Quantcast
Last updated on April 18, 2014 at 9:23 EDT

Promyelocytic Leukemia Nuclear Bodies Provide a Scaffold for Human Polyomavirus JC Replication and Are Disrupted After Development of Viral Inclusions in Progressive Multifocal Leukoencephalopathy

April 25, 2008

By Shishido-Hara, Yukiko Higuchi, Kayoko; Ohara, Sinji; Duyckaerts, Charles; Hauw, Jean-Jacques; Uchihara, Toshiki

Abstract Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder due to human polyomavirus JC infection in which there are viral inclusions in enlarged nuclei of infected oligodendrocytes. We report that the pathogenesis of this disease is associated with distinct subnuclear structures known as promyelocytic leukemia nuclear bodies (PML-NBs). Postmortem brain tissues from 5 patients with the disease were examined. Affected cells with enlarged nuclei contained distinct dot-like subnuclear PML-NBs that were immunopositive for PML protein and nuclear body protein Sp100. Major and minor viral capsid proteins and proliferating cell nuclear antigen, an essential component for DNA replication, colocalized with PML-NBs. By in situ hybridization, viral genomic DNA showed dot-like nuclear accumulation, and by electron microscopy, virus-like structures clustered in subnuclear domains, indicating that PML-NBs are the site of viral DNA replication and capsid assembly. Molecules involved in the ubiquitin proteosome pathway (i.e. ubiquitin and small ubiquitin-like modifier 1) did not accumulate in the nuclei with viral inclusions, indicating that cell degeneration may not be dependent on this pathway. When viral progeny production was advanced, PML-NBs were disrupted. These data suggest that: 1) PML-NBs allow for efficient viral propagation by providing scaffolds, 2) disruption of PML-NBs is independent of the ubiquitin-proteasome pathway, and 3) this disruption probably heralds oligodendrocyte degeneration and the resulting demyelination.

Key Words: JC virus, Progressive multifocal leukoencephalopathy, Promyelocytic leukemia nuclear bodies, Sp100, SUMO-1, Ubiquitin.

INTRODUCTION

Progressive multifocal leukoencephalopathy is a fatal demyelinating disorder of the central nervous system due to human polyomavirus JC (JCV) infection. Oligodendrocytes in affected tissues display markedly enlarged nuclei in which JCV was identified in early electron microscopy studies as round and filamentous structures (1, 2). The virus has an icosahedral capsid composed of a major capsid protein, VP1, and minor capsid proteins, VP2/VP3; double-stranded circular DNA comprises its genome (3, 4). Based on electron microscopic analyses, when virions infect oligodendrocytes and first reach the nucleus, the nuclear volume increases. Then viral progeny appears in the vicinity of the inner nuclear membrane (5). Even after more than a quarter of a century, however, the molecular mechanisms that underlie the development of viral inclusions in oligodendrocytes remain unclear.

Promyelocytic leukemia nuclear bodies (PML-NBs; also known as PML oncogenic domains or nuclear domain 10) are ubiquitous subnuclear structures in the interphase nucleus (6-8). Promyelocytic leukemia nuclear bodies were first identified in acute promyelocytic leukemia in which PML-NBs are disrupted due to the synthesis of fusion proteins of PML/retinoic acid receptor-alpha in association with a t(15;17) chromosomal translocation (9, 10). Promyelocytic leukemia nuclear bodies in proliferating cells typically appear as 10 to 30 small dots with diameters of 0.2 to 1 [mu]m. The numbers and sizes of PML-NBs vary throughout the cell cycle; the smallest average number is seen in G^sub 0^; the numbers slowly increase during progression to G^sub 1^; the highest number is observed in the S phase (11). More than 50 proteins have been reported to localize either transiently or consistently to PML-NBs. Constitutive components of PML-NBs include PML protein and the nuclear body Sp100 protein. Promyelocytic leukemia protein is a tumor suppressor (12- 17), and Sp100 influences transcription and chromatin dynamics (18, 19).

Using COS-7 cells, we found that the JC virus capsid proteins accumulate and are efficiently assembled into virus-like structures in PML-NBs (20). Moreover, in addition to JCV, other DNA viruses cause PML-NB-associated degeneration or transformation of host cells (21, 22). For example, herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus all disrupt PML-NBs so that they can replicate efficiently in early stages of the viral replication cycle (23-25). By contrast, human papillomavirus type 33 reorganizes components of PML-NBs, using these bodies as a site for efficient progeny production during the late stage of replication (26, 27). Promyelocytic leukemia nuclear bodies are also related to multiple histologic types of solid cancers, including prostate, thyroid, colon, breast, lung, and brain (28-33); decreased expression of PML protein has been reported in most of these cancer cell types. In addition, PML-NBs are reorganized in neurodegenerative disorders such as polyglutamine diseases. In these diseases, the disease-related proteins such as ataxin or huntingtin accumulate at PML-NBs with modification of small ubiquitin-like modifier 1 (SUMO-1). This results in the formation of ubiquitin- positive neuronal intranuclear inclusions, and involvement of the ubiquitin-proteasome pathway has therefore been suggested as the mechanism of cell degeneration (34-42). Thus, PML-NBs are involved in the pathogenesis of a wide variety of human diseases, but the role of PML-NBs in progressive multifocal leukoencephalopathy, a disorder caused by infection of the highly oncogenic DNA virus JCV, is not known.

