JC Virus Induces Nonapoptotic Cell Death of Human Central Nervous System Progenitor Cell-Derived Astrocytes

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JOURNAL OF VIROLOGY, May 2004, p. 4884–4891 0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.9.4884–4891.2004

Vol. 78, No. 9

JC Virus Induces Nonapoptotic Cell Death of Human Central Nervous System Progenitor Cell-Derived Astrocytes Pankaj Seth,1 Frank Diaz,1 Jung-Hwa Tao-Cheng,2 and Eugene O. Major1* Laboratory of Molecular Medicine and Neuroscience1 and Electron Microscopy Facility,2 National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892 Received 3 September 2003/Accepted 23 December 2003

ciency virus (HIV)-associated dementia (13), and complications arising from neurotropic virus infections, including glial activation in influenza encephalopathy (22) and herpes simplex virus-associated acute focal encephalitis (24). Cell death, however, is not restricted only to the apoptotic pathway; it may occur through necrosis as well (12, 17, 38). Necrosis is typically a pathological process resulting from acute infections or injuries, while apoptosis is more indicative of a chronic disease. The hallmarks of necrosis include mitochondrial swelling, dissolution of organelles, extensive vacuolization, watery cytoplasm, and condensation of chromatin toward the periphery of the nucleus. Apoptosis, on the other hand, involves cytoplasmic shrinkage and the condensation and fragmentation of nuclear chromatin, along with significant membrane alterations (25). Although it may seem detrimental to the host to destroy its cells, such elimination of infected cells actually spares surrounding cells from the spread of the virus, effectively containing the viral infection. However several emerging reports suggest that viruses are evolving to produce specific agents or to trigger signals that enable them to evade this defense mechanism (2, 6, 8, 10, 32), rendering the task of complete viral eradication nearly impossible. To further improve our understanding of the nature of JCV infection in cell types supporting productive viral infection, we used a human central nervous system (CNS) progenitorderived astrocyte cell culture model (18). The human CNS progenitor cells were isolated from a fetal brain at an early gestational age and characterized by specific human nestin antibody staining. These CNS progenitor cells possess multipotential properties, as they can be differentiated into either astrocyte or neuronal pathways based on the growth factors and other supplements provided in culture media (18). The present study utilized a purified population of astrocytes derived from these progenitor cells. The focus of our study was to investigate the mechanism of

The human polyomavirus, JC virus (JCV), is a doublestranded DNA virus with a worldwide distribution of more than 80% seropositivity in the normal human population (15). Ultrastructural and X-ray diffraction studies have revealed that JCV particles are 40 to 45 nm in diameter and possess icosahedral symmetry similar to that of other, related polyomaviruses. The supercoiled circular DNA codes for the two nonstructural proteins, large T and small t, as well as early proteins T⬘135, T⬘136, and T⬘165 (34). Also encoded are three structural capsid proteins—VP-1, VP-2, and VP-3—and another regulatory protein, agnoprotein (28). Although JCV was initially described as having a restricted neurotropism, recent reports have expanded the host range to include a variety of human cell types (15, 21, 28). Viral infection can lead to host cell death via necrotic or apoptotic pathways. In apoptosis, the infected cells undergo distinctive alterations at the morphological and biochemical levels (2, 25). Extracellular and/or intracellular stimuli culminate in sequential activation of cysteine-aspartyl proteases, collectively called caspases, that result in DNA fragmentation and other structural hallmarks of apoptosis (27, 33, 35). Apoptotic pathways also involve members of the bcl-2 family genes, which maintain a balance between pro- and antiapoptotic signals. The bcl-2 family of genes is also crucial for the release of cytochrome c from the mitochondria, thereby triggering the caspase cascade through a series of steps (36). Apoptosis is a feature of both acute and chronic neurological diseases (7, 16, 17, 40). Specifically, apoptosis has been shown to play a role in a wide variety of neurodegenerative diseases such as multiple sclerosis (30), human immunodefi* Corresponding author. Mailing address: Chief, Laboratory of Molecular Medicine and Neuroscience, NINDS, NIH, Building 36, Room 5W21, 36 Convent Dr., Bethesda, MD 20892-4164. Phone: (301) 4961635. Fax: (301) 594-5799. E-mail: [email protected] 4884

