Fig. 4: BKPyV stimulates urothelial cell cycle re-entry by inactivating phosphorylation of retinoblastoma protein.

a Schematic summary of proposed BKPyV cell cycle regulation. pRb Retinoblastoma protein, DP Dimerization partners, P Phosphorylation. b The RB1 transcript that encodes pRB showed no changes in response to infection or IFNγ (Supplementary Fig. 11a); however, western blotting of pRb phosphorylated at serine 807/811 showed a significant infection-associated increase (mean 3.0-fold) that was suppressed by IFNγ. A representative blot from a single donor is shown here, with full blots for all donors in Supplementary Fig. 11b and the β-actin loading controls in Supplementary Fig. 2. c BKPyV infection also led to a significant mean 88-fold increase in phosphorylated-pRb labelling index (p < 0.01; representative images are shown in Supplementary Fig. 12). When cells from infected cultures were split into LT-Ag positive and negative populations, there was a mean 63.1-fold ( ± 57.1) increase in phosphorylated-pRb labelling of LT-Ag negative cells from BKPyV-infected cultures from all donors compared to non-infected controls, supporting the proposed field effect. The red colouration for LT-Ag positive cells applies only to panel c. d mRNAseq showed significant induction of the pRb-regulated E2F1 transcription factor, which was induced more prominently than other members of the E2F family (Supplementary Fig. 13). e Comparison of genes significantly 2-fold induced by BKPyV-infection with those reported to possess proximal E2F1 [21] and FOXM1 [23] ChIP-seq peaks, suggested 40% of the BKyV-induced transcriptome may be activated by these transcription factors. E2F1 ChIPseq peaks [21] and BKPyV-induced genes had a significant overlap of 82 genes (exact hypergeometric probability p < 4.95 × 10⁻¹⁰⁶; representation factor = 38.6). There was a 60-gene overlap (exact hypergeometric probability p < 1.30 × 10⁻⁸⁰; representation factor = 43.8) between genes up-regulated upon BKPyV-infection and those associated with FOXM1 binding [23]. Gene lists provided as Supplementary Fig. 14. Increased E2F and FOXM1-activity was further supported by GSEA (Supplementary Fig. 10d–f and 10h). f and g mRNAseq and western blotting respectively show a BKPyV-mediated increase in expression of the DREAM complex member EZH2. EZH2 joins the polycomb repressive complex 2 (PRC2) dimerization partners to drive transcription as shown by GSEA (Supplementary Fig. 10g). A representative blot from a single donor is shown here, with full EZH2 blots shown in Supplementary Fig. 15 and the β-actin loading controls in Supplementary Fig. 2. h mRNAseq shows significant induction of FOXM1 transcription by BKPyV-infection.