In this study, we investigated the ways that JCV interacts with PML-NBs in the human brain in cases of progressive multifocal leukoencephalopathy. We characterized the expression of PML-NB- related proteins and their association with JCV capsid proteins in different stages of lesion development. The findings indicate that JCV interactions with PML-NBs in oligodendrocytes with enlarged nuclei are associated with degeneration of these cells and demyelination.

MATERIALS AND METHODS

Case Material

Five autopsied cases (3 men, 2 women) of progressive multifocal leukoencephalopathy were studied. The mean age at death was 60 years (range, 35-85 years). Underlying diseases included AIDS (n = 2) and leukemia (n = 2); the remaining patient was an elderly woman without any associated underlying diseases.

Antibodies

Rabbit polyclonal antibody against the potential BC loop structure of VP1 (anti-VP1BC antibody) has been described previously (4). VP1-HI antibody was generously provided by Prof. Nagashima of Hokkaido University, Japan. Rabbit polyclonal antibody against VP2/ VP3 was prepared against the C-terminal sequence RKEGPRASSKTSYKR as previously described (20). Antibodies to the following proteins were purchased: PML protein (MBL, Nagoya, Japan); Sp100 protein (Chemicon, Temecula, CA); proliferating cell nuclear antigen (PCNA) (Novocastra, Newcastle, UK); SUMO-1 (Zymed, San Francisco, CA); and ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA).

Histology and Immunohistochemistry

Formalin-fixed paraffin-embedded sections of human brain tissue were stained using hematoxylin and eosin and Luxol fast blue. For immunostaining, 5-[mu]m-thick sections were deparaffinized in xylene then rehydrated in 90%, 70%, and 50% ethanol. For antigen retrieval, the sections were autoclaved in buffered citrate at 120[degrees]C for 10 minutes. Primary antibodies were incubated at 4[degrees]C for approximately 12 hours. The sections were then washed and incubated using biotinylated secondary antibodies, followed by avidin-biotin- peroxidase complex from a Vectastain avidin-biotin-peroxidase complex kit (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as the chromogen. After immunostaining, the sections were counterstained with hematoxylin.

Immunofluorescence

After deparaffinization, sections were rinsed in phosphate- buffered saline (PBS) containing 0.05% Tween-20 then blocked in PBS containing 5% normal goat serum at room temperature for 30 minutes. Sections were then incubated with primary antibodies for 1 hour, washed in PBS containing 0.05% Tween-20, then further incubated using the appropriate secondary antibodies conjugated with either Alexa Fluor 488-conjugated anti-rabbit immunoglobulin G (IgG) or Alexa Fluor 568-conjugated anti-rabbit IgG (Molecular Probes, Invitrogen, Carlsbad, CA). After 3 washes in PBS, samples were mounted in VectaShield (Vector Laboratories). Fluorescent images were captured using a TCS-SP confocal laser microscope (Leica, Heidelberg, Germany).

In Situ Hybridization

Paraffin-embedded tissue sections were deparaffinized and rehydrated as above. The sections were pretreated by autoclave heating in buffered citrate at 120[degrees]C for 10 minutes, followed by incubation in 0.01% pepsin in 0.2 N HCl for 20 minutes at 26[degrees]C. The sections were then dehydrated through an increasing ethanol series and air-dried for 2 days. The appropriate probe mix 1 [mu]g/ml was applied to the tissue section and then covered with a coverslip. The target DNA was denatured at 90[degrees]C for 5 minutes and incubated at 45[degrees]C for 60 minutes, 40[degrees]C for 60 minutes, and 35[degrees]C for more than 6 hours. Posthybridization washes were performed according to the procedures for human papillomavirus in use of GenPoint System (Dako, Carpinteria, CA). The sections were treated with 0.3% H^sub 2^O^sub 2^ in methanol at 26[degrees]C, and diaminobenzidine was used as the chromogen. The sections were then counterstained with hematoxylin. The single-strand DNA probe was targeted to the VP2 coding regions 5′-CTCCAGTAATTACAGCATATGT TTCAGGAGTAAGGCCTATTGCAGCTATAGCCTCAGAGGTACTTGTAAT-3′. Transfection of COS-7 Cells and Immunocytochemistry

COS-7 cells were incubated at 37[degrees]C under 5% CO2 in Dulbecco’s modified Eagle’s minimum essential medium supplemented with 10% fetal bovine serum. The cells were transfected with the AVP231-SRalpha expression vector as described previously (4, 20, 43, 44) and then harvested 3 days after transfection. For immunofluorescence, transfected cells were fixed in 4% paraformaldehyde in PBS for 15 minutes at room temperature then permeabilized with 0.5% Triton X-100 in PBS for 20 minutes. Cells were subsequently rinsed in PBS containing 0.05% Tween-20 then blocked with PBS containing 5% normal goat serum at room temperature for 30 minutes. Lastly, the cells were incubated with antibodies using the same methods as for formalin-fixed paraffin-embedded tissues.

Western Blotting

COS-7 cells were transfected with AVP231-SRalpha or VP231- SRalpha as described previously (4, 20, 43, 44) then harvested 3 days after transfection. The cells were homogenized in sodium dodecyl sulfate sample buffer and reduced by boiling at 95[degrees]C for 5 minutes. The protein was separated by electrophoresis on 12.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane in 50 mmol/L Tris buffer containing 145 mmol/ L glycine. The membrane was blocked for 60 minutes at room temperature with 5% skim milk and then reacted with the anti-VP2/ VP3 primary antibody. Peroxidase-labeled anti-rabbit IgG was used as the secondary antibody. The Western Lightning Chemiluminescence Reagent Plus Kit (PerkinElmer LAS, Inc., Boston, MA) was used for detection.

