JC virus propagation is potentiated by glial replication and accelerated by demyelination-associated glial proliferation [scRNA-seq]

Progressive multifocal leukoencephalopathy (PML) is a demyelinating infection of the brain of immunosuppressed individuals, mediated by the gliotropic polyomavirus JCV. JCV replicates in mitotically-competent human glial progenitor cells and astrocytes, which are triggered to divide in the setting of viral T antigen-mediated cell cycle entry, allowing viral replication; the death of mitotically-incompetent oligodendrocytes occurs secondarily, largely through T antigen-mediated apoptosis. This observation suggested that JCV infection might be potentiated by astrocytic replication, and hence accelerated in the setting of mitotic gliogenesis. To test this hypothesis, we tagged dividing human glia in vitro with bromodeoxyuridine (BrdU), then infected them with JCV MAD1, and confirmed that proliferating human astrocytes are more supportive of JCV propagation than mitotically quiescent cells. In vitro, scratch assays confirmed that viral propagation was greatly enhanced in peri-scratch regions of dividing glia. JCV infection of human glial chimeras established that infection was greatly accentuated by cuprizone-mediated demyelination, which was associated with increased glial progenitor cell proliferation. JCV infection in vivo was associated with caspase3-defined death of uninfected as well as infected oligodendrocytes, suggesting the contribution of bystander death to JCV-associated demyelination. These results suggest that JCV propagation in PML may be potentiated by glial cell division, and that the accentuated glial cell division and hence DNA replication attending acute demyelination might provide an especially favorable environment for JCV propagation and PML progression. These data thus argue for the aggressive prevention of new demyelinating events in patients at risk for PML. Infected primary fetal astrocytes were collected at different stages. At three different time points (day 0, day 4, and day 14 post infection), cells were washed with HBSS-/- and detached with TryplE at 37ºC for 5-10min. We transferred all the cells to 50 mL tube and set the volume to 30 mL. Aliquots of 5e5 cells in 15 mL tubes were preserved for single cell RNA-seq. Cells were suspended in 100 µL Miltenyi washing buffer and incubated with cell hashing antibody (1 µL, BioLegend, USA). Cells from different groups were independently stained with one of the barcodes labeled antibody pools. Samples were pooled at equal concentration, then loaded on a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion (GEMs). Libraries were prepared using Chromium Single Cell v3 Reagent Kit (10x GENOMICS) according to the manufacturer’s protocols.