope (Olympus, Tokyo, Japan).

Preparation of VLPs—VLPs composed of wtVP1 or {Delta}NLS-VP1 were prepared as previously described (19), with slight modifications. The expression plasmids for wtVP1 and {Delta}NLS-VP1 were separately introduced into competent E. coli BL21(DE3)/pLys cells (Stratagene, La Jolla, CA) by transformation, and expression of the recombinant proteins was induced by incubation of the cells for 4 h at 30 °C with 1 mM isopropyl-{beta}-D-thiogalactopyranoside. The cells were then separated by centrifugation for 10 min at 4,000 x g and resuspended in 20 ml of reassociation buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM CaCl2) containing lysozyme (1 mg/ml). After incubation on ice for 30 min and the addition of sodium deoxycholate to a final concentration of 2 mg/ml, the cells were incubated for an additional 10 min on ice and then lysed by five cycles of sonication (15-s bursts), and genomic DNA was digested with DNase I (Amersham Biosciences). The lysate was then centrifuged through a layer of 20% (w/v) sucrose at 100,000 x g for 2 h at 4 °C. The VLPs in the resulting pellet were purified further by CsCl density gradient centrifugation at 100,000 x g for 16 h at 16 °C. All of the gradient fractions were assayed by the hemagglutination test, and those containing the highest activity were pooled (~0.5 ml) and dialyzed for 24 h at 4 °C against two changes of reassociation buffer (1000 ml).

For conjugation with fluorescein isothiocyanate (FITC), VLPs (2 mg) were dissolved in 400 µl of phosphate-buffered saline (PBS), mixed with 40 µl of 1 M carbonate/bicarbonate buffer (pH 9.0) and 156 µl of FITC (Sigma) solution (1 mg/ml in 0.1 M carbonate/bicarbonate buffer (pH 9.0)), and incubated at room temperature for 2 h in the dark. After centrifugation of the mixture at 100,000 x g for 1 h at 4 °C, the pellet was dissolved in PBS and centrifuged overnight at 12,000 x g and 4 °C. The final pellet was resuspended in PBS. The purified VLPs composed of either wtVP1 or {Delta}NLS-VP1 possessed hemagglutination activity (data not shown) and yielded a prominent band of ~45 kDa on SDS-PAGE and staining with Coomassie Brilliant Blue (Fig. 1A). The electrophoretic mobility of {Delta}NLS-VP1 was slightly greater than was that of wtVP1, probably because of the difference in electrical charge between the two proteins.



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  		FIG. 1.
SDS-PAGE and electron microscopic analyses of VLPs composed of wtVP1 or {Delta}NLS-VP1. A, purified wtVLPs or {Delta}NLS-VLPs were subjected to SDS-PAGE and Coomassie Brilliant Blue staining. The positions of the wtVP1 and {Delta}NLS-VP1 proteins (~45 kDa) are indicated (arrow). B and C, electron micrographs of wtVLPs and {Delta}NLS-VLPs, respectively. Scale bars, 60 nm. D, SDS-PAGE analysis of wtVLPs and {Delta}NLS-VLPs containing packaged Cy3. The VLPs were subjected to SDS-PAGE, and the resulting gel was stained with Coomassie Brilliant Blue to detect VP1 (left panel) and examined with a fluorescence image analyzer to detect Cy3 (right panel).

 
Electron Microscopic Analysis of VLPs—VLPs (5 µl) were transferred to polyvinyl formal-coated grids and left for 5 min. After removal of residual solution with filter paper, 10 drops of 2.5% phosphotungstic acid were placed onto each grid and allowed to air dry. The VLPs were then observed with a model H-800 electron microscope (Hitachi, Tokyo, Japan). Electron microscopy revealed that both wtVLPs and {Delta}NLS-VLPs exhibited virion particle-like structures with a diameter of 40-50 nm (Fig. 1, B and C). A similar morphology was evident for VLPs after labeling with FITC or packaging of Cy3 (data not shown).



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  		FIG. 10.
Limited proteolysis of the VP1 monomer and wtVLPs. A, both the VP1 monomer and wtVLPs that had been purified by gel filtration were subjected to limited digestion with trypsin for the indicated times, after which the protein fragments were fractionated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining. Closed arrowheads indicate full-length VP1, open arrowheads indicate ~30-kDa fragments, and the arrow indicates an ~40-kDa fragment. B, the NH2-terminal amino acid sequences of the ~30-kDa tryptic fragments of both the VP1 monomer and wtVLPs were determined and are shown compared with the sequence of the first 18 residues of JCV VP1. The NLS motif is underlined.

