Human glial chimeric mice reveal astrocytic dependence of JC virus infection

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease triggered by infection with the human gliotropic JC virus (JCV). Due to the human-selective nature of the virus, there are no animal models available to investigate JCV pathogenesis. To address this issue, we developed mice with humanized white matter by engrafting human glial progenitor cells (GPCs) into neonatal immunodeficient and myelin-deficient mice. Intracerebral delivery of JCV resulted in infection and subsequent demyelination of these chimeric mice. Human GPCs and astrocytes were infected more readily than oligodendrocytes, and viral replication was noted primarily in human astrocytes and GPCs rather than oligodendrocytes, which instead expressed early viral T antigens and exhibited apoptotic death. Engraftment of human GPCs in normally myelinated and immunodeficient mice resulted in humanized white matter that was chimeric for human astrocytes and GPCs. JCV effectively propagated in these mice, which indicates that astroglial infection is sufficient for JCV spread. Sequencing revealed progressive mutation of the JCV capsid protein VP1 after infection, suggesting that PML may evolve with active infection. These results indicate that the principal CNS targets for JCV infection are astrocytes and GPCs and that infection is associated with progressive mutation, while demyelination is a secondary occurrence, following T antigen–triggered oligodendroglial apoptosis. More broadly, this study provides a model by which to further assess the biology and treatment of human-specific gliotropic viruses.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating condition characterized by the degenerative loss of cerebral white matter after infection by JC virus (JCV; also known as JCPyV), a normally latent polyoma virus that becomes virulent in the setting of immunosuppression (1, 2). JCV is gliotropic and associated with oligodendrocytic loss in humans, but the human-selective nature of its infectivity and glial pathology has prevented the establishment of informative animal models of PML. As a result, prior studies have focused on modeling the systemic spread of JCV infection in mice with humanized immune systems (3) and in mice in which preinfected cells were delivered to the brain (4), but no experimental studies have yet achieved infection of the adult CNS by JCV or permitted modeling of the progressive demyelination of PML. On that basis, we asked whether mice neonatally engrafted with human glial progenitor cells (GPCs), whose forebrain glial populations become substantially humanized with age, might be able to support JCV infection and manifest the clinical hallmarks of PML, white matter gliosis and demyelination. We therefore engrafted newborn immunodeficient and myelin-deficient homozygous shiverer (Rag2^–/– Mbp^shi/shi) mice with bipotential GPCs isolated from fetal human brain tissue, thereby generating human glial-chimeric mouse brains (5, 6).

The donor human GPCs with which these mice were engrafted were bipotential oligodendrocyte-astrocyte progenitor cells, identified by their expression of CD140a (also known as PDGFRα) as well as by gangliosides and chondroitin sulfate proteoglycans recognized by the A2B5 and NG2 antibodies, respectively. In the neonatal mouse brain environment, the human GPCs preferentially expand and broadly migrate, so that over a several-month period postnatally, most murine glia are replaced by human glia, including astrocytes and their progenitors, as well as oligodendrocytes when congenitally hypomyelinated recipients are used as hosts (7). These mice develop a largely humanized glial environment; in particular, Rag2^–/– Mbp^shi/shi mice develop entirely humanized central myelin. We postulated that the effective glial humanization of these mice might permit their productive in vivo infection by cell type– and species-specific human gliotropic viruses. To address this hypothesis, once our human glial chimeric Rag2^–/– Mbp^shi/shi animals grew to maturity, we injected them intracerebrally with live JCV of several distinct virulent strains, including type 1A (referred to herein as Mad-1 JCV) and the type 2A JCV archetype, and several patient-isolated mutant isoforms thereof (8). We then assessed the consequent JCV infection of GPCs, astrocytes, and oligodendrocytes using immunolabeling for both early viral T antigen (T-Ag) and VP1 capsid protein.

The JCV-injected human glial chimeric Rag2^–/– Mbp^shi/shi mice developed widespread infection of their integrated human glia, and this process was accompanied by local demyelination in association with regions of frank gliosis. The resultant humanized rodent model of JCV infection allowed us to then ask a number of hitherto unapproachable questions regarding the genesis of PML. What is the phenotypic selectivity of JCV infection in vivo? Are oligodendrocytes indeed the principal target phenotype of the virus? What is the principal reservoir of infection? What are the kinetics of temporal spread of the virus? Using what cellular hosts, and by what anatomic pathways, does it propagate and spread? Is the virus genetically stable during replication in the host? Does infectivity vary by viral genotype? Beyond addressing these questions in vivo, we also infected cultures of human fetal GPCs and their progeny with JCV in order to assess the cellular mechanisms of JCV toxicity as concurrent functions of time, cell cycle, and phenotype. We found that the principal targets of JCV were GPCs and astrocytes; that oligodendroglia were also infected, but later and less efficiently; that the virus actively mutated with viral spread; and, most remarkably, that infected oligodendroglia were not even necessary for viral propagation and spread. Our data thus indicate that JCV is principally a disease of astrocytes and their progenitors, with oligodendrocytic loss and demyelination a pathogenic but unnecessary concomitant to viral infection and spread.