Electron Microscopy

For conventional electron microscopy, brain tissues were fixed using 2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.3) and postfixed in 1.0% osmium tetroxide. After dehydration through a graded ethanol series, 50%, 70%, 80%, 90%, 95%, and 100% 3 times each, the tissues were embedded in epoxy resin. Ultrathin sections were prepared, stained using uranyl acetate and lead citrate, and examined using an H-7100 electron microscope (Hitachi, Tokyo, Japan). For immunoelectron microscopy, transfected COS-7 cells were fixed at 72 hours posttransfection in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 15 minutes. After dehydration through a graded ethanol series as previously described, the cells were embedded in LR White resin (Structure Probe, West Chester, PA). Ultrathin sections were prepared and mounted on nickel grids. After incubation with 10% normal goat serum for 10 minutes, sections were incubated overnight at 4[degrees]C with anti-VP1HI antibody. After washing with PBS, sections were incubated with goat anti-rabbit IgG conjugated to 15- nm gold particles (1:30; British BioCell International, Cardiff, UK) for 30 minutes at room temperature. Sections were then stained with uranyl acetate and examined by electron microscopy.

RESULTS

Dot-Like Subnuclear Structures (PML-NBs) Appear in Cells With Enlarging Nuclei

There were multiple demyelinated lesions in brains of patients with progressive multifocal leukoencephalopathy predominantly located in the cerebral hemispheric white matter (Fig. 1A). Numerous abnormal cells were apparent, most frequently at the periphery of demyelinated lesions (Fig. 1B). At the corticomedullary borders (i.e. areas with relatively intact myelin), the myelin sheaths were stained with Luxol fast blue (*1 in Fig. 1A, B). Cells with small but slightly enlarged nuclei were scattered in these areas (Fig. 1C). These cells usually displayed bright nuclei with marginated chromatin, which included small dots or fine granular structures stained with hematoxylin (Fig. 1D). The appearance of these cells was distinct from that of normal oligodendrocytes with condensed chromatin. In partially demyelinated areas, only degenerated myelin debris stained with Luxol fast blue, and myelin fiber sheaths were no longer seen (*2 in Fig. 1A, B). Numerous cells in these areas had nuclei that were approximately 3- or 4-fold larger than those of normal oligodendrocytes (Fig. 1E). Abnormal cells exhibited 2 distinct intranuclear staining patterns. Some showed amphophilic staining throughout the entire nucleus, whereas other cells showed bright nucleoplasm with marginated chromatin in which frequent dot- shaped structures that were well stained with hematoxylin were present at the nuclear periphery (arrows in Fig. 1F). In the completely demyelinated lesions that lacked staining with Luxol fast blue (*3 in Fig. 1A, B), the density of cells with enlarged nuclei was decreased, and foamy macrophages appeared.

Accumulation of JCV Capsid Proteins in PML-NBs

Immunolocalization of JCV-related epitopes was examined using antibodies against viral capsid proteins VP2/VP3 (Fig. 2). Because expression of VP2/VP3 has previously been reported only in transfected cell lines, this is the first demonstration of VP2/VP3 expression in human progressive multifocal leukoencephalopathy. At the corticomedullary borders (i.e. areas with relatively intact myelin), many small, slightly enlarged oligodendrocyte-like nuclei lacked immunoreactivity for viral capsid proteins (Fig. 2A[1], B[1]). As in hematoxylin and eosin stains, cells with enlarged nuclei in neighboring regions with mild demyelination displayed 2 distinct staining patterns. Some showed accumulation of capsid proteins in the dot-like structures along the inner periphery of the bright nucleoplasm (Fig. 2A[2], B[2]), and others displayed intense and diffuse staining for capsid proteins throughout the entire nucleus; in these cells, very little dot-like staining was seen (Fig. 2A[3], B[3]).

The specificity of the VP2/VP3 antibody was confirmed by Western blotting using cultured cells expressing JCV capsid proteins. VP3 of approximately 26 kD was clearly detected, but the least capsid protein, VP2 of approximately 37 kD, was less than the detection threshold (Fig. 2C).

The dot-like subnuclear structures were identifiable as PML-NBs because they expressed both PML protein (Fig. 2D[1]) and Sp100 protein (Fig. 2D[2]). Frequently, the enlarged nuclei contained approximately 2 to 5 PML-NBs that were up to 2.0 [mu]m in diameter. In contrast, normal oligodendrocyte-like nuclei rarely showed immunoreactivity for either PML or Sp100 proteins.

PML-NBs Can Be Sites of Viral Progeny Production

When double staining was performed for JCV major capsid protein VP1 and PML protein, some cells showed colocalization of VP1 and PML protein in dot-like subnuclear structures, indicating VP1 localization in the PML-NBs (Fig. 3A). Proliferating cell nuclear antigen, an essential component of DNA replication during S phase, also showed dot-like staining in some enlarged nuclei and was colocalized with PML protein (Fig. 3B). Both VP1-positive and PCNA- positive PML-NBs were frequently seen along the inner nuclear periphery.