 
Packaging of Cy3 into FITC-labeled VLPs—The fluorescent dye Cy3 (Amersham Biosciences) contains a monofunctional NHS group for conjugation to the amino groups of proteins. To quench this reactive group, we dissolved Cy3 in 50 mM monoethanolamine and incubated the resulting solution on ice overnight. The quenched Cy3 was then packaged into FITC-labeled VLPs by the method described for the packaging of propidium iodide into JCV VLPs (23). In brief, purified FITC-labeled VLPs (0.5 mg) were dissociated in 1 ml of dissociation buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM EGTA, 5 mM dithiothreitol) by incubation for 1 h at room temperature. Cy3 (100 µg) in 10 µl of 50 mM ethanolamine-HCl (pH 8.0) was added to the dissociated VP1 pentamers, and the resulting mixture was dialyzed overnight at 4 °C in the dark against 1,000 ml of reassociation buffer with a membrane that had an exclusion size of 14 kDa. After the addition of 6 mM MgCl2 and 10 units of DNase I, the FITC-labeled VLPs containing Cy3 were incubated for 1 h at 37 °C, isolated by centrifugation at 100,000 x g for 1 h at 4 °C, and resuspended in 200 µl of PBS. To confirm that Cy3 was indeed packaged into the VLPs, we inoculated cells with a mixture of dissociated VLPs and Cy3 that had not been subjected to subsequent treatment with reassociation buffer. In addition, we subjected the FITC-labeled VLPs containing packaged Cy3 to SDS-PAGE and analyzed the resulting gel by Coomassie Brilliant Blue staining for VP1 and with a fluorescence imager (FLA 3000; Fuji Film, Tokyo, Japan) for Cy3. The fact that VP1 and Cy3 migrated at different positions in the gel confirmed that Cy3 did not bind covalently to VP1 (Fig. 1D).

Packaging of DNA into VLPs and Detection of DNA by PCR—To examine whether VLPs with packaged DNA are able to enter the nucleus, we introduced the pBluescript II SK+ plasmid (Stratagene) containing the replication origin of JCV into VLPs by the same method as that used for packaging of Cy3. HeLa or SVG cells were seeded into 60-mm plates, cultured for 24 h, and inoculated for 3 h at 37 °C either with VLPs containing DNA or with JCV in 2 ml of DMEM supplemented with 10% fetal bovine serum. Total DNA was then extracted with the use of a DNA extraction kit (Nucleobond AX; Macherey-Nagel, Easton, PA) either from total lysates of the inoculated cells or from a nuclear fraction thereof prepared as described (7).

The PCR was performed with a Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA) in a 50-µl reaction mixture containing 1.0 µg of template DNA, 0.2 mM of each deoxynucleoside triphosphate, and 0.1 mM of each primer. For detection of the JCV genome, the amplification protocol included an initial incubation at 95 °C for 5 min; 30 cycles of 95 °C for 45 s, 61 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. For detection of the packaged plasmid containing the JCV origin of replication, the protocol comprised an initial incubation at 95 °C for 5 min; 30 cycles of 95 °C for 30 s and 55 °C for 30 s; and a final incubation at 72 °C for 7 min. The primers for detection of the JCV genome (nucleotides 1828 to 2039) were 5'-TGTGCACTCTAATGGGCAAGC-3' (forward) and 5'-CTAGCTACGCCTTGTGCTCTG-3' (reverse); those for detection of the packaged plasmid were 5'-GTAAAACGACGGCCAG-3' (forward) and 5'-CAGGAAACAGCTATGAC-3' (reverse). The PCR products were separated by electrophoresis on an agarose gel containing ethidium bromide and visualized with ultraviolet illumination.