JCV efficiently infects astroglia and their progenitors in culture. Although PML has traditionally been viewed as a disease of oligodendrocytes, both astrocyte and GPC infection have been reported in vitro (9–13). Therefore, we first sought to assess the phenotypic selectivity of viral infection and propagation among the different phenotypes of human macroglia. We used either A2B5-directed immunoselection after PSA-NCAM depletion or CD140a-targeted selection to isolate human GPCs from second-trimester fetal human brain. The resultant isolates were then aliquoted, with some cultures maintained as human GPCs in serum-free media supplemented with FGF2 and PDGF, while others were switched to tri-iodothyronine (T3) to bias oligodendroglial differentiation. In parallel, CD44-based immunomagnetic sorting was used to select phenotypically restricted astroglia from matched samples. After at least 1 week in vitro, cultures of each phenotype were separately exposed to either JCV or vehicle control. At 3, 5, or 10 days thereafter, the cultures were immunostained for either T-Ag or VP1 (n = 4 experiments per marker per time point). At least 2,000 cells in total were analyzed for each phenotype at each time point (>500 cells in n = 4 human samples per experimental group). In the JCV-exposed CD140a- and CD44-sorted cultures, both CD140a⁺ human GPCs and glial fibrillary acidic protein–expressing (GFAP⁺) astrocytes, respectively, were infected quickly and efficiently. Within days, each manifested robust expression of both early viral T-Ag and VP1 (Figure 1, A and B), neither of which was detected in vehicle controls. In contrast to the rapid course of astroglial infection, oligodendrocytic infection in vitro was delayed and initially of relatively low efficiency (Figure 1C; see Supplemental Figure 1 for individual immunolabels; supplemental material available online with this article; doi:10.1172/JCI76629DS1). At both 3 and 5 days postinfection (DPI), the incidence of T-Ag infection was significantly higher in cultured astrocytes than in oligodendrocytes (P < 0.01, 2-way ANOVA with Bonferroni post-hoc comparisons). VP1-defined viral replication was also significantly less common in oligodendroglia than in astrocytes at 3 and 5 DPI (P < 0.01, 2-way ANOVA with Bonferroni post-hoc comparisons); indeed, VP1⁺ oligodendroglia were rarely noted in the first week after infection. Nonetheless, by 10 DPI, significant numbers of VP1⁺ oligodendroglia appeared as well, following the delayed rise in T-Ag–associated oligodendroglial infection (Figure 1, C–E). In astrocytes and oligodendrocytes alike, VP1-defined viral replication was associated with significant increments in nuclear size and DNA content (Supplemental Figure 2).

Figure 1

Human astroglia are most efficiently infected by JCV in vitro. Human GPCs and astrocytes were readily infected by JCV in vitro, with robust expression within days of both the early viral large T-Ag and the later VP1 capsid protein. (A) Both T-Ag and VP1 were expressed by CD140a⁺ GPCs grown in suspension culture, and both were more abundantly expressed at 10 DPI than at 3 DPI. (B) In CD44-sorted GFAP⁺ astrocytes, T-Ag was expressed as early as 1 DPI, whereas VP1 was first expressed at 3 DPI. (C) In contrast, O4⁺ oligodendrocyte infection in vitro was both delayed and of relatively low efficiency, showing weak T-Ag expression without VP1 at 5 DPI (arrowheads), with VP1⁺ oligodendroglia (arrows) appearing only at 10 DPI. (D) Representative images of a JCV-infected T-Ag⁺VP1⁺ oligodendrocyte at 10 DPI; nuclear hypertrophy (arrows) was apparent. (E) At 3 and 5 DPI, T-Ag⁺ oligodendroglial infection was of significantly lower efficiency than that of astrocytes, while oligodendrocytic VP1⁺ JCV replication was even less frequent. By 10 DPI, VP1⁺ oligodendroglia as well as astrocytes began to accumulate. Data are presented as percentage of cells of each phenotype at 3, 5, and 10 DPI. Scale bars: 20 μm. *P < 0.05; ***P < 0.01.

JCV initially infects astroglia in vivo. Since astrocytic infection was surprisingly more robust than that of oligodendrocytes in vitro, we next sought to define the relative phenotype-selective tropism and infectivity of JCV in vivo. Since JCV infects only human glia, we established human glial chimeric mice so as to provide an in vivo model for JCV infection and JCV-dependent demyelination. We injected neonatal Rag2^–/– Mbp^shi/shi mice with 2 × 10⁵ human GPCs, delivered as 10⁵ cells/hemisphere in 2 intracallosal injections per side of 5 × 10⁴ cells each. Myelination by neonatally engrafted human GPCs has been well characterized in human glial chimeric Rag2^–/– Mbp^shi/shi mice, which first achieve dense callosal and capsular myelination only after 3 months of age (6, 14). As a result, we assessed the effects of JCV in Rag2^–/– Mbp^shi/shi mice first injected with virus at 2, 3, or 4 months of age (n = 5, 10, and 3, respectively). At those time points, we delivered Mad-1 (type 1A) JCV to the human glial chimeras by stereotaxic intracallosal injection. The animals (n = 18 total) were all killed at 20 ± 1 weeks of age, i.e., at 3–4, 6–8, or 11–12 weeks after infection (n = 3, 10, and 5, respectively). Their brains were then assessed for both early and late JCV antigens, as well as for cellular pathology and myelin integrity (see Supplemental Table 1 for details of all mice assessed histologically).

We found that JCV induced the expression of the major early and late viral gene products (T-Ag and the VP1 capsid protein, respectively) in oligodendrocytes, astrocytes, and GPCs throughout the chimeric Rag2^–/– Mbp^shi/shi corpus callosum (Figure 2A). Infected astrocytes and GPCs were often magnocellular, with overtly enlarged nuclei, while processes of infected astrocytes manifested a bizarre fibrotic morphology (Figure 2B), as described previously in human PML (15). In contrast, at these early time points, relatively few infected myelin basic protein–expressing (MBP⁺) oligodendroglia were noted, most of which expressed T-Ag rather than VP1 (Figure 2C and see below), suggestive of both relatively later infection and less viral replication compared with astrocytes. Importantly, infection was restricted to human cells; no murine cells expressed either early or late viral genes, and nonchimeric, unengrafted mouse controls manifested no evidence of infection after JCV injection (Figure 2D).