FIGURE 1. Dot-shaped subnuclear structures in enlarged nuclei of oligodendrocytes in progressive multifocal leukoencephalopathy. (A, B) Demyelinating lesions in cerebral white matter of patients with progressive multifocal leukoencephalopathy. Corticomedullary borders are areas in which predominantly intact myelin sheaths are stained with Luxol fast blue (*1). Partially demyelinated lesions in which myelin debris is stained with Luxol fast blue, but intact fiber bundles and tracts are no longer apparent (*2). Lesions with distinct demyelination in which staining with Luxol fast blue is not evident (*3). (C, D) In area *1, there are scattered cells with small but slightly enlarged nuclei. (D) is a higher magnification of (C). (E, F) In area *2, the nuclei of affected cells are approximately 3- or 4-fold larger than those of normal oligodendrocytes. Some cells exhibit dot-like structures at the inner periphery of the nucleus (arrows), whereas other cells have amphophilic inclusions throughout the nucleoplasm. (F) is a higher magnification of (E). Luxol fast blue; scale bar = (A) 650 [mu]m. Hematoxylin and eosin; scale bar = (B) 650 [mu]m. Hematoxylin and eosin; scale bar = (C, E) 100 [mu]m. Hematoxylin and eosin; scale bar = (D, F) 20 [mu]m.

The presence and localization of intranuclear viral DNA were also examined by in situ hybridization using a DNA probe complementary to the viral capsid proteins. The JCV DNA was detected as nuclear dot- like signals in enlarged nuclei of infected cells, indicating that replication of viral DNA molecules occurs at distinct subnuclear domains. Although JCV DNA signals were hardly seen at the corticomedullary borders (intact myelin), in areas with mild demyelination, large dot-like DNA signals were roughly or densely aligned in the nuclei (Fig. 3C).

By electron microscopy, JCV virions were seen as round or filamentous structures in enlarged nuclei. Although intranuclear amounts and distributions of virions varied in individual cells, many cells contained virions at the periphery, rather than in the center of the nucleus (Fig. 3D[1]). We previously established an expression system to produce virus-like structures in COS-7 cells (4, 44), and in these cells, colloidal gold particles representing the VP1 epitope were similarly clustered along the inner nuclear periphery and clusters of numerous virus-like particles with both round and filamentous structures were evident (Fig. 3D[2]). Therefore, the intranuclear distribution was quite similar to that of the viruses observed in human brain tissue.

FIGURE 2. Accumulation of human polyomavirus JC (JCV) capsid proteins at promyelocytic leukemia nuclear bodies (PML-NBs). (A, B) Nuclear features of cells and intranuclear localization of JCV VP2/ VP3. (A) Hematoxylin and eosin; (B) VP2/VP3 immunostaining. Cells in corticomedullary borders (largely intact myelin) exhibit small but slightly enlarged nuclei but lack immunoreactivity for VP2/VP3 (1). In demyelinated white matter, some cells show dot-like structures at the inner periphery of enlarged nuclei that are immunopositive for VP2/VP3 (2). Cells with amphophilic inclusions throughout the nucleoplasm correspond to those with a diffuse distribution of VP2/ VP3 immunoreactivity in the entire nucleus (3). (C) Western blot using the VP2/VP3 antibody for detecting JCV capsid proteins. Lane 1, untransfected COS-7 cells; lane 2, COS-7 cells transfected with AVP231-SRalpha; lane 3, COS-7 cells transfected with VP231-SRalpha. (D) Identification of PML-NBs. Promyelocytic leukemia protein staining (1); Sp100 protein staining (2). These observations support the idea that the PML-NBs are the sites of JCV progeny production, and that in addition to capsid assembly, viral DNA replication can occur in the PML-NBs. Promyelocytic leukemia nuclear bodies thus seem to provide a scaffold for efficient viral replication, and the cells in which the virus particles are propagated seem to be in an S- phase-like state with expression of PCNA.

Distribution of the PML-NB-Related Proteins SUMO-1 and Ubiquitin

Promyelocytic leukemia nuclear bodies are known to be sites of protein degradation. In polyglutamine diseases and in other DNA virus infections, proteins modified with SUMO-1 (SUMOylation) are degraded through a ubiquitin-proteasome pathway. To investigate cell degeneration in JCV-infected cells, we next examined expression of SUMO-1 and ubiquitin. At corticomedullary borders, immunoreactivity for SUMO-1 was apparent in the nuclei of cortical neurons but not in oligodendrocyte-like nuclei (Fig. 4A[1]). In the white matter with mild demyelination, some cells showed faint dot-like staining in enlarged nuclei, but these were relatively sparse (Fig. 4A[2]). Enlarged nuclei with full basophilic inclusions were minimally immunoreactive for SUMO-1 (Fig. 4A[3]).

Ubiquitin distribution also varied in different cells. At the corticomedullary borders, slightly enlarged oligodendrocyte-like nuclei showed dot-shaped immunostaining for ubiquitin. Most neurons exhibited diffuse cytoplasmic staining and/or nuclear dot-like staining (Fig. 4B[1]). In mildly demyelinated white matter, only small oligodendrocyte-like nuclei were immunoreactive for ubiquitin, but there was none in markedly enlarged nuclei (Fig. 4B[2]). Interestingly, ubiquitin immunoreactivity was present in the cytoplasm in cells containing viral inclusions throughout the nucleus (Fig. 4B[3]).