Laser-scanning Confocal Microscopy—HeLa or SVG cells (2 x 104) were transferred to 35-mm glass-bottom dishes (Iwaki, Chiba, Japan) in DMEM supplemented with 10% fetal bovine serum and incubated for 24 h at 37 °C. The cells were then inoculated for 1 h at 4 °C in DMEM with FITC-labeled VLPs with or without packaged Cy3 (0.256 to 2.56 units of hemagglutination activity per cell). After three washes with PBS, the cells were incubated at 37 °C in DMEM and examined at various times with a laser-scanning confocal microscope (Olympus). For determination of the frequency of import of VLPs into the nucleus, the cells were fixed 3 h after inoculation by incubation for 10 min at room temperature with 3% paraformaldehyde and were then stained for 5 min with propidium iodide (0.2 µg/ml). The number of cells in which FITC-labeled VLPs were detected in the nucleus (as revealed by propidium iodide staining) was counted with the laser-scanning confocal microscope and expressed as a percentage of total cells.

In Vitro Nuclear Transport Assay—HeLa or SVG cells were transferred to 8-well coverslips for 24 h, washed twice with a solution containing 20 mM Hepes-NaOH (pH 7.3), 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, and 1 mM EGTA, and then permeabilized for 5 min on ice with digitonin (90 µg/ml) in the wash solution supplemented with 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, as well as leupeptin, pepstatin, and aprotinin each at 1 µg/ml (transport buffer). This concentration of digitonin was selected as optimal from several different concentrations (40, 60, 90, and 120 µg/ml) tested. The cells were then washed three times with transport buffer and incubated first for 5 min on ice with the same solution to deplete cytosolic factors and then for 15 min at room temperature with 10 µl of transport buffer in the absence or presence of either WGA (50 µg/ml) or a mouse monoclonal antibody (200 µg/ml) to the NPC (Co-vance, Richmond, CA). They were washed twice with transport buffer before the addition of 10 µl of transport buffer as well as 1 µl of an ATP-regenerating system (1 mM ATP, 5 mM creatine phosphate, 20 units of creatine phosphokinase) and 10 µl of either a cytosolic fraction (68 µg of protein) or recombinant importins {alpha} or {beta} (0.3 µg of each). After inoculation with 5 µl of FITC-labeled VLPs, the cells were incubated for 30 min at 37 °C, washed extensively with transport buffer, fixed for 10 min with 3% paraformaldehyde, and examined by laser-scanning confocal microscopy.

A cytosolic fraction was prepared as previously described (7). In brief, HeLa or SVG cells in the exponential phase of growth were harvested by centrifugation for 10 min at 900 x g and washed twice with ice-cold PBS. They were then washed with a solution containing 10 mM Hepes-NaOH (pH 7.3), 10 mM potassium acetate, 2 mM magnesium acetate, and 2 mM dithiothreitol, resuspended in 5 volumes of lysis buffer (5 mM Hepes-NaOH (pH 7.4), 10 mM potassium acetate, 2 mM magnesium acetate, 2 mM dithiothreitol, 20 µM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride, and aprotinin, leupeptin, and pepstatin each at 1 µg/ml), incubated for 10 min on ice, and homogenized in a steel homogenizer (10 strokes). The homogenate was centrifuged at 100,000 x g for 40 min at 4 °C, and the resulting supernatant was dialyzed against transport buffer and then concentrated with a membrane that eliminates proteins of <10 kDa (Millipore). Recombinant importins {alpha} and {beta} were produced in and purified from E. coli as glutathione S-transferase (GST) fusion proteins as described previously (24, 25).

VLP Overlay Assay—A VLP overlay assay was performed as previously described (4). Recombinant GST-importin {alpha}, GST-importin {beta}, both importins, or GST alone were separated by SDS-PAGE on an 8% gel and then transferred to a polyvinylidene difluoride membrane. Nonspecific binding sites on the membrane were blocked by incubation for 1 h with 5% skim milk in Tris-buffered saline (TBS: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl) containing 0.1% Tween 20 (TBS-T). The membrane was then incubated for 2 h at 4 °C with purified VLPs (5 µg/ml) suspended in blocking solution. After four washes with TBS-T, the membrane was incubated for 1 h at 4 °C with rabbit polyclonal antibodies (1:1000 dilution in TBS-T) to VP1 (26, 27) or GST (Amersham Biosciences). The membrane was again washed with TBS-T and then incubated for 1 h at 4 °C with horseradish peroxidase-conjugated goat F(ab')2 (1:3000 dilution) to rabbit immunoglobulin (BioSource Int., Camarillo, CA). Immune complexes were detected with ECL reagents (Amersham Biosciences) and a LAS-1000 Plus image analyzer (Fuji Film).