Figure 2

JCV replicates more rapidly and efficiently in astroglia than in oligodendrocytes in vivo. JCV induced the expression of the major early and late viral gene products (T-Ag and VP1 protein, respectively) throughout the corpus callosum of human glial chimeric Rag2^–/– Mbp^shi/shi mice. (A) Confocal images of infected oligodendrocytes, astrocytes, and GPCs in Rag2^–/– Mbp^shi/shi mice neonatally engrafted with human GPCs and infected with type 1A (Mad-1) JCV for 12 weeks. (B) By 12 weeks, infected astrocytes were highly abundant and largely magnocellular, with overtly enlarged nuclei and bizarrely fibrotic processes. (C) In contrast, substantial human oligodendrocytic loss was evident by 12 weeks after infection, and most of the infected remainder expressed T-Ag (arrowheads), as exemplified by the T-Ag⁺MBP⁺ oligodendroglia shown; only human oligodendroglia expressed MBP in Rag2^–/– Mbp^shi/shi brain. (D) Infection was restricted to human cells; in this example, unengrafted mouse corpus callosum manifested no evidence of infection 12 weeks after JCV injection. Scale bars: 20 μm (A); 50 μm (B and C); 100 μm (D).

JCV infection in vivo results in demyelination of human chimeric white matter. To better understand the means by which demyelination occurs in JCV infection, we next examined the myelination of JCV-infected human glial chimeric Rag2^–/– Mbp^shi/shi mouse brains. By 4 weeks after JCV infection, focal regions of demyelination and infection-associated astrogliosis were noted in the forebrain white matter of infected mice, typically in discrete foci abutting the callosal and fimbrial walls of the lateral ventricle (Figure 3, A and B). Importantly, while areas of demyelination were associated with dense aggregations of VP1⁺ astrocytes, VP1⁺ oligodendroglia were rare, despite overt oligodendrocytic loss and demyelination (Figure 3, A–D). By 12 weeks postinfection, demyelination was widespread and associated with regions of focal gliosis embedded within demyelinated loci. At these longer postinfection time points, diffuse hypomyelination of the callosa and capsules of infected chimeric Rag2^–/– Mbp^shi/shi mice was noted (Figure 3, C and D); oligodendrocytic VP1 expression remained unusual, but T-Ag immunolabeling revealed large numbers of infected oligodendrocytes (Figure 3D). Human chimeric controls by that time point exhibited dense human GPC-derived myelination (Figure 3E).

Figure 3

JCV infection of human glial chimeras triggers both focal and diffuse demyelination. (A and B) At 4 weeks after viral infection, focal regions of demyelination (A, arrows) and infection-associated astrogliosis (B, arrow) were noted in the forebrain white matter of infected mice, typically in discrete foci abutting the callosal wall of the lateral ventricle. hGFAP, human GFAP. (C and D) By 11 weeks after infection, diffuse hypomyelination of the callosa and capsules of infected chimeric mice was noted. (E) Uninfected human chimeric controls by 20 weeks after transplantation exhibited dense GPC-derived myelination, as did JCV-injected non-xenografted murine controls (not shown). hN, human nuclear antigen. Scale bars: 200 μm (A and C); 100 μm (B, D, and E).

These observations suggest that the principal direct targets of JCV infection in the adult CNS are astrocytes and their progenitors, as cellular expression of the VP1 capsid protein, which is expressed by mature postreplication virions, was largely limited to astroglia and GPCs. Thus, astrocytes and GPCs may be the principal reservoirs for intracerebral viral propagation. In contrast, VP1⁺ human oligodendrocytes were uncommon, and inevitably appeared dying or pyknotic (Figure 2A); most infected oligodendroglia that could be identified as such in vivo were T-Ag⁺, but VP1^–. This finding indicated that oligodendroglia are initially infected, but die or are lost before the VP1-identifiable completion of viral replication.

JCV infection in vivo spreads in different cell types at different rates. As a result of the human-selective nature of JCV infection, and the lack of early or phenotype-specific radiographic surrogates, the dynamics of viral propagation in the infected CNS remain unknown. We thus investigated the pattern of JCV spread in human glial chimeras, as a function of time after infection. We tracked the expansion and spread of both infected glia and of the fraction harboring replicating virus by immunostaining for T-Ag and VP1, respectively. Both T-Ag⁺ and VP1⁺ human cells were progressively more numerous and widespread as a function of time after infection, with infection progressing from the site of viral injection at 4 weeks to include much of the central white matter at 12 weeks (Figure 4A). Notably, despite ample evidence of astrocytic viral replication, as defined by VP1 expression, by astrocytes and GPCs alike (Figure 4, B, D, and E), a high proportion of T-Ag⁺ glia remained viable but VP1^–; indeed, the ratio of T-Ag⁺ glia to VP1⁺ glia increased with time over the 12-week observation period (Figure 4B), suggestive of an accelerated rate of infection. While these T-Ag⁺VP1^– infectants included oligodendrocytes, most were astroglial; both the density and relative proportions of T-Ag⁺MBP⁺ oligodendrocytes were significantly lower than those of T-Ag⁺ astrocytes and GPCs at all time points (P < 0.05, ANOVA with post-hoc comparisons as well as linear regression of T-Ag⁺ cell density on length of infection; Figure 4C). Interestingly, while the T-Ag⁺ infection rate of both GPCs and astrocytes was significantly higher than that of oligodendrocytes, consistent with the substantially greater infectivities of both GPCs and astrocytes noted in vitro, VP1⁺ astrocyte and GPC density in vivo remained relatively low and did not differ from that of oligodendrocytes (Figure 4, D–F). This appeared to reflect the rapid clearance of infected cells once they reached the stage of viral replication; VP1⁺ astroglia and GPCs were lost due to viral-induced hypertrophy and lysis and appeared never to accumulate as such, despite their higher incidence and density of infection. Together, these data indicated that JCV infection and replication were significantly less robust in oligodendroglia than in GPCs and astrocytes, and that the latter proved more efficient as vehicles for viral spread. As postmitotic cells, oligodendrocytes might then be merely the victims of JCV infection, and not significant contributors to viral propagation and spread.