FIGURE 3. Promyelocytic leukemia nuclear bodies (PML-NBs) can be the site of human polyomavirus JC (JCV) progeny production. (A) Double staining for JCV major capsid protein VP1 (green) and PML protein (red). Colocalization of VP1 and PML protein to PML-NBs in the periphery of enlarged nuclei. (B) Double staining for proliferating cell nuclear antigen (PCNA; green) and PML (red). In dot-like PML-NBs, PCNA also colocalized with PML protein. (C) In situ hybridization of JCV genomic DNA. At the corticomedullary borders (relatively intact myelin), JCV DNA is barely detected in cells with small, but slightly enlarged, nuclei (1). In white matter with partial demyelination, replicated DNA is detected as granular or roughly aligned dot-like signals (2). In some cells, dot-like signals of JCV DNA were densely aligned, indicating full inclusions (3). (D) Electron micrograph of JCV-infected cells in the human brain. Human polyomavirus JC virions of both round and filamentous structures are clustered along the inner nuclear periphery (1). Electron micrograph of COS-7 cells transfected with the vector encoding the viral genome (2). Colloidal gold particles representing the VP1 viral capsid protein are clustered along the inner nuclear periphery, where clusters of virus-like particles are also apparent. Asterisks (*) indicate clusters of viruses or virus-like particles located on the inner nuclear periphery. Scale bar = (D[1] and D[2]) 1 [mu]m. Cyt, cytoplasm; nm, nuclear membrane; Nuc, nucleoplasm.

FIGURE 4. Distribution of small ubiquitin-like modifier 1 (SUMO- 1) and ubiquitin. (A) Immunoreactivity of SUMO-1. In cells in corticomedullary borders, immunoreactivity for SUMO-1 is seen in neurons but not in oligodendrocyte-like nuclei (1). In white matter with partial demyelination, a few cells with enlarged nuclei show distinct dot-like SUMO-1 staining (2). In cells with full inclusions, SUMO-1 immunoreactivity is not seen (3). (B) Immunoreactivity for ubiquitin. At the corticomedullary borders, immunoreactivity for ubiquitin is seen in both cortical neurons and slightly enlarged oligodendrocyte-like nuclei (1). In white matter with partial demyelination, ubiquitin is immunoreactive in small nuclei but not in markedly enlarged nuclei of infected cells (2). Ubiquitin immunoreactivity in cytoplasm of cells with full intranuclear viral inclusions (3).

These observations indicate that the localizations of SUMO-1 and ubiquitin are variable. Accumulation of SUMO-1 or ubiquitin in nuclei was not observed in cells with fully developed viral inclusions, suggesting that the degradation of JCV-infected oligodendrocytes is not tightly linked to the ubiquitin-proteasome pathway.

PML-NB Structures Are Disrupted in Fully Demyelinated Lesions

Finally, the morphologic features of PML-NBs were examined in association with degrees of JCV-induced demyelination. Brain tissues were double-stained for JCV VP1 and PML protein to assess cell degeneration after production of progeny virions. Promyelocytic leukemia protein immunoreactivity was seen over a relatively broad area. Promyelocytic leukemia protein and VP1 were colocalized to PML- NBs in both partially demyelinated areas (Fig. 5A) and in areas with more marked demyelination (Fig. 5B). In the center of large demyelinated lesions, infected cells were severely degraded. In these, VP1 and PML protein barely showed dot-like staining and instead exhibited more marginated staining in degenerated nuclei (Fig. 5C). In severely degenerated lesions, VP1 and PML stained rather nonspecifically in cells that seemed to be close to death (Fig. 5D). These observations indicate that PML-NBs are progressively disrupted with viral infection in a process associated with the oligodendrocyte degeneration and consequent demyelination.

DISCUSSION

This is the first comprehensive study to demonstrate targeted localization of JCV capsid proteins to PML-NBs in oligodendrocyte- like cells within and around demyelinated foci in human progressive multifocal leukoencephalopathy. Because viral DNA accumulate in enlarged nuclei of these cells, it is likely that PML-NBs provide a structural support for JCV replication in these cells. Disruption of PML-NBs occurs when there is oligodendrocyte degeneration and subsequent demyelination. These observations indicate that PML-NBs in progressive multifocal leukoencephalopathy function in a manner partly similar to other viral infections and, to some extent, cancer, but that their function is somehow distinct from polyglutamine diseases.

FIGURE 5. Promyelocytic leukemia nuclear bodies (PML-NBs) are disrupted after the development of viral inclusions. Human polyomavirus JC-infected human brain tissues were double-stained for VP1 (green) and PML protein (red). Promyelocytic leukemia protein displayed dot-like staining with VP1 in the periphery of large demyelinated lesions. Both VP1 and PML are diffusely dispersed in the center and show nonspecific staining in necrotic areas. Diffuse distribution of PML protein may represent disruption of PML-NB structures. (A) Periphery of lesions with mild demyelination. (B) Intermediate zone. (C) Center of lesions with distinct demyelination. (D) Necrotic area.

Human polyomavirus JC is unique because both major capsid protein VP1 and minor capsid proteins VP2/VP3 cooperatively target PML-NBs for efficient production of progeny virions (20). Expression of PCNA in the inner nuclear periphery suggests that these intranuclear viral inclusions are formed in an S-phase-like state of the cell cycle. Some cells exhibit dot-like accumulations of PCNA at the inner nuclear periphery (Fig. 3). This is similar to dividing cells in S phase in which dot-like accumulations of PCNA align along the nuclear periphery (45) and are associated with PML-NBs (46). Moreover, PML-NBs have been reported to be sites of DNA replication in several DNA viruses (47-51). Thus, viral replication is tightly associated with the host cell machinery, and viral DNA replication and capsid assembly seem to occur simultaneously at PML-NBs. In addition to PCNA, JCV-infected oligodendrocytes show very high expression of MIB-1 and are also positive for cyclins A and B1, which are expressed during S-G2-M phases (52-55). Therefore, the cell cycle of the infected oligodendrocytes likely is highly activated until formation of viral inclusions and provides a cellular environment that promotes viral infection. It remains unclear, however, why activated oligodendrocytes are degraded, rather than undergo mitotic division.