Limited Proteolysis of VLPs and VP1—VLPs and VP1 were further purified by gel filtration on a column of HiLoad 16/60 Superdex 200 (Amersham Biosciences). VLPs were applied to the column after its equilibration with TBS and eluted in the void volume with the same solution at a flow rate of 0.5 ml/min. For purification of VP1, VLPs were dialyzed against dissociation buffer overnight at 4 °C and then applied to the same column after its equilibration with dissociation buffer. VP1 monomers eluted in fractions corresponding to a molecular size of ~70 kDa. The purified VLPs and VP1 monomers were dialyzed against TBS and dissociation buffer, respectively.

For limited proteolysis, VLPs or VP1 monomers (1 mg/ml) were digested at room temperature with trypsin (10 µg/ml) (sequencing grade; Roche Diagnostics) in TBS or dissociation buffer, respectively. The reaction was terminated after various times by the addition of phenylmethylsulfonyl fluoride at a final concentration of 2 mM. The digestion products were fractionated by SDS-PAGE on a 15% gel, transferred to an Immobilon-P membrane (Millipore), and stained with Coomassie Brilliant Blue, and selected bands were excised, destained with methanol, and subjected to amino acid sequence analysis with a Procise49X cLC Protein Sequencer (Applied Biosystems).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Entry of VLPs into the Nucleus—The function of the amino acid stretch at the NH2 terminus of JCV VP1 was examined by transfecting SVG cells with expression vectors for either FLAG-wtVP1 or FLAG-{Delta}NLS-VP1. The electrophoretic mobility of FLAG-{Delta}NLS-VP1 was little greater than that of FLAG-wtVP1, which was similar to the band previously shown in Fig. 1A, and the expression level of FLAG-wtVP1 was slightly higher than that of FLAG-{Delta}NLS-VP1 (Fig. 2A). FLAG-wtVP1 was localized predominantly in the nucleus of SVG cells, however, FLAG-{Delta}NLS-VP1 was mostly restricted to the cytoplasm (Fig. 2B), suggesting that the basic amino acid region at the NH2 terminus of VP1 functions as an NLS.



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  		FIG. 2.
Localization of wtVP1 and {Delta}NLS-VP1 in transfected SVG cells. Twenty-four hours after transfection with expression vectors encoding either FLAG-wtVP1 or FLAG-{Delta}NLS-VP1, the cells were harvested with RIPA buffer and subjected to immunoblot analysis (A) or immunofluorescence analysis (B) with antibodies to FLAG. The positions of the wtVP1 and {Delta}NLS-VP1 proteins (~45 kDa) are indicated (arrow). Nuclei are stained red with propidium iodide (PI) in the lower panels of B.

 
Nuclear entry of JCV VLPs was assayed by inoculating HeLa or SVG cells, which are nonpermissive and permissive, respectively, for JCV infection, with FITC-labeled VLPs. The FITC signal of wtVLPs was detected in the nucleus of HeLa cells after subsequent incubation of the cells for 1 h at 37 °C (Fig. 3A). Nuclear fluorescence increased in intensity during incubation for an additional 2 h, with 95.6% of HeLa cells containing wtVLPs in the nucleus 3 h post-inoculation. In contrast, the nuclear entry of {Delta}NLS-VLPs was markedly inhibited in HeLa cells, even though these particles were able to enter the cytoplasm (Fig. 3A). Only 2.2% of HeLa cells contained {Delta}NLS-VLPs in the nucleus 3 h post-inoculation. Similar results were obtained with SVG cells (Fig. 3B). Whereas 95.4% of SVG cells manifested wtVLPs in the nucleus 3 h post-inoculation, the corresponding value for {Delta}NLS-VLPs was only 6.1%. These results indicated that the NLS of VP1 is important for the nuclear translocation of VLPs in host cells.



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  		FIG. 3.
Comparison of the abilities of wtVLPs and {Delta}NLS-VLPs to enter the nucleus of HeLa or SVG cells. HeLa (A) or SVG (B) cells were inoculated with FITC-labeled wtVLPs (upper panels) or {Delta}NLS-VLPs (lower panels). The cells were examined by confocal microscopy after 1- or 2-h subsequent incubation at 37 °C. After 3 h, the cells were fixed and then stained with propidium iodide to identify nuclei; the percentage of cells exhibiting FITC fluorescence in the nucleus was determined and is indicated in the panels on the right. Scale bars, 10 µm.