Figure 4

Viral propagation exhibits cell type–selective spread. JCV spread in vivo was tracked by immunostaining human glial chimeric Rag2^–/– Mbp^shi/shi brains for both T-Ag and VP1, each as a function of time after infection. In these chimeric Rag2^–/– Mbp^shi/shi mice, a large proportion of oligodendrocytes, as well as GPCs and astrocytes, were human. (A) Sagittal sections of 3 different infected chimeras at each of 3 time points; individual VP1⁺ cells are dot-mapped. VP1⁺ human cells became progressively more widespread with time, with JCV infection progressing from the site of viral injection to include much of the forebrain white matter by 12 weeks postinfection, with marked cortical spread by that point as well. (B) T-Ag⁺ and VP1⁺ cells, representing all JCV-infected cells and those in which viral replication has occurred, respectively, both accumulated as a function of time. (C) The number of T-Ag⁺ astrocytes (GFAP⁺) and GPCs (NG2⁺) was significantly higher than that of T-Ag⁺ oligodendroglia (MBP⁺) at all time points examined (P < 0.01, repeated-measures 1-way ANOVA). Furthermore, the in vivo rates of accumulation of T-Ag⁺ astrocytes and GPCs (reflecting the rate of infection among each cell type, estimated by regression line slope) were each significantly higher than that of oligodendrocytes (P < 0.01, linear regression). (D–F) Despite their marked differences in T-Ag–defined infectivity and spread, astrocytes and oligodendrocytes did not differ in their rates of accumulation of VP1⁺ infectants, likely reflecting the rapid lytic loss of cells at that stage. *P < 0.05; **P < 0.01; ***P < 0.001.

JCV does not require oligodendrocytes for viral infection and spread. Since astroglia appeared sufficient for both viral infection and propagation, we next asked whether oligodendrocytes were even necessary for viral spread in vivo. To this end, we injected Mad-1 JCV into the callosa of 16-week-old immunodeficient but myelin WT human glial chimeric Rag1^–/– mice (n = 5); these normally myelinated mice recruit few, if any, oligodendrocytes from the engrafted progenitor pool, so that their human cell complement remains limited to GPCs and astrocytes (7). At 12 weeks after viral injection (i.e., 28 weeks of age), the expansion of VP1⁺ infected cells within these chimeric Rag1^–/– hosts was grossly similar to that of human glial Rag2^–/– Mbp^shi/shi mice (Figure 5A; compare with Figure 4A). Importantly, however, the compartmental pattern of viral spread differed in the recipient phenotypes. In Rag1^–/– chimeras, in which human donor cells integrated only as astrocytes and GPCs, but not oligodendrocytes, JCV infection predominated in the cortex (Figure 5, B–E). This sharply contrasted the pattern of viral spread in the chimeric Rag2^–/– Mbp^shi/shi mice, in which human cells also engrafted as oligodendrocytes and fibrous astrocytes, and in which JCV infection was noted to preferentially spread in the white matter (Figure 5F). These observations indicated that oligodendroglia are not necessary for JCV propagation in vivo and that astroglia are sufficient to support viral infection and spread. Thus, these data strongly support the notion that astrocytes and GPCs serve as the principal reservoirs for JCV in vivo.

Figure 5

Astrocytes and GPCs are sufficient to support viral replication and spread in vivo. (A) JCV introduced into myelin WT Rag1^–/– mice (colonized with human GPCs and astrocytes, but not oligodendrocytes) yielded viral propagation and geographic spread as rapid and extensive as that in human glial chimeric Rag2^–/– Mbp^shi/shi mice (in which human oligodendroglia were densely represented). Shown are distributions of T-Ag⁺ and VP1⁺ cells mapped in 14-μm sagittal sections of 3 different Rag1^–/– mice injected with JCV as adults, 12 weeks previously. Infected human cells were widely distributed, despite the absence of human oligodendroglia. (B) Sagittal section along the callosal length of a chimeric myelin WT Rag1^–/– mouse 12 weeks after infection, showing widespread infection and VP1 expression by GFAP⁺ subcortical human astrocytes and GFAP^– cortical human astrocytes and GPCs. (D and E) Higher-magnification views showing the predominance of infected cells (arrowheads) in Rag1^–/– cortical grey, including both T-Ag⁺ (D) and VP1⁺ (E) glia, manifesting the typical hypertrophic nuclei of cells that have undergone viral replication. (F) Conversely, VP1⁺ glia in the corpus callosum of a human glial chimeric Rag2^–/– Mbp^shi/shi mouse 12 weeks after type 1A (Mad-1) JCV infection showed the predominant white matter spread of virus in these mice, which manifested both oligodendrocytic and astrocytic infection (compare with the Rag1^–/– section in C). Notably, since JCV can only infect human cells, the potential volume of infection in these mice was limited by the geographic dispersal of the resident human oligodendrocyte progenitor cells. Since these mice were given only forebrain injections so as to produce forebrain-only human glial chimeras, all JCV infection in these mice was limited to the forebrain. Cx, cerebral cortex; CC, corpus callosum. Scale bars: 100 μm (B, C, and F); 50 μm (D and E).