The mechanisms of oligodendrocyte degeneration after the production of viral progeny are not clear. Promyelocytic leukemia nuclear bodies are also known to be the site of regulated protein degradation through a ubiquitin-proteasome pathway. Herpes simplex virus and human cytomegalovirus abrogate SUMOylated PML through a proteasome-dependent pathway, resulting in the disruption of PML- NBs (56-58). Similar mechanisms may explain neurodegeneration in polyglutamine diseases with ubiquitin-positive neuronal intranuclear inclusions (34, 37). In JCV infection, however, SUMO-1 and ubiquitin were not found to accumulate in the nucleus with full inclusions (Fig. 4). Although PML-NBs of JCV-infected oligodendrocytes were disrupted along with development of viral inclusions and resulting demyelination (Fig. 5), the mechanisms of PML-NB disruption seem to be unrelated to the ubiquitin-proteasome pathway. We currently speculate that JCV-infected cells would burst in an M-phase-like state. In normal-dividing cells, PML-NBs disappear during M phase. Many JCV-infected cells positive for cyclin A showed degraded nuclear membrane with scant and unclear cytoplasm, as if they were bursting, rather than undergoing mitosis (data not shown). In conclusion, PML-NBs may play important roles in progressive multifocal leukoencephalopathy. Promyelocytic leukemia nuclear bodies likely provide a scaffold for the replication of viral progenies, providing a cancer-like cellular environment, and these are finally disrupted after the development of viral inclusions. It remains unclear as to what determines whether a cell is to undergo degeneration or transformation. Human polyomavirus JC is highly oncogenic and is known to induce brain tumors in various animal models (59-61); the presence of the JCV genome has also been reported in different types of human cancers (62-65). It is hoped that future investigations will clarify the dynamic pathogenic roles of PML-NBs, leading to either cell degeneration or transformation, in association with JCV.

ACKNOWLEDGMENTS

The authors thank Michiru Umino, Akiko Kitazawa, Ayumi Sumiishi, Kaoruko Kojima, and Kazuko Nakayama of the Department of Pathology at Kyorin University School of Medicine for technical assistance.

REFERENCES

1. Silverman L, Rubinstein LJ. Electron microscopic observations on a case of progressive multifocal leukoencephalopathy. Acta Neuropathol (Berl) 1965;5:215-24

2. Zu Rhein GM, Chou S-M. Particles resembling Papova viruses in human cerebral demyelinating disease. Science 1965;11:1477-79

3. Frisque RJ, Bream GL, Cannella MT. Human polyomavirus JC virus genome. J Virol 1984;51:458-69

4. Shishido-Hara Y, Hara Y, Larson T, et al. Analysis of capsid formation of human polyomavirus JC (Tokyo-1 strain) by a eukaryotic expression system: Splicing of late RNAs, translation and nuclear transport of major capsid protein VP1, and capsid assembly. J Virol 2000;74:1840-53

5. Mazlo M, Tariska I. Morphological demonstration of the first phase of polyomavirus replication in oligodendroglia cells of human brain in progressive multifocal leukoencephalopathy (PML). Acta Neuropathol 1980;49:133-43

6. Maul GG, Negorev D, Bell P, et al. Review: Properties and assembly mechanisms of ND10, PML bodies, or PODs. J Struct Biol 2000;129:278-87

7. Borden KL. Pondering the promyelocytic leukemia protein (PML) puzzle: Possible functions for PML nuclear bodies. Mol Cell Biol 2002;22:5259-69

8. Hodges M, Tissot C, Howe K, et al. Structure, organization, and dynamics of promyelocytic leukemia protein nuclear bodies. Am J Hum Genet 1998;63:297-304

9. Melnick A, Licht JD. Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia. Blood 1999;93:3167-3215

10. Dyck JA, Maul GG, Miller WH Jr, et al. A novel macromolecular structure is a target of the promyelocyte-retinoic acid receptor oncoprotein. Cell 1994;76:333-43

11. Terris B, Baldin V, Dubois S, et al. PML nuclear bodies are general targets for inflammation and cell proliferation. Cancer Res 1995;55:1590-97

12. Kakizuka A, Miller WH Jr, Umesono K, et al. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell 1991;66:663-74

13. Goddard AD, Borrow J, Freemont PS, et al. Characterization of a zinc finger gene disrupted by the t(15;17) in acute promyelocytic leukemia. Science 1991;254:1371-74

14. de The H, Lavau C, Marchio A, et al. The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell 1991;66:675-84

15. Salomoni P, Pandolfi PP. The role of PML in tumor suppression. Cell 2002;108:165-70

16. Guo A, Salomoni P, Luo J, et al. The function of PML in p53- dependent apoptosis. Nat Cell Biol 2000;2:730-36

17. Zhong S, Salomoni P, Pandolfi PP. The transcriptional role of PML and the nuclear body. Nat Cell Biol 2000;2:E85-90

18. Seeler JS, Marchio A, Sitterlin D, et al. Interaction of SP100 with HP1 proteins: A link between the promyelocytic leukemia- associated nuclear bodies and the chromatin compartment. Proc Natl Acad Sci U S A 1998;95:7316-21