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JC virus (JCV), a human neurotropic polyomavirus, demonstrates a selective glial cell tropism that causes cell death through lytic infection. Whether these cells die via apoptosis or necrosis following infection with JCV remains unclear. To investigate the mechanism of virus-induced cell death, we used a human central nervous system progenitor-derived astrocyte cell culture model developed in our laboratory. Using in situ DNA hybridization, immunocytochemistry, electron microscopy, and an RNase protection assay, we observed that astrocytes support a progressive JCV infection, which eventually leads to nonapoptotic cell death. Infected astrocyte cell cultures showed no difference from noninfected cells in mRNA expression of the caspase family genes or in any ultrastructural features associated with apoptosis. Infected cells demonstrated striking necrotic features such as cytoplasmic vacuolization, watery cytoplasm, and dissolution of organelles. Furthermore, staining for caspase-3 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling were not detected in infected astrocyte cultures. Our findings suggest that JCV-induced cell death of these progenitor cell-derived astrocytes does not utilize an apoptosis pathway but exhibits a pattern of cell destruction consistent with necrotic cell death.

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Downloaded from http://jvi.asm.org/ on December 27, 2014 by guest FIG. 1. Composite figure showing differences between uninfected and JCV-infected astrocyte morphologies. (A and B) Light microscopy of uninfected (A) and JCV-infected (B) human progenitor-derived astrocytes viewed with Hoffman interference optics. Infected astrocytes show a distinct morphology at 7 to 10 days p.i., including enlarged nuclei compared to those of uninfected controls. (C and D) In situ DNA hybridization of uninfected (C) and infected (D) astrocytes demonstrates active viral DNA replication in the majority of infected cells, detected by a JCV-specific biotinylated probe and DAB (3,3⬘-diaminobenzidine) visualization (brown). (E) Immunocytochemistry demonstrated that more than 95% of astrocytes in culture (infected and uninfected) were positive for the astrocyte marker GFAP (green). (F) Immunocytochemistry for the late viral protein VP-1 in infected astrocytes. An overlay of the nuclear stain bisbenzimide (blue) and VP-1 (red) shows that infected astrocytes are positive for the JCV capsid protein (purple). This figure represents results from three independent experiments.

cell death in JCV-infected astrocytes. Using in situ hybridization, hemagglutination, immunocytochemistry, electron microscopy, and an RNase protection assay (RPA), we observed that astrocytes unequivocally support a progressive JCV infection, which eventually leads to necrotic cell death.

MATERIALS AND METHODS Astrocyte cultures and JCV infection. Human CNS progenitor cells were isolated from an 8-week-gestation human fetal brain according to National Institutes of Health (NIH) guidelines. The isolation and cell culture conditions of the progenitor cells, and subsequent selective differentiation into homoge-

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FIG. 2. Viral multiplication in JCV-infected astrocytes. Human CNS progenitor cell-derived astrocytes were adsorbed with JCV and examined for virion production over a period of 12 days by hemagglutination activity. Virion production increased threefold from day 7 to day 12 p.i. Data represent results obtained from at least three different JCV infection experiments. temperature. After PBS washes and a brief exposure to the equilibrium buffer, the terminal deoxynucleotidyltransferase (tdt) enzyme mix was added for 1 h at 37°C in a humidified chamber, followed by a stop/wash solution. The tdt enzyme added digoxigenin-labeled nucleotides to the 3⬘ OH ends of DNA. After PBS washes, a fluorescein-conjugated anti-digoxigenin antibody was added for 30 min at room temperature. After nuclear labeling, cells were mounted and observed by using the same equipment described above. As a positive control, astrocyte cultures were exposed to DNase I (Roche) (10 U/ml). For negative controls, an enzyme solution without tdt was added.