JCV-infected oligodendrocytes enter the cell cycle. Polyoma large T-Ag can trigger cell cycle entry and S-phase initiation by various mitotically quiescent somatic cell types, via binding to retinoblastoma protein (Rb); the resultant progression to S-phase both permits and accelerates viral replication (16). On that basis, we next asked whether cell cycle initiation occurs in JCV-infected oligodendrocytes in PML, and if so, whether the induction of cell cycle in postmitotic human oligodendroglia is sufficient to trigger their death. We immunolabeled infected oligodendrocytes in the engrafted Rag2^–/– Mbp^shi/shi mouse for the mitosis-associated antigen Ki67 and found that 31.0% ± 8.4% of infected mature MBP⁺ oligodendrocytes were indeed Ki67⁺, whereas no Ki67⁺MBP⁺ oligodendrocytes were found in matched uninfected controls (Figure 6, A and B).

Figure 6

JCV-infection leads to cell cycle entry-associated with oligodendrocytic death. (A and B) Mad-1 JCV-infected MBP⁺ oligodendrocytes were frequently noted to coexpress Ki67, a marker of mitotic entry, suggesting the aberrant entry of these typically postmitotic cells into cell cycle. (A) Representative T-Ag⁺Ki67⁺MBP⁺ oligodendrocyte in the corpus callosum of a human glial chimeric Rag2^–/– Mbp^shi/shi mouse 12 weeks after infection. (B) Whereas roughly one-third of all infected MBP⁺ oligodendroglia were Ki67⁺ at this time point, no Ki67⁺MBP⁺ oligodendrocytes were noted in uninfected controls. (C and D) T-Ag⁺Ki67⁺O4⁺ oligodendrocytes were common in vitro (C), and most T-Ag⁺ oligodendroglia were Ki67⁺, while few if any uninfected (T-Ag^–) O4⁺ oligodendroglia were Ki67⁺ (D). In culture, JCV infection significantly reduced the number of O4⁺ oligodendrocytes at 10 DPI (E) by inducing TUNEL-defined apoptosis (F). At 10 DPI, of infected TUNEL⁺O4⁺ oligodendrocytes, 43.5% ± 2.1% were T-Ag⁺, whereas only 4.6% ± 0.3% were VP1⁺ (G), indicative of the failure of most infected oligodendroglia to progress to VP1-defined viral replication before dying. (H) TUNEL expression by T-Ag⁺ pyknotic O4⁺ oligodendrocytes was typical. *P < 0.05, ***P < 0.001, paired 2-tailed Student’s t test. (B) n = 3 per group. (D–G) n = 4 runs, triplicate wells; >2,000 scored cells/group. Scale bars: 10 μm (A); 20 μm (C and H).

To further investigate the relationship of JCV infection to aberrant oligodendrocytic cell cycle entry, we then infected cultured oligodendrocytes derived from CD140a⁺ human fetal GPCs. The plated GPCs were differentiated in vitro into O4⁺ oligodendroglia over 7 days, then infected with Mad-1 JCV at 10⁵ genome equivalents per cell (GE/cell). For each experiment, 4 matched runs were performed, each in triplicate for a total of 12 scored wells, which represented a minimum of 2,000 cells per group. When assessed at 5 DPI, 26.5% ± 2.2% of O4⁺ oligodendrocytes expressed T-Ag (Figure 6C), whereas only 1.3% ± 0.6% expressed VP1. When reassessed at 10 DPI, 58.4% ± 2.0% of the T-Ag⁺ JCV-infected O4⁺ oligodendrocytes coexpressed Ki67, indicative of their aberrant entry into cell cycle; conversely, uninfected T-Ag^– oligodendrocytes in the same cultures exhibited no appreciable Ki67 expression (Figure 6D). These findings support the in vivo observation of Ki67⁺MBP⁺ oligodendroglia in JCV-injected human glial chimeric Rag2^–/– Mbp^shi/shi mice (Figure 6, A and B) and suggest that otherwise-postmitotic human oligodendroglia may be aberrantly induced into cell cycle entry by JCV infection.

JCV-induced cell cycle entry is associated with oligodendrocytic death. We next asked whether the T-Ag–associated induction of oligodendrocytic cell cycle entry is sufficient to trigger oligodendrocytic death, since ectopic cell cycle entry has been associated with cell death in a variety of otherwise-postmitotic phenotypes. In particular, an analogous process of cell cycle activation–induced death has been described extensively in neurons, in which it has been explored as a mechanism of neurodegenerative cell loss (17–19), and apoptotic oligodendrocytic death has been observed in response to JCV (20, 21).

In vitro, we found that the number of viable O4⁺ oligodendrocytes was significantly decreased at 10 DPI with type 2A JCV compared with otherwise-matched uninfected controls (Figure 6E); the diminished oligodendrocytic numbers of infected cultures were accompanied by a significant increase in the number of infection-associated dying oligodendrocytes, as defined by TUNEL (Figure 6F and ref. 22). Whereas 43.5% ± 2.1% of TUNEL⁺O4⁺ oligodendrocytes expressed T-Ag at 10 DPI, only 4.6% ± 0.3% expressed VP1 (P < 0.001, n = 4 each; Figure 6, G and H). These in vitro data indicate that the vast majority of oligodendrocytes killed by JCV infection die in association with T-Ag expression, never progressing to the point of VP1-defined viral replication. Furthermore, in JCV-infected human glial chimeras, we identified sporadic apoptotic oligodendroglia by TUNEL labeling in vivo and found that a large proportion of these TUNEL⁺O4⁺ oligodendroglia expressed the mitotic marker Ki67, indicating that their deaths occurred in temporal association with aberrant cell cycle entry. Such apoptotic loss was not observed in infected astrocytes, which instead assumed the hypertrophic appearance of infected pre-lytic cells prior to death. Together, these data strongly suggested that the oligodendrocytic death that ensues after JCV infection is primarily apoptotic and occurs in response to T-Ag–triggered forced cell cycle entry, rather than to replication-associated cytolysis.