19. Seeler JS, Marchio A, Losson R, et al. Common properties of nuclear body protein SP100 and TIF1alpha chromatin factor: Role of SUMO modification. Mol Cell Biol 2001;21:3314-24

20. Shishido-Hara Y, Ichinose S, Higuchi K, et al. Major and minor capsid proteins of human polyomavirus JC cooperatively accumulate to nuclear domain 10 for assembly into virions. J Virol 2004;78:9890-9903

21. Everett RD. DNA viruses and viral proteins that interact with PML nuclear bodies. Oncogene 2001;20:7266-73

22. Moller A, Schmitz ML. Viruses as hijackers of PML nuclear bodies. Arch Immunol Ther Exp (Warsz) 2003;51:295-300

23. Adamson AL, Kenney S. Epstein-Barr virus immediate-early protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia bodies. J Virol 2001;75:2388-99

24. Ahn JH, Hayward GS. The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML- associated nuclear bodies at very early times in infected permissive cells. J Virol 1997;71:4599-613

25. Everett RD, Maul GG. HSV-1 IE protein Vmw110 causes redistribution of PML. EMBO J 1994;13:5062-69

26. Florin L, Schafer F, Sotlar K, et al. Reorganization of nuclear domain 10 induced by papillomavirus capsid protein L2. Virology 2002;295:97-107

27. Florin L, Sapp C, Streeck RE, et al. Assembly and translocation of papillomavirus capsid proteins. J Virol 2002;76:10009-14

28. Gurrieri C, Capodieci P, Bernardi R, et al. Loss of the tumor suppressor PML in human cancers of multiple histologic origins. J Natl Cancer Inst 2004;96:269-79

29. Gambacorta M, Flenghi L, Fagioli M, et al. Heterogeneous nuclear expression of the promyelocytic leukemia (PML) protein in normal and neoplastic human tissues. Am J Pathol 1996;149:2023-35

30. Szendefi M, Walt H, Krasieva TB, et al. Association between promyelocyte protein and small ubiquitin-like modifier protein and the progression of cervical neoplasia. Obstet Gynecol 2003;102:1269- 77

31. Zhang H, Melamed J, Wei P, et al. Concordant down-regulation of proto-oncogene PML and major histocompatibility antigen HLA class I expression in high-grade prostate cancer. Cancer Immun 2003;3:14

32. Tian XX, Chan JY, Pang JC, et al. Altered expression of the suppressors PML and p53 in glioblastoma cells with the antisense- EGF-receptor. Br J Cancer 1999;81:994-1001

33. Yu E, Lee KW, Lee HJ. Expression of promyelocytic leukaemia protein in thyroid neoplasms. Histopathology 2000;37:302-8

34. Skinner PJ, Koshy BT, Cummings CJ, et al. Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures. Nature 1997;389:971-74

35. Dovey CL, Varadaraj A, Wyllie AH, et al. Stress responses of PML nuclear domains are ablated by ataxin-1 and other nucleoprotein inclusions. J Pathol 2004;203:877-83

36. Kumada S, Uchihara T, Hayashi M, et al. Promyelocytic leukemia protein is redistributed during the formation of intranuclear inclusions independent of polyglutamine expansion: An immunohistochemical study on Marinesco bodies. J Neuropathol Exp Neurol 2002;61:984-91

37. Yamada M, Sato T, Shimohata T, et al. Interaction between neuronal intranuclear inclusions and promyelocytic leukemia protein nuclear and coiled bodies in CAG repeat diseases. Am J Pathol 2001;159:1785-95

38. Chai Y, Koppenhafer SL, Shoesmith SJ, et al. Evidence for proteasome involvement in polyglutamine disease: Localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro. Hum Mol Genet 1999;8:673-82

39. Takahashi J, Fukuda T, Tanaka J, et al. Neuronal intranuclear hyaline inclusion disease with polyglutamine-immunoreactive inclusions. Acta Neuropathol (Berl) 2000;99:589-94

40. Takahashi J, Tanaka J, Arai K, et al. Recruitment of nonexpanded polyglutamine proteins to intranuclear aggregates in neuronal intranuclear hyaline inclusion disease. J Neuropathol Exp Neurol 2001;60:369-76

41. Takahashi J, Fujigasaki H, Zander C, et al. Two populations of neuronal intranuclear inclusions in SCA7 differ in size and promyelocytic leukaemia protein content. Brain 2002;125:1534-43

42. Takahashi J, Fujigasaki H, Iwabuchi K, et al. PML nuclear bodies and neuronal intranuclear inclusion in polyglutamine diseases. Neurobiol Dis 2003;13:230-37

43. Shishido-Hara Y, Nagashima K. Synthesis and assembly of polyomavirus virions. In: Khalili K, Stoner GL, eds. Human Polyomaviruses: Molecular and Clinical Perspectives. New York: John Wiley & Sons, Inc., 2001:149-77

44. Shishido Y, Nukuzuma S, Mukaigawa J, et al. Assembly of JC virus-like particles in COS7 cells. J Med Virol 1997;51:265-72

45. Leonhardt H, Rahn HP, Weinzierl P, et al. Dynamics of DNA replication factories in living cells. J Cell Biol 2000;149:271-80