 
To exclude the possibility that our detection of the FITC signal of wtVLPs in the nucleus of inoculated host cells actually represented the nuclear translocation of FITC-labeled VP1 monomers contaminating the VLP preparation, we inoculated cells with FITC-labeled wtVLPs containing packaged Cy3. The FITC (green) and Cy3 (red) signals exhibited similar temporal and spatial patterns of accumulation in the nucleus of either HeLa (Fig. 4A) or SVG (Fig. 4B) cells, indicating that FITC and Cy3 entered the nucleus as surface and internal components, respectively, of intact wtVLPs. Consistent with the results obtained with FITC-labeled {Delta}NLS-VLPs (Fig. 3), the FITC and Cy3 signals derived from FITC-labeled {Delta}NLS-VLPs containing packaged Cy3 overlapped in the cytoplasm of HeLa (Fig. 4C) or SVG (Fig. 4D) cells but did not enter the nucleus to a marked extent.



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  		FIG. 4.
Entry of FITC-labeled wtVLPs containing packaged Cy3 into the nucleus of host cells. A and B, HeLa or SVG cells, respectively, were inoculated with FITC-labeled wtVLPs containing packaged Cy3 and monitored for FITC (green) and Cy3 (red) fluorescence by confocal microscopy after subsequent incubation at 37 °C for the indicated times. The merged FITC and Cy3 signals are shown in yellow. C and D, HeLa or SVG cells, respectively, were inoculated with FITC-labeled {Delta}NLS-VLPs containing packaged Cy3 and monitored as in A and B. E and F, as a negative control, FITC-labeled wtVLPs were incubated in dissociation buffer for 1 h at room temperature and then mixed with Cy3 without reassociation treatment. The mixture was then used to inoculate HeLa (E) or SVG (F) cells, which were subsequently monitored for FITC and Cy3 fluorescence. Scale bars, 10 µm.

 
We also inoculated cells with a mixture of dissociated FITC-labeled VP1 and Cy3 that had not been subjected to reassociation treatment. The Cy3 signal was not detected within either HeLa (Fig. 4E) or SVG (Fig. 4F) cells under these conditions, indicating that Cy3 did not bind to the dissociated VP1.

Role of Importins in the Nuclear Entry of VLPs Through the NPC—It has been generally accepted that the NLS motif binds to importin {alpha}, which also interacts with importin {beta}, and the resulting tripartite complex is translocated into nuclei through the NPC (28). We next examined the role of importins and the NPC in the entry of VLPs into the nucleus with digitonin-permeabilized HeLa and SVG cells. In the presence of an ATP-regenerating system, FITC-labeled wtVLPs were not able to enter the nucleus of either HeLa or SVG cells without the addition of a cytosolic fraction prepared from the corresponding intact cells (Fig. 5, A, B, F, and G), suggesting that cytosolic factors play an important role in the nuclear entry of VLPs. To determine whether such cytosolic factors might include importins, we examined the effects of GST fusion proteins of importin {alpha} or {beta} in this system. Whereas neither importin {alpha} or {beta} alone did not induce the nuclear translocation of wtVLPs (Fig. 5, C, D, H, and I), the combination of the two proteins did (Fig. 5, E and J).



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  		FIG. 5.
Role of importins in transport of wtVLPs into the nucleus of digitonin-permeabilized cells. HeLa (A-E) or SVG (F-J) cells were permeabilized with digitonin and then incubated for 30 min at 37 °C with FITC-labeled wtVLPs and an ATP-regenerating system in the absence (A and F) or presence of a corresponding cytosolic fraction (B and G), GST-importin {alpha} (C and H), GST-importin {beta} (D and I), or both GST-importins (E and J). FITC fluorescence was then examined by confocal microscopy. Scale bars, 10 µm.

 
We next tested the effect of WGA, which binds to the NPC and inhibits the import of proteins into the nucleus of intact cells (29). The nuclear import of wtVLPs apparent in digitonin-permeabilized HeLa or SVG cells in the presence of a cytosolic fraction was prevented by WGA (Fig. 6, A, B, D, and E). Furthermore, the translocation of wtVLPs into the nucleus was also completely blocked by antibodies to the NPC (Fig. 6, C and F). Together, these results thus suggested that wtVLPs entered the nucleus of host cells through the NPC. The role of the NLS of VP1 in this process was confirmed by the observation that {Delta}NLS-VLPs did not enter the nucleus of permeabilized HeLa or SVG cells even in the presence of a cytosolic fraction or both importin {alpha} and importin {beta} (Fig. 7).



