Fig. 2: Expression of viral genes and proteins during BKPyV infection of normal human urothelium.

a mRNAseq analysis of BKPyV gene expression at 14 days post infection (dpi) showed late promoter genes including Agnoprotein were the most expressed viral genes, whilst early promoter genes such as LT-Ag were less transcriptionally-active. All viral gene expression was significantly suppressed by the indirect actions of IFNγ on the urothelium; however, the efficacy was widely variable between donors. Statistically-significant differences are indicated by stars with the mean log₂ fold change in gene expression reported beneath (n = 6 or 7 independent donors). b BKPyV genome map showing the non-coding control region (NCCR) which regulates both the early and late genes that are expressed in opposing orientations. c RT-qPCR analysis of BKPyV LT-Ag and VP1 transcript abundance in normal human urothelial (NHU) cell cultures. Data is displayed as log₂ fold-change normalised to abundance at 7 dpi. LT-Ag and VP1 abundance increased significantly between 7 and 14 dpi. The addition of IFNγ led to a significant reduction in LT-Ag and VP1 abundance at 14 dpi. d 14 dpi western blot densitometry for large T antigen (LT-Ag) showing significant reduction by IFNγ (mean log₂ fold change −1.77), with exemplar blot image from a representative patient shown below the x-axis. Truncated LT-Ag (truncT-Ag) was also expressed; densitometry analysis of truncT-Ag and whole blots for all donor lines probed with LT-Ag can be found in Supplementary Fig. 1 and the β-actin loading controls in Supplementary Fig. 2. e 14 dpi western blot densitometry for viral capsid protein 1 (VP1) showing significant (mean log₂ fold change −0.64) reduction by IFNγ, with exemplar blot image from a representative patient shown below x-axis. Full VP1 blots for all donor lines are shown as Supplementary Fig. 3 and the β-actin loading controls in Supplementary Fig. 2 (n = 5 independent donors). f LT-Ag indirect immunofluorescence labelling index found a mean of 29.8% of urothelial cells expressed detectable protein. n > 2900 cells per condition per donor. g Exemplar LT-Ag indirect immunofluorescence image from Donor 3 BKPyV-infected urothelial cells (all conditions shown in Supplementary Fig. 4). Scale bar denotes 100 μm. Significance was assessed in panels a by LRT test and c–f by paired t-test.