46. Grande MA, van der Kraan I, van Steensel B, et al. PML- containing nuclear bodies: Their spatial distribution in relation to other nuclear components. J Cell Biochem 1996;63:280-91

47. Swindle CS, Zou N, Van Tine BA, et al. Human papillomavirus DNA replication compartments in a transient DNA replication system. J Virol 1999;73:1001-9

48. Day PM, Roden RB, Lowy DR, et al. The papillomavirus minor capsid protein, L2, induces localization of the major capsid protein, L1, and the viral transcription/replication protein, E2, to PML oncogenic domains. J Virol 1998;72:142-50

49. Roberts S, Hillman ML, Knight GL, et al. The ND10 component promyelocytic leukemia protein relocates to human papillomavirus type 1 E4 intranuclear inclusion bodies in cultured keratinocytes and in warts. J Virol 2003;77:673-84

50. Tang Q, Bell P, Tegtmeyer P, et al. Replication but not transcription of simian virus 40 DNA is dependent on nuclear domain 10. J Virol 2000;74:9694-9700 51. Jul-Larsen A, Visted T, Karlsen BO, et al. PML-nuclear bodies accumulate DNA in response to polyomavirus BK and simian virus 40 replication. Exp Cell Res 2004;298:58-73

52. Ariza A, Mate JL, Fernandez-Vasalo A, et al. p53 and proliferating cell nuclear antigen expression in JC virus-infected cells of progressive multifocal leukoencephalopathy. Hum Pathol 1994;25:1341-45

53. Ariza A, Mate JL, Isamat M, et al. Overexpression of Ki-67 and cyclins A and B1 in JC virus-infected cells of progressive multifocal leukoencephalopathy. J Neuropathol Exp Neurol 1998;57:226- 30

54. Ariza A, von Uexkull-Guldeband C, Mate JL, et al. Accumulation of wild-type p53 protein in progressive multifocal leukoencephalopathy: A flow of cytometry and DNA sequencing study. J Neuropathol Exp Neurol 1996;55:144-49

55. Lammie GA, Beckett A, Courtney R, et al. An immunohistochemical study of p53 and proliferating cell nuclear antigen expression in progressive multifocal leukoencephalopathy. Acta Neuropathol (Berl) 1994;88:465-71

56. Muller S, Dejean A. Viral immediate-early proteins abrogate the modification by SUMO-1 of PML and Sp100 proteins, correlating with nuclear body disruption. J Virol 1999;73:5137-43

57. Everett RD, Freemont P, Saitoh H, et al. The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms. J Virol 1998;72:6581-91

58. Chelbi-Alix MK, de The H. Herpes virus induced proteasome- dependent degradation of the nuclear bodies-associated PML and Sp100 proteins. Oncogene 1999;18:935-41

59. Nagashima K, Yasui K, Kimura J, et al. Induction of brain tumors by a newly isolated JC virus (Tokyo-1 strain). Am J Pathol 1984;116:455-63

60. Walker DL, Padgett BL, ZuRhein GM, et al. Human papovavirus (JC): Induction of brain tumors in hamsters. Science 1973;181:674- 76

61. London WT, Houff SA, Madden DL, et al. Brain tumors in owl monkeys inoculated with a human polyomavirus (JC virus). Science 1978;201:1246-49

62. Del Valle L, Gordon J, Enam S, et al. Expression of human neurotropic polyomavirus JCV late gene product agnoprotein in human medulloblastoma. J Natl Cancer Inst 2002;94:267-73

63. Rencic A, Gordon J, Otte J, et al. Detection of JC virus DNA sequence and expression of the viral oncoprotein, tumor antigen, in brain of immunocompetent patient with oligoastrocytoma. Proc Natl Acad Sci U S A 1996;93:7352-57

64. Del Valle L, Gordon J, Assimakopoulou M, et al. Detection of JC virus DNA sequences and expression of the viral regulatory protein T-antigen in tumors of the central nervous system. Cancer Res 2001;61:4287-93

65. Krynska B, Del Valle L, Croul S, et al. Detection of human neurotropic JC virus DNA sequence and expression of the viral oncogenic protein in pediatric medulloblastomas. Proc Natl Acad Sci U S A 1999;96:11519-24

Yukiko Shishido-Hara, MD, PhD, Kayoko Higuchi, MD, PhD, Sinji Ohara, MD, PhD, Charles Duyckaerts, MD, Jean-Jacques Hauw, MD, and Toshiki Uchihara, MD, PhD

From Department of Neurology (YS-H, TU), Tokyo Metropolitan Institute for Neuroscience; Department of Pathology (YS-H), Kyorin University School of Medicine, Tokyo; Section of Pathology (KH), Aizawa Hospital, Nagano; Department of Neurology (SO), National Chushin-Matsumoto Hospital, Matsumoto Japan; and Laboratoire Raymond Escourole (CD, J-JH), Service de Neuropathologie, Association Claude Bernard, Groupe hospitalier Pitie-Salpetriere, Paris, France.

Send correspondence and reprint requests to: Yukiko Shishido- Hara, MD, PhD, Department of Pathology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo 181-8611, Japan. E-mail: shishido-hara@y6.dion.ne.jp

This study was supported by the Prion Disease and Slow Virus Infection Research Committee of the Ministry of Health, Labour, and Welfare of Japan.

Copyright Lippincott Williams & Wilkins Apr 2008

(c) 2008 Journal of Neuropathology and Experimental Neurology. Provided by ProQuest Information and Learning. All rights Reserved.