RESULTS Human CNS progenitor cell-derived astrocytes support JCV infection. Human CNS progenitor cell-derived astrocytes were differentiated for these studies following a 3-week process in an appropriate serum-containing medium. The resulting populations were 95% or more GFAP positive (Fig. 1E) (18, 19). The ability of the astrocytes to support JCV infection was also verified by exposing the astrocytes to JCV at an MOI of 1, based on quantitation using an HA. The morphology of the astrocytes began to indicate infection as early as day 4 p.i., when some astrocytes appeared swollen. At day 10 p.i., the majority of astrocytes exhibited a swollen morphology, with some cells detaching from the monolayer (Fig. 1B). This was not observed in uninfected astrocytes (Fig. 1A). Astrocytes show evidence for active viral replication, assembly, and productive infection. ISH was performed on samples collected on days 7 and 12 p.i. in order to look for active viral DNA replication in astrocytes. As seen in Fig. 1D (brown), there were significant numbers of JCV DNA hybridizationpositive nuclei in infected astrocyte cultures, indicating that the JCV genome was being actively replicated in these cells. Results obtained from three independent experiments revealed that at day 7 p.i., 30.827% ⫾ 6.67% (mean ⫾ standard deviation) of the astrocytes exposed to JCV infection were positive for JCV DNA ISH. The presence of JCV in infected astrocytes was further confirmed by immunostaining for the viral structural protein VP-1. As shown in a representative micrograph (Fig. 1F), we observed that at day 7 p.i., 15.48% ⫾ 4.60% (mean ⫾ standard deviation) of astrocytes in the infected cultures were positive for VP-1 staining (purple). The number of VP-1-positive actrocytes increased to 53.26% ⫾ 5.44% (mean ⫾ standard deviation) at day 10, confirming that astrocytes harbor JCV at

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neous astrocyte populations, have been reported recently (18). Briefly, to induce astrocyte differentiation, the progenitor cell culture medium was replaced with an astrocyte medium containing Eagle’s minimal essential medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM), gentamicin (50 ␮g/ml), and penicillin-streptomycin (100 IU/ml). The differentiation process lasted for 20 days, until a 95% or higher culture of glial fibrillary acidic protein (GFAP)-positive astrocytes was obtained. Cells were grown on poly-D-lysinecoated plasticware. Human astrocytes were exposed to 200 hemagglutination units of the Mad-1 strain of JCV/106 cells in a minimum amount of serum-free astrocyte medium to cover the cells, at a multiplicity of infection (MOI) of 1.0. Ninety minutes after JCV exposure, the medium was supplemented with fresh complete astrocyte medium. All tests were performed on cells 7 to 12 days postinfection (p.i.). JCV detection. The presence of JCV in astrocyte cultures was detected by DNA-DNA in situ hybridization (ISH) using a biotinylated JCV DNA probe (ENZO Biochemical), as previously described (20), or by immunofluorescence against the structural protein VP-1 (see “Immunofluorescence” below). Data were presented as percentages (means ⫾ standard deviations) of astrocytes positive for VP-1 immunostaining or as JCV DNA ISH results, as seen under five different fields with at least 50 cells in each field, from three different experiments. HA. A hemagglutination assay (HA) was conducted to measure virion production during infection of human astrocytes and was performed as previously described (23). Briefly, infected astrocytes were harvested on day 7 or day 12 p.i. and treated with 0.25% deoxycholic acid (DOC) for 60 min at 37°C to release the JC virions. Hemagglutination titers were expressed as the reciprocal of the final dilution of JCV resulting in hemagglutination of human type-O erythrocytes. Electron microscopy. Cells were grown in plastic chamber slides, fixed with 4% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 1 to 2 h at room temperature, washed in buffer, treated with 1% OsO4 for 1 h, mordanted en bloc with 0.25% uranyl acetate overnight, washed and dehydrated through a graded series of ethanol, and embedded in epoxy resin. Thin sections were counterstained with uranyl acetate and lead citrate and examined under a JEOL 1200 transmission electron microscope. Images were captured with a digital camera system (XR-100 charge-coupled device; AMT, Danvers, Mass.). RNA isolation and gene expression by RPA. An RPA was used to detect and quantitate the mRNA levels of caspase family genes in human CNS progenitorderived astrocytes. For each time point, uninfected and JCV-infected astrocytes were collected and total RNA was isolated by using the Qiagen RNAeasy minikits according to the manufacturer’s protocol. RPA was performed by using the RiboQuant RNase Protection Assay kit (BD Biosciences). This kit contained cDNA templates for caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10a (hAPO1c); the bcl-2 family genes bcl-w, bcl-x (L,S), bfl-1, bid, bik, bak, bax, bcl-2, and mcl-1 (hAPO2b); and cDNA templates for ribosomal protein L32 and glyceraldehyde3-phosphate dehydrogenase (GAPDH) as internal controls. Labeled antisense RNA probes were synthesized from these cDNA templates, by using [␣-32P]UTP (Perkin-Elmer Sciences) in an in vitro transcription reaction mixture which was then hybridized with 10 ␮g of total RNA extracted both from the control as well as JCV-infected astrocytes. Probe synthesis, hybridization, and proteinase K and RNase digestion were carried out according to the protocol supplied by BD Biosciences or as previously described (29). Samples were resolved on 5% denaturing acrylamide gels that were dried at 80°C under a vacuum. The gels were then positioned on autoradiographic film and exposed at ⫺80°C for 6 to 24 h prior to development. The resulting bands were scanned and quantitated by using Adobe PhotoShop and Scion Corporation (NIH Image) software. The band intensity was normalized to that of either GAPDH or L32 in the same reaction. Immunofluorescence. Cultured cells were fixed in 4% paraformaldehyde and permeabilized with 0.02% Triton for 15 min. After three phosphate-buffered saline (PBS) washes, fixed cells were incubated with rabbit polyclonal antibodies against GFAP (1:100; Dako), active caspase-3 (1:1,000; R&D Systems), or VP-1 (1:200) diluted in 2% bovine serum albumin (Sigma) at room temperature for 60 min. Rhodamine- or fluorescein-conjugated goat anti-rabbit secondary antibodies (1:100; Jackson Immunoresearch) were added for 60 min at room temperature, followed by three PBS washes. Bisbenzimide (5 ng/ml; Calbiochem) was then added for 10 min to stain the nuclei of all cells. Fluorescent cells were examined by using a Zeiss Axiovert inverted microscope with corresponding fluorescence filters. In situ detection of apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling [TUNEL]). An Apoptag fluorescein detection kit was used according to the manufacturer’s instructions (Serologicals Corporation). Briefly, cells were fixed in 4% paraformaldehyde for 30 min at room temperature. Fixed cells were then permeabilized with 0.5% Triton for 10 min at room

