Fig 2: PML and Sp100 association with HPV16 E1/E2 replication foci increases in the presence of replicating viral DNA.A. HFK 1A cells cultured on glass coverslips were transfected with HPV16 E1 and E2 expression vectors (400 ng each) and 50 ng of pKS (empty vector), pKS-16Ori, or recircularized HPV16 genome. E1 and E2 expression was induced 24 hours post-transfection with 3 μM CdSO4 for four hours and cells were fixed with 4% PFA for analysis by indirect immunofluorescence. Cells were stained with EE (E1, green), PML (red) or Sp100 (cyan) antibodies. Nuclei (outlined in light blue) were detected with DAPI counterstain. Images are single optical slices collected by confocal microscopy, and are representative of three independent transfections. B. E1-foci positive cells were scored for association with PML by visually assessing PML’s proximity to the E1-foci. Results were calculated from a minimum of 20 cells per experiment derived from three independent experiments (Total N = 60). Error bars represent +/-SEM.

Fig 3: SAND domain containing isoforms of Sp100 repress viral transcription and replication.A. qPCR was performed on cDNA prepared from HFKs coelectroporated with 2µg of recircularized HPV18 genome and 1µg of an empty expression vector or a vector expressing either Sp100A, B, C, HMG or the Sp100B-DBD mt. Samples were harvested 72 hours post DNA transfection. The spliced viral messages E1^E4 (solid bars) or E6*I (hashed bars) were detected using specific primers for the spliced messages. Data displayed are averages of three independent experiments using four different HFKs donor strains (n = 4 replicates). All mRNA levels were normalized to TBP. B. Southern blot analysis of whole cell DNA extracted from the same coelectroporated cells graphed in A. DNA was separated by agarose gel electrophoresis and probed with 32P-labeled HPV18 genome 72 hours after DNA transfection. Prior to electrophoresis, DNA was digested with the restriction endonucleases DpnI to remove input, un-replicated DNA and EcoRI to linearize the viral genome. The arrow indicates the linear form of the viral DNA. Blot was performed using DNA from one of the two replicates shown in panel A. C. Phosphorimage quantitation of Southern blot assays from two independent experiments, except Sp100B-Q sample was tested only once. Error bars represent SEM. The paired student t-test was used to determine statistical significance between the EYFP-transfected control and each Sp100 isoform for panel A and C. n.s., not statistically significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Fig 4: Sp100 represses late HPV mRNA transcription.A. Timeline of experiment. CIN612-9E cells were transfected with 20 nM siRNA to Sp100 and grown as shown. RNA was isolated at T = 0, 2, 4 and 6 days. B. Cellular transcripts for Sp100, involucrin and filaggrin were measured at the time points indicated. C. Viral transcripts were measured using qPCR with primers that overlapped the splice sites of E6*I, E2 877^2646, E1^E4 and L1. All results were obtained from three independent experiments, each of which contained two technical replicates. Error bars represent +/- SEM. *, P<0.05; **, P<0.005; ***, P<0.0005; ****, P<0.00005; ns, not statistically significant.

Fig 5: Sp100 represses differentiation dependent genome amplification.A. Southern blot analysis of 9E cell DNA extracted from cells treated with either control, PML or Sp100 siRNA for two days before addition of 1.5 mM CaCl2 to induce differentiation. DNA was collected at 0 or 96 hours post addition of CaCl2. Prior to electrophoresis, DNA was digested with a restriction enzyme that left supercoiled viral DNA intact (-), or with HindIII to linearize the viral genome (+). DNA was separated by agarose gel electrophoresis and probed with 32P-labeled HPV 31 genome. The blot shown is representative of four total Southern blots. B. DNA qPCR was performed on CIN612-9E cells treated according to the scheme shown in Fig 6A. Results shown were averaged from three independent experiments, each of which contained two technical replicates. Error bars represent +/- SEM. *, P<0.05.