Cell cycle arrest at the G2/M transition in astrocytes and GPCs. Polyoma viral replication requires S-phase progression on the part of infected host cells. In particular, previous studies had revealed that JCV genome replication may be facilitated by virally induced cell cycle arrest in G2, prior to the G2/M transition (23). In light of the apparent delay in viral replication in human oligodendroglia, relative to astrocytes and GPCs, we next asked whether infected astrocytes and oligodendrocytes differ in their efficiency of S/G2 traversal.

Since our data indicated that astrocytes and GPCs are the principal vectors of viral spread in vivo, we first investigated the cell cycle status of JCV-infected astroglia by focusing on cyclin B1, which is normally expressed in the cytoplasm in G2 and enters the nucleus only at the initiation of M-phase. Among cells infected with type 2A JCV, T-Ag⁺ glia expressed nuclear cyclin B1, regardless of their mitotic stage (Figure 7, A–D). In GPCs as well as in astrocytes, cyclin B1 immunoreactivity was localized to the hypertrophic nuclei of infected GPCs (Figure 7, A and E). Furthermore, DAPI staining revealed the frequent presence of T-Ag⁺ JCV-infected cells with nuclear cyclin B1⁺ that were not mitotic (Figure 7, C and D), which suggests that JCV infection was associated with cell cycle arrest.

Figure 7

Cell cycle arrest at G2/M in infected glia. T-Ag⁺ glia expressed the mitosis-associated nuclear protein cyclin B1, as well as the DNA damage and cell cycle arrest–associated phospho-p53(Ser15), regardless of their mitotic stage. (A and B) Vehicle-treated and uninfected (T-Ag^–) GPCs expressed intranuclear cyclin B1 only when mitotic and in M phase (the latter as assessed by DAPI; arrows), whereas the nuclei of JCV-infected (T-Ag⁺) GPCs admitted cyclin B1 in a temporally promiscuous fashion (arrowheads). (C) Nuclear phospho-p53(Ser15) and cyclin B1 were coexpressed by mitotically arrested infected astrocytes, but not by uninfected astrocytes. (D) Like control GPCs, uninfected astrocytes (T-Ag^–GFAP⁺) expressed intranuclear cyclin B1 only in M phase, whereas JCV-infected astrocytes expressed nuclear cyclin B1 even when not dividing. (E and F) JCV-infected astrocytes (T-Ag⁺GFAP⁺) coexpressed phospho-p53(Ser15), associated with G/2M arrest, whereas neither vehicle-treated nor T-Ag^– astroglia in infected cultures did so to any significant degree. (G and H) Similarly, JCV-infected GPCs (T-Ag⁺CD140a⁺) coexpressed phospho-p53(Ser15), as well as did infected oligodendrocytes (T-Ag⁺O4⁺) (I and J); for both oligodendrocytes and their progenitors, T-Ag⁺ cells were significantly more likely to express phospho-p53(Ser15). All cultures were assessed at 10 DPI with type 2A JCV (Mad-1 NCCR). Scale bars: 20 μm. *P < 0.05; ***P < 0.001.

To further define the possibility of phenotype-selective cell cycle arrest by JCV, we investigated the state of p53 phosphorylation in JCV-infected human oligodendrocytes, GPCs, and astrocytes. Because p53 is phosphorylated at Ser15 when DNA damage is detected, phospho-p53(Ser15) expression may be used as a marker of DNA damage, as well as its associated cell cycle arrest at G2/M (23–25). We found that a significantly higher proportion of T-Ag⁺ astrocytes coexpressed phospho-p53(Ser15) than did uninfected T-Ag^– cells (Figure 7, E and F). Similarly, among CD140a⁺ GPCs infected with type 2A JCV, 36.1% ± 6.1% expressed phospho-p53(Ser15), while only 3.2% of uninfected T-Ag^– GPCs expressed phospho-p53(Ser15)⁺ (P < 0.001; Figure 7, G and H). The expression of phospho-p53(Ser15) by T-Ag⁺ cells was even more pronounced in O4⁺ oligodendrocytes, among which the majority of T-Ag⁺ cells expressed phospho-p53(Ser15) (Figure 7, I and J). Indeed, among T-Ag⁺O4⁺ oligodendroglia, 83.6% ± 0.9% (n = 4) were phospho-p53(Ser15)⁺, compared with 1.3% ± 0.8% of uninfected T-Ag^–O4⁺ cells in the same plates (P < 0.001, 2-tailed Student’s t test). Together, these data strongly suggest that JCV infection was associated with G2/M checkpoint arrest, in all 3 glial phenotypes assessed.

To confirm the possibility of T-Ag–associated mitotic arrest, we next conducted cell cycle analysis of JCV-infected human astroglia, which were derived from GPCs exposed to high serum for 10 days, then exposed to JCV and analyzed 14 days later. We noted that the JCV-infected T-Ag⁺ astrocytes indeed exhibited marked accumulation of cells in G2/M relative to their uninfected controls (P < 0.001; Supplemental Figure 2A). Interestingly, a fraction of these infected astrocytes appeared hyperploid, suggestive of ongoing DNA replication in infected astroglia, despite their relative cell cycle arrest (Supplemental Figure 2B). Together, these results suggest that JCV infection of human glia triggers phosphorylation of p53 at Ser15 with concomitant cell cycle arrest at G2/M. Whereas in oligodendrocytes this event appeared to trigger apoptotic cell death, in astrocytes and GPCs it was associated with viral replication leading to cell lysis.