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  		FIG. 6.
Role of the NPC in the nuclear import of wtVLPs in digitonin-permeabilized cells. Permeabilized HeLa (A-C) or SVG (D-F) cells were pretreated in the absence (A and D) or presence of WGA (B and E) or antibodies to the NPC (C and F) and then incubated for 30 min at 37 °C with FITC-labeled wtVLPs in the presence of an ATP-regenerating system and a cytosolic fraction. Scale bars, 10 µm.

 



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  		FIG. 7.
Role of the NLS of VP1 in the nuclear import of wtVLPs in digitonin-permeabilized cells. Permeabilized HeLa (A-D) or SVG (E-H) cells were incubated for 30 min at 37 °C with FITC-labeled wtVLPs (A, B, E, and F) or {Delta}NLS-VLPs (C, D, G, and H) in the presence of an ATP-regenerating system and either a cytosolic fraction (A, C, E, and G) or both GST-importin {alpha} and GST-importin {beta} (B, D, F, and H). Scale bars, 10 µm.

 
We investigated the possible binding of wtVLPs or {Delta}NLS-VLPs to importins with the use of a VLP overlay assay (4). Both GST-importin {alpha} and GST-importin {beta} interacted with wtVLPs but not with {Delta}NLS-VLPs; no interaction was apparent between GST and VLPs (Fig. 8). Together, these results indicate that the nuclear import of wtVLPs is mediated by interaction of the NLS motif of VP1 with both importins {alpha} and {beta}.



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  		FIG. 8.
Interaction of wtVLPs with importins {alpha} and {beta} in vitro. Recombinant GST-importin {alpha}, GST-importin {beta}, both GST-importin {alpha} and GST-importin {beta}, or GST alone were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was incubated with either wtVLPs or {Delta}NLS-VLPs and then subjected to immunoblot analysis (IB) with antibodies to VP1 ({alpha}-VP1) or GST. Open arrowheads indicate signals corresponding to GST-importin {beta} or GST; closed arrowheads indicate GST-importin {alpha}. The reactivity of the antibodies to VP1 was confirmed by immunoblot analysis of a lysate of JCV-producing cells (JCI) as well as of purified wtVLPs and {Delta}NLS-VLPs (bottom panel).

 
Nuclear Entry of VLPs Containing Packaged DNA—We also investigated whether VLPs containing packaged DNA, specifically a plasmid containing the JCV origin of replication, were able to enter the nucleus of HeLa or SVG cells. JCV DNA was detected in both total lysates and the nuclear fraction of HeLa or SVG cells after incubation of the cells for 3 h with wtVLPs containing the plasmid; the viral DNA was not detected in cell lysates after incubation of cells with a mixture of wtVLPs and nonpackaged plasmid (Fig. 9A). Although JCV DNA was detected in cell lysates after inoculation of cells with {Delta}NLS-VLPs containing the packaged plasmid, it was not detected in the nuclear fraction. As a control, we showed that JCV DNA was present in both the total lysates and nuclear fraction of cells after infection with JCV (Fig. 9B). Together, these results suggest that VLPs with packaged DNA are able to enter the nucleus of host cells.



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  		FIG. 9.
Entry of wtVLPs containing packaged DNA into the nucleus of host cells. HeLa or SVG cells were inoculated with either wtVLPs or {Delta}NLS-VLPs containing a packaged plasmid that includes the JCV origin of replication (A) or were infected with JCV (B). Total cell lysates or the nuclear fractions thereof were then subjected to PCR to detect JCV DNA. As a control in A, cells were inoculated with a mixture of wtVLPs and plasmid without packaging treatment.

 
Limited Proteolysis of VLPs and VP1—With the use of limited proteolysis, we next investigated whether the NLS motif of VP1 is exposed on the surface of wtVLPs. Incubation of VP1 monomer (purified by gel filtration) with trypsin, which cleaves proteins on the COOH-terminal side of Arg or Lys residues, resulted in the generation of a fragment of ~30 kDa (Fig. 10A). Digestion of purified wtVLPs with trypsin yielded several polypeptides, including an ~30-kDa fragment that appeared similar to that generated from purified VP1 as well as a 40-kDa fragment (Fig. 10A).