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postreplication stages where the late viral structural proteins are being produced and assembled into new virion particles. To further confirm that JCV infection of astrocytes resulted in productive infection, we performed an HA to measure virion particle production. The tests revealed a significant increase in viral titers at day 12 versus day 7 p.i., indicating that the astrocytes not only are infected but also are consistently producing more virions with subsequent days p.i. (Fig. 2). Ultrastructural studies demonstrate virion production and necrotic death in infected astrocytes. Because electron microscopy provides the most reliable method for recognizing morphological changes at the cell organelle level, we carefully examined the control as well as JCV-infected astrocytes for the

presence of virions and patterns of cell death. Cells were examined systematically for every grid opening, and every cell encountered was scored for the presence of virions. Infected cells showed progressive signs of viral infection from day 7 to day 10, whereas no viral particles were found in the control cells (a total of 73 cells examined). The presence of 40- to 45-nm-diameter viral particles was prominent in nuclei as clusters in typical crystalline array structure (Fig. 3A). These clusters were of variable distribution and size. Virions were also seen in the cytoplasm, where they were present in specialized endoplasmic reticulum compartments termed annulated lamellae. The viral particles were membrane bound in these specialized organelles (Fig. 3B).

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FIG. 3. Electron micrographs of a JCV-infected human CNS progenitor-derived astrocyte. (A) Clustered virion assemblies (arrows) in the nucleus (Nuc) of an infected astrocyte (day 8 p.i.). Arrowheads indicate characteristic astroglial intermediate filaments in the cytoplasm. (B) Viral particles (arrows) located inside the specialized endoplasmic reticulum compartments (annulated lamellae) in the cytoplasm of an infected astrocyte (day 8 p.i.). (C) Infected-astrocyte (day 10 p.i.) necrotic morphology. Note the watery cytoplasm surrounding the nucleus and numerous vacuoles (long arrows) in the cytoplasm. Cellular debris (small arrows) is also evident at the cell periphery. (Inset) Enlarged image of viral particles in annulated lamellae near the nucleus.