JCV VP1 rapidly mutates in vivo. In PML, point mutations in the gene encoding VP1 capsid have been reported at multiple sites of the sialic acid binding region, yet neither the genesis nor the pathogenic role of these mutations has been clear (26, 27). Because the propagation and spread of JCV in the human glial chimeric Rag2^–/– Mbp^shi/shi brains was so robust, we next asked whether JCV mutation might be identified during its replication and spread in this system. To assess viral evolution during active infection in vivo, we sampled 3 sections from each of 5 JCV-infected brains from mice that had been killed 3–11 weeks after viral infection; all mice had been neonatally implanted with human GPCs and were given intracallosal injections of Mad-1 JCV between 4 and 16 weeks of age. Of the 5 mice, 4 were Rag2^–/– Mbp^shi/shi (in which donor human GPCs, astrocytes, and oligodendrocytes coexisted) and 1 was Rag1^–/– (chimeric only for human donor-derived astrocytes and GPCs). TA PCR cloning was used to clone and sequence JCV VP1 DNA from a total of 32 viral clones per sampled section, or 96 per mouse. The VP1 segment of each clone was sequenced and aligned with WT Mad-1 JCV, whose sequence was validated and confirmed in matched aliquots of the injected virus. Among the 480 clones sequenced from the 5 JCV-injected mice, 155 (32%) VP1 genomic mutations were noted compared with the WT Mad-1 sequence; 125 of these yielded mutations in protein sequence. Of these muteins, only 15 distinct loci were represented twice or more in the overall set (Supplemental Table 2); these included 2 mutations — S123C and D66G — which have been described previously in JCV isolated from patients with active PML (26, 27). Each of these lies within the sialic acid binding site of VP1, which is particularly notable as VP1 mutants may lose sialic acid dependence for their binding to host cells; this suggests that specific JCV VP1 mutants may acquire selective advantage during host infection (26). This in turn suggests that capsid protein mutations occurring during viral propagation may dynamically shift the infectivity and phenotypic specificity of JCV in vivo over the course of a single host’s infection.

VP1 mutants are infective, but show no phenotypic preferences. On the basis of these observations, we next asked whether JCV mutants carrying PML-associated VP1 mutations could infect human glia as well as parental JCV, and if so, whether their relative infectivities differed in either virulence or phenotypic specificity. We therefore assessed the infectivities of 3 VP1 mutations of type 2A JCV (all with PML-associated Mad-1 NCCR; see Methods) both in vitro and in vivo. These VP1 capsid mutants included L55F, K60E, and S269F, each isolated from a different PML patient. We first assessed each mutant in vitro and found that each robustly infected human oligodendrocytes, astrocytes, and GPCs (Supplemental Figure 3).

We next assessed the in vivo infectivities of the 2 most frequent PML-associated mutations, L55F and S269F. Either WT VP1 JCV or mutant VP1 type 2A JCV was injected into the corpus callosum of human glial Rag2^–/– Mbp^shi/shi chimeras at 14 weeks of age, and mice were killed 5 weeks later. Histological analysis revealed that each mutant elicited robust infection in the host, with a recapitulation of both glial infection and local demyelination. At the single time point assessed, no overt differences were noted in the respective infectivities of GPCs, astrocytes, or oligodendrocytes by WT VP1 JCV relative to the 2 VP1-mutant variants (Supplemental Figure 4); all 3 viruses manifested robust infection in the human glial chimeric Rag2^–/– Mbp^shi/shi mice.

In this study, we used human glial chimeric mice to establish an in vivo model of JCV viral infection and demyelination, mimicking the salient features of PML. We found that in neonatally engrafted Rag2^–/– Mbp^shi/shi mice (chimeric for human oligodendrocytes as well as astrocytes), oligodendrocytic death and demyelination followed astrocytic infection. In vitro, JCV infection of astrocytes was significantly more rapid and efficient than that of oligodendrocytes, whereas in vivo, JCV appeared to propagate primarily via GPCs and astroglia, and much less robustly so in central oligodendrocytes. Indeed, astrocytes proved sufficient for viral propagation in vivo, as shown by rapid and robust viral spread in Rag1^–/– mice chimeric only for human GPCs and astrocytes, and utterly lacking human oligodendrocytes.

While human oligodendrocytes exhibited much less frequent viral replication than did astrocytes and progenitors, their infection-associated loss and concomitant demyelination was profound; all macroglial phenotypes were targeted and lost after intracerebral injection of JCV. Yet whereas astrocytic death was associated with cellular lysis in the setting of viral replication and spread, oligodendrocytic death was instead presaged by the aberrant cell cycle entry of these otherwise-postmitotic cells, leading to their arrest in G2 and subsequent apoptotic death. Indeed, this finding provides a mechanistic base for previous observations of apoptotic death by JCV-infected oligodendroglia (20, 28). The oligodendrocytic loss and demyelination of JCV-infected human glial chimeras appeared to result from T-Ag–dependent cell cycle induction leading to oligodendrocytic death before viral replication. As a result, when assessed at 5 weeks postinfection, a large proportion of T-Ag⁺ oligodendrocytes coexpressed Ki67, while oligodendrocytic expression of the replication-associated VP1 protein was uncommon. Our data are therefore suggestive of viral propagation and amplification in an astrocytic reservoir, followed by oligodendrocytic infection, apoptotic death, and demyelination, but with little oligodendroglial viral replication. Although we posit that this concept reflects the typical pattern and pathobiology of JCV’s spread in adult humans, it is important to note that the astrocytic and GPC predominance of JCV infection in these brains may also reflect the unique environment of these human glial chimeras; one might readily envision mechanisms by which the relative infectivities of astrocytes and oligodendrocytes might be influenced by cell type–specific adaptations of the human donor cells to the murine brain environment.