Finally, we subjected the ~30-kDa tryptic fragments of VP1 and wtVLPs to amino acid sequencing. This analysis revealed that trypsin cleaved the VP1 monomer between Arg6 and Lys7, whereas it cleaved wtVLPs between Arg10 and Lys11 of VP1 (Fig. 10B). Both of these cleavage sites are located within the NLS, indicating that the NLS of VP1 is exposed on the surface of wtVLPs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present experiments, inoculation of cells that are permissive (SVG) or nonpermissive (HeLa) for JCV infection with wtVLPs revealed that the VLPs entered the nucleus of both cell types. We had previously shown that wtVLPs are transported to the nucleus of various other cell types (19). These observations indicate that 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 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. These results suggested that the virion structure of wtVLPs was preserved during their transport to the nucleus.

The nuclear import of wtVLPs in digitonin-permeabilized cells supplemented with an ATP-regenerating system and a cytosolic fraction was completely inhibited by WGA or by antibodies to the NPC, indicating that wtVLPs of JCV entered the nucleus through the NPC, as do SV40 virions (12). Digitonin permeabilizes the plasma membrane but leaves the nuclear envelope intact. Cells permeabilized with digitonin thus retain import-competent nuclei but are largely depleted of cytosolic components. The transport of wtVLPs to the nucleus of digitonin-permeabilized HeLa or SVG cells in the presence of an ATP-regenerating system was restored not only by a cytosolic fraction but also by recombinant importins {alpha} and {beta}. We also demonstrated the ability of wtVLPs to bind to both importin {alpha} and {beta} with an overlay assay, suggesting that both importins are required for the nuclear import of wtVLPs, as has previously been shown to be the case for SV40 VP3 (30).

Proteins containing a classical NLS are transported into the nucleus after the formation of a complex with importins {alpha} and {beta}. The nuclear entry of {Delta}NLS-VLPs was greatly inhibited in both intact and permeabilized cells. Unlike wtVLPs, {Delta}NLS-VLPs did not bind to importins {alpha} or {beta}, suggesting that nuclear import of VLPs is mediated by interaction between importins and the NLS. The outer shell of the SV40 virion is thought to consist of 72 pentamers of VP1, with the NH2-terminal region of VP1 extending across the inside of the pentamer beneath the clockwise neighboring subunit (31). In the intact SV40 virion, the NLS of VP1 is bound to the viral minichromosome and is not exposed on the virion surface (31). Rather, interaction of the NLS of VP3 in the virion with importins mediates the nuclear entry of SV40 in infected cells.

The JCV virion also comprises 72 pentamers of VP1, but its crystal structure has not been determined. With the use of limited tryptic digestion, we have now shown that the NLS motif at the NH2 terminus of JCV VP1 appears to be exposed on the surface of VLPs consisting exclusively of this protein. We were not able to synthesize JCV VLPs that include VP2 and VP3 in addition to VP1. Given the important role of the NLS of SV40 VP3 in the nuclear translocation of SV40 (30), however, it is possible that the NLS of JCV VP3 might also contribute to the entry of JCV into the nucleus.

In summary, we have shown that the nuclear translocation of JCV VLPs is dependent on the interaction between the NLS motif of VP1 and cellular importins and occurs through the NPC. These findings may serve as a basis for the development of new therapeutic strategies to combat JCV infection.


   FOOTNOTES
 
* This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan and the Ministry of Health, Labor, and Welfare, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, N15, W7, Kita-ku, Sapporo 060-8638, Japan. Tel.: 81-11-706-5053; Fax: 81-11-706-7806; E-mail: h-sawa{at}patho2.med.hokudai.ac.jp.

1 The abbreviations used are: JCV, JC virus; SV40, simian virus 40; NPC, nuclear pore complex; NLS, nuclear localization signal; WGA, wheat germ agglutinin; VLP, virus-like particle; wt, wild-type; DMEM, Dulbecco's minimum essential medium; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; GST, glutathione S-transferase; TBS, Tris-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank M. Satoh and M. Sasada for technical assistance and valuable suggestions as well as Y. Abe (Center for Instrumental Analysis, Hokkaido University) for amino acid sequencing.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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