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Of all 62 nuclei examined from the infected samples, none showed any signs of apoptosis, which would have been marked by condensed chromatin materials and aggregated mitochondria. In contrast, many cells showed signs of necrosis, especially at day 10 (Fig. 3C). These cells had large cytoplasmic vacuoles, watery cytoplasm, and a notable amount of cellular debris nearby (Fig. 3C). JCV-infected astrocytes do not overexpress caspase family genes. We studied the expression of all the relevant human caspases, including caspase-1, -2, -3, -4, -5, -6, -7, -8, -9, and -10a, at the mRNA level by an RPA. Although JCV infection of astrocytes was confirmed by robust viral titers, immunohistochemistry for JCV structural proteins, electron microscopy, and a bizarre morphology compared with that of uninfected controls, the caspase mRNA levels remained unchanged over a wide range of time points ranging from very early (6, 12, 24, 48, and 72 h p.i.) (data not shown) to late (days 7 and 12 p.i.), as seen in Fig. 4A. The subtle differences that may be seen in Fig. 4 were not found significant when the bands were quantitated for day 12 p.i. by using the NIH Image software and normalized with the respective GAPDH controls to account for any differences in sample loading onto the gel (Fig. 4B). Since the bcl-2 family of genes works upstream of the caspase cascade and is important in the regulation of caspase

activation, we also studied the expression profile of the bcl-2 genes in RNA isolated from astrocytes following JCV infection. None of the genes in the bcl-2 family, including the proapoptotic bcl-x, bax, and bak genes, showed altered expression by RPA following JCV infection of astrocytes (data not shown). Human CNS progenitor cell-derived astrocytes undergo apoptosis with chemical inducers. The absence of caspase activation prompted an investigation of whether these human CNS progenitor-derived astrocytes have intact and functional apoptotic machinery. Astrocytes were treated with the chemical agents camptothecin (10 ␮M) and cycloheximide (200 ␮M), which are known to induce apoptosis. Although active caspase3 was rarely seen in the untreated and infected cultures, a considerable number of active caspase-3-positive cells were seen after a 48-h treatment with campothecin (10 ␮M) or cycloheximide (200 ␮M) (Fig. 5B). Also, following a 24-h treatment with apoptosis inducers, the proapoptotic genes bcl-x and bax were overexpressed, confirming that these cells are capable of undergoing apoptosis (data not shown). JCV-infected astrocytes are negative for TUNEL and active caspase-3. Evidence for the presence of JC virions in astrocytes by positive immunostaining for the structural viral protein VP-1, as well as by electron microscopy, provides strong support for progressive viral infection in these astrocytes. How-

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FIG. 4. (A) RPA autoradiograms showing the expression of various apoptosis regulatory genes. mRNA expression of caspase genes in astrocytes remained unchanged following JCV infection. Arrow indicates the unaltered gene expression of caspase-3, which is the main downstream effector of the apoptosis pathway. (B) Densitometric ratios of genes of interest to the housekeeping gene GAPDH, for day 12 p.i. Data are means ⫾ standard deviations from three different experiments. The subtle differences seen in the figure were not statistically significant.

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ever, the absence of caspase regulation and of alterations in expression of the pro- and antiapoptotic genes of the bcl-2 family calls for an apoptotic end point assessment to confirm the mRNA results, before one can suggest a nonapoptotic cell death pathway following JCV infection. TUNEL staining was performed as an end point marker for apoptosis. JCV-infected astrocytes at days 7 and 10 have little or no TUNEL or active caspase-3 staining (Fig. 5C and D insets, respectively). The results confirm that the same cells showing clear evidence for JCV infection, as assessed by positive VP-1 staining, have no signs of apoptosis, and that cell death most likely does not occur via an apoptotic pathway. DISCUSSION These data show that human CNS progenitor-derived astrocytes support progressive JCV infection, as demonstrated by active viral-genome replication, structural-protein production,

viral assembly, and virion multiplication. In normal brains, astrocytes perform many regulatory functions, which suggests that nonfunctional astrocytes might be involved directly or indirectly in a range of neurodegenerative disorders. Astrocytes have also been suggested to serve as viral reservoirs in HIV-associated dementia (4). Although the documented cytopathic effects of JCV infection include bizarre-looking astrocytes with lobulated hyperchromatic nuclei and astrocytes containing mitotic bodies, the role of human astrocytes in JCV infection and the induction of cell death remains unclear. To further study the nature of JCV infection, we used a homogeneous GFAP-positive astrocyte population which was differentiated from human fetal brain progenitor cells (18). We propose that these human CNS progenitor-derived astrocytes are a viable system for the study of CNS infections of the human brain, particularly by viruses with a glial-cell tropism. Not only are these astrocyte cultures highly homogeneous and