Importantly, viral spread within infected brains was associated with rapid and progressive mutation, presumably within the astrocytic reservoir of infection. This process of serial and progressive mutation may provide a selective advantage to the most virulent viral mutants within individual brains, and thus result in the acceleration of viral spread and cytopathogenicity with time. One might then anticipate the emergence and selection of dominant JCV mutants with time during disease progression in any given patient. While JCV mutations have been noted to arise during the course of disease and have been well documented in VP1 (26, 27), mutation in JCV’s noncoding regulatory regions may be especially critical to disease progression (29–31). Together, these observations suggest that the clonal selection of more infective mutants may occur naturally in human hosts (8); if so, such clonal evolution might account for the terminal acceleration of demyelination often observed in PML patients.

T-Ag can induce aberrant cell cycle entry and S-phase initiation in a variety of somatic phenotypes, through its binding to the tumor suppressor retinoblastoma protein (Rb) and consequent derepression of Rb targets (16, 32–35). Whereas some mitotic phenotypes may undergo neoplastic transformation by this process (and the polyomaviruses have been causally linked to oncogenesis in various species and cell types; refs. 36–41), in postmitotic phenotypes, as in human oligodendrocytes, Rb derepression may instead trigger p53-dependent apoptotic cell death (18). Our present data thus suggest that JCV-induced T-Ag may initiate apoptotic oligodendrocytic death via aberrant cell cycle induction, followed by p53-dependent apoptotic cell death before viral replication can be completed. While our in vitro and in vivo data collectively suggest that this is the dominant mechanism of JCV-induced oligodendrocytic death, a minority of infected oligodendroglia did progress to viral replication and VP1 expression, which suggests that at least some oligodendroglia may activate mechanisms for apoptotic escape, such as the expression of apoptosis inhibitors (including survivin), which may dominate in astroglia (28). It remains unclear why some JCV-infected oligodendroglia progress to viral replication, whereas most die before reaching that stage, although we posit that this might be a function of the developmental stage at which individual oligodendroglia — or their progenitors — are infected.

In addition to these avenues of death by infected oligodendroglia, our frequent identification of dying oligodendrocytes absent any antigenic evidence of viral infection suggests the concurrent incidence of additional mechanisms for oligodendrocytic loss in JCV-infected brains. Recent studies have highlighted the dependence of oligodendrocytes on local astrocytes (42) and that of neurons upon oligodendroglia (43, 44). Given the marked astroglial pathology so evident in JCV-infected brains, the withdrawal of metabolic support of oligodendrocytes by infected astroglia, as well as the latter’s potential paracrine cytotoxicity (42), might contribute substantially to both oligodendrocytic loss and its associated neuropathology. Oligodendrocytic loss and demyelination in PML may then represent secondary events, reflecting a combination of paracrine toxicity by infected astroglia, loss of astroglial support of local oligodendrocytes, and apoptotic oligodendrocytic death after direct infection; complicating matters further, each of these mechanisms may be dynamically modulated by concurrent JCV mutation, occurring within a contiguous astrocytic reservoir of virus.

Besides identifying astrocytes and their progenitors as principal substrates for JCV propagation in vivo, with oligodendrocytic death a secondary consequence of viral propagation, this astroglial-centric view of PML pathogenesis may have significant clinical implications for both presentation and treatment of disease. Patients with PML frequently manifest encephalopathic confusion and cognitive deficits long before frank radiographic evidence of demyelination is noted. Our results suggest that the clinical deterioration in these patients may reflect progressive astrocytic dysfunction, especially given the strong influence of astrocytes on synaptic coordination and plasticity (7, 45, 46). Even a profound degree of astrocytic pathology may not have been previously noted in PML patients, since MRI of the brain is highly biased toward identifying signal abnormalities in the white matter; contemporary MRI has been relatively insensitive to gray matter disruption. Similarly, little focused investigation of astrocytes has been performed in studies of human pathological samples, which have emphasized oligodendrocytic loss and demyelination (21, 47–51), although the early infection of astrocytes in PML has previously been noted (52, 53). Together, our present results suggest that astrocytes may be both necessary and sufficient for JCV infection of the brain and that disease-associated astrocytic dysfunction and loss may be at least as important to the neurological deterioration of JCV-infected patients as their progressive demyelination. More broadly, our findings also introduce the human glial chimeric Rag2^–/– Mbp^shi/shi mouse brain as a unique preparation by which the natural history of pathogens specific to the human brain may be assessed in vivo, both longitudinally and in real time, thereby providing fundamentally new opportunities for their mechanistic dissection and therapeutic targeting.

View Supplemental data

This work was supported by Biogen Idec, NINDS grant R01NS75345, the New York State Stem Cell Research Program (NYSTEM), and the National Multiple Sclerosis Society. We thank Richard Frisque and Ian Duncan for gifts of antibodies; Margot Brickelmaier, Jeff Thompson, Ken Simon, Wanyong Zeng, Eric Baetscher, Kathryn Doyle, Christopher Tonkin, Michele McAuliffe, and John Anderson of Biogen for technical support; Adam Cornwell for statistical advice and support; and Abdellatif Benraiss for imaging assistance.

Address correspondence to: Steven A. Goldman, Center for Translational Neuromedicine, University of Rochester Medical Center, Rochester, New York 14642, USA. Phone: 585.275.9550; E-mail: steven_goldman@urmc.rochester.edu.

Conflict of interest: This work was supported in part by Biogen Idec, of which A. Kazarov and L. Gorelik are employees and for which S.A. Goldman has served as a paid consultant.

Reference information: J Clin Invest. 2014;124(12):5323–5336. doi:10.1172/JCI76629.

See the related article at Human glial chimeric mice reveal astrocytic dependence of JC virus infection.

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