Fig 1.
Schematic map and the working principle of the Renilla luciferase-encoding marker genomes.
The coding sequences of Renilla luciferase and the 2A peptide of FMDV are inserted in frame into the E2 ORF of HPV immediately after the overlapping region of E1 and E2, and the full-length E2 gene follows. Translation results in functional Renilla luciferase and a full-length E2 proteins separated by the “cleavage” of the 2A peptide. The Renilla luciferase protein contains additional amino acids of the N-terminal E2 protein, as shown. The 20–22 amino acids in the N-terminus of E2 protein are thus present in both the Renilla protein as well as the full-length E2 protein. Due to the effects of 2A peptide, an additional amino acid proline is present in the beginning of the E2 protein. Renilla luciferase expression is controlled by viral transcription. Modified regions of the viral genome are shown in red and green.
Fig 2.
Analysis of the transient replication of HPV18, HPV16 and HPV5 marker genomes in the U2OS GFP2-Fluc #10.15 cell line.
U2OS #10.15 cells were transfected with 2 μg of HPV18 wt, HPV18-RlucE2, HPV18-RlucE2-K490A, HPV16 wt, HPV16-RlucE2, HPV5 wt and HPV5-RlucE2. The genomic DNA was extracted 3, 5 and 7 days after transfection, the HPV DNA was linearized and bacterially produced input DNA was digested with DpnI. Replication signals were visualized by Southern blot, and the position of the replicated DNA is shown with an arrow. Replication signals were also quantitated by qPCR and are expressed relative to either HPV18, HPV16 or HPV5 wt at the 3-day timepoint. Both Renilla (from HPV marker genomes) and Firefly (from U2OS genome) luciferase were measured in a dual-luciferase assay from the same experiments, and the results are expressed as the Rluc/Fluc ratio (Luciferase) relative to the 3-day timepoint from either HPV18-RlucE2 (A and B), HPV16-RlucE2 or HPV5-RlucE2 (C–E). Wt (and HPV18-RlucE2-K490A) genomes serve as negative controls for Renilla expression. Error bars represent standard deviations from at least two independent experiments.
Fig 3.
Analyses of stable replication and late amplification of monoclonal U2OS #10.15 cell lines containing an episomal HPV18-Rluc-E2 marker genome.
A: The expression of Renilla (right axis) and Firefly (left axis) luciferases from two clones of U2OS #10.15 cells stably maintaining an episomal HPV18-Rluc-E2 genome (#2B3 and #2G10), measured on two different days. Renilla expression resembles differences in the HPV18-RlucE2 copy number in these cell lines. Firefly expression resembles the growth of the cells. Viral copy numbers are indicated. B: To evaluate the stability of these subclones, #2B3 and #2G10 were thawed from the cell banks generated and cultivated in subconfluent conditions for 30 days. HPV-RlucE2 copy number (by qPCR) and Rluc/Fluc ratios were measured in every 5 days and the values are expressed relative to the 1-day timepoint. C and D: To measure late amplification, the cells were seeded in 6-well plates and grown for the indicated number of days without splitting. C: To measure viral replication, the genomic DNA was extracted 0, 4, 8 and 12 days after seeding the cells, linearized and quantitated by qPCR. Replication signals obtained from the subclones are expressed relative to the signals obtained from the respective 0-day timepoint. D: Both Renilla (from HPV18 marker genome) and Firefly (from U2OS genome) levels from the same experiment were measured in a dual-luciferase assay, and the values obtained from either subclone are expressed relative to the signals obtained from the respective 0-day timepoint.
Fig 4.
Inhibition of initial amplification of the HPV18 genome by the compounds identified from HTS of NCI Diversity Set IV.
U2OS cells were transfected with 2 μg of an HPV18 wt minicircle genome and grown in the presence of compounds at the indicated concentrations for 5 days. Genomic DNA was extracted, HPV18 DNA was linearized with BglI and bacterially produced input DNA was digested with DpnI. HPV18 genome replication signals were detected using Southern blot analyses