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FIG. 5. JCV-infected astrocytes immunostained for the active form of caspase-3. Human CNS progenitor-derived astrocytes were treated with 10 ␮M campthothecin, a topoisomerase inhibitor, and 200 ␮M cycloheximide to assess if our in vitro system has functional apoptosis machinery. After 48 h, there was significant overexpression of the proapoptotic enzyme active caspase-3 (red and purple) (B), compared to untreated control astrocytes, where only the blue nuclear stain was seen (A). However, though JCV-infected astrocytes are positive for the viral capsid protein VP-1 (red) (D), they are negative for active caspase-3 (d, inset) and TUNEL (c, inset). When treated with DNase I (10 U/ml), the majority of positive-control astrocytes are TUNEL positive (green) (C), indicating that the technique works well with positive controls.

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apoptosis in astrocytes by using electron microscopy. There were no signs of the typical hallmarks of apoptosis. Electron microscopy revealed that these cells had extensive vacuolization and watery cytoplasm, thus confirming our results suggesting that JCV-infected astrocytes die via necrosis as a result of the lytic infection. Other known or unknown apoptotic mechanisms, as well as indirect mechanisms, may need to be considered in light of a recent report suggesting that SV40 large T antigen may sensitize cells to apoptosis (5). Evaluation of additional tissues and further studies will be necessary in order to further explore apoptosis in JCV-infected oligodendrocytes and PML. However, such studies are impeded by the need for a reliable, primary human oligodendrocyte culture system, which would make it possible to specifically test for apoptosis pathways and to perform other functional assays, including caspase inhibition. Collectively, the data from this study confirm that human astrocytes effectively support a progressive JCV infection that is similar to sequential events observed in oligodendrocytes. Furthermore, the data indicate that JCV infection of astrocytes leads to necrotic cell death in this population of cells. ACKNOWLEDGMENTS We thank Jean Hou for critical review and editing of the manuscript. We also thank Virginia Crocker at the NINDS Electron Microscopy facility for technical help. F. Diaz is supported in part by the Undergraduate Scholarship Program (UGSP) and the Office of Loan Repayment and Scholarship at NIH. REFERENCES 1. Ariza, A., J. L. Mate, A. Fernandez-Vasalo, C. Gomez-Plaza, J. Perez-Piteira, M. Pujol, and J. J. Navas-Palacios. 1994. p53 and proliferating cell nuclear antigen expression in JC virus-infected cells of progressive multifocal leukoencephalopathy. Hum. Pathol. 25:1341–1345. 2. Benedict, C. A., P. S. Norris, and C. F. Ware. 2002. To kill or be killed: viral evasion of apoptosis. Nat. Immunol. 3:1013–1018. 3. Boldorini, R., S. Cristina, L. Vago, A. Tosoni, S. Guzzetti, and G. Costanzi. 1993. Ultrastructural studies in the lytic phase of progressive multifocal leukoencephalopathy in AIDS patients. Ultrastruct. Pathol. 17:599–609. 4. Brack-Werner, R. 1999. Astrocytes: HIV cellular reservoirs and important participants in neuropathogenesis. AIDS 13:1–22. 5. Cole, S. L., and M. J. Tevethia. 2002. Simian virus 40 large T antigen and two independent T-antigen segments sensitize cells to apoptosis following genotoxic damage. J. Virol. 76:8420–8432. 6. Everett, H., and G. McFadden. 1999. Apoptosis: an innate immune response to virus infection. Trends Microbiol. 7:160–165. 7. Friedlander, R. M. 2003. Apoptosis and caspases in neurodegenerative diseases. N. Engl. J. Med. 348:1365–1375. 8. Hardwick, J. M. 2001. Apoptosis in viral pathogenesis. Cell Death Differ. 8:109–110. 9. Hartmann, A., S. Hunot, P. P. Michel, M. P. Muriel, S. Vyas, B. A. Faucheux, A. Mouatt-Prigent, H. Turmel, A. Srinivasan, M. Ruberg, G. I. Evan, Y. Agid, and E. C. Hirsch. 2000. Caspase-3: a vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc. Natl. Acad. Sci. USA 97:2875–2880. 10. Hay, S., and G. Kannourakis. 2002. A time to kill: viral manipulation of the cell death program. J. Gen. Virol. 83:1547–1564. 11. Hengartner, M. O. 2000. The biochemistry of apoptosis. Nature 407:770– 776. 12. Kanduc, D., A. Mittelman, R. Serpico, E. Sinigaglia, A. A. Sinha, C. Natale, R. Santacroce, M. G. Di Corcia, A. Lucchese, L. Dini, P. Pani, S. Santacroce, S. Simone, R. Bucci, and E. Farber. 2002. Cell death: apoptosis versus necrosis. Int. J. Oncol. 21:165–170. 13. Kaul, M., G. A. Garden, and S. A. Lipton. 2001. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 410:988–994. 14. Krynska, B., J. Gordon, J. Otte, R. Franks, R. Knobler, A. DeLuca, A. Giordano, and K. Khalili. 1997. Role of cell cycle regulators in tumor formation in transgenic mice expressing the human neurotropic virus, JCV, early protein. J. Cell. Biochem. 67:223–230.

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capable of large-scale expansion, but our in vitro ultrastructural studies also clearly demonstrate striking similarities to documented in vivo results obtained from actual progressive multifocal leukoencephalopathy (PML) patients (3, 37). Interestingly, virion particles outside the nucleus were seen in association with a membrane that appears to be an extension of the endoplasmic reticulum. We are currently investigating the observation of intracytoplasmic virions in detail due to very similar findings for PML patients, some reported more than 3 decades ago (3, 37). We believe that the presence of these virions in the cytoplasm in annulated lamellae may indicate a novel method of viral dissemination. In an effort to elucidate the molecular mechanism of cell death in JCV-infected cells, it was necessary to go beyond identifying apoptosis versus necrosis. We studied the expression of proapoptotic genes that included the whole caspase family. Due to reported apoptosis-related specificity and implications in many neurodegenerative disorders, we also tested for expression of the active form of caspase-3 by immunostaining (9, 31). No difference in the expression of apoptosis-related genes or active caspase-3 was detected between our JCV-infected astrocytes and noninfected cells. Infected human astrocytes were also negative for TUNEL, further confirming that JCV-induced cell death is a result of necrosis and not apoptosis. These results parallel JCV biology in vivo, where the virus is the etiologic agent responsible for the fatal demyelinating disease PML. The pathology of PML results from the lytic destruction of glial cells, which is necessary to continue virus propagation and for the progression of multifocal lesions. Furthermore, the JCV early protein large T antigen, which displays close homology to the simian virus 40 (SV40) T protein, has also been suggested to form complexes and possibly inactivate the tumor suppressor protein p53 (1, 14, 15). T protein might thus inhibit cells from undergoing p53-dependent apoptosis, as has been suggested for other viruses that evade the apoptotic machinery, such as SV40 (2). Two recent studies have looked for markers of apoptosis in brain biopsy specimens from PML patients (26, 39). In agreement with our results, infected astrocytes did not show signs of apoptosis in these studies, but oligodendrocytes did show some evidence. Studies of JCV infection and PML are limited by the lack of a reliable in vitro human oligodendrocyte culture system. Based on our in vitro data, we believe that JCV does not induce apoptosis, at least in astrocytes; however, the biochemical environment in the PML brain is much more complex. Thus, apoptosis in PML cannot be eliminated as a possibility. A possible explanation for the lack of any apoptosis markers in our in vitro system could be that the astrocytes used in this study were unable to undergo apoptosis through known mechanisms. To test this possibility, chemical inducers of apoptosis were added to the astrocytes in order to note active caspase-3 staining and Bax mRNA overexpression (data not shown). Although apoptosis is a complex series of events and the induced pathway most likely differs from that of an infection, overexpression of bax and active caspase-3 is necessary in the early and late stages of apoptosis, respectively (11). Since morphology remains critical in identifying apoptosis and necrosis, we were able to further assess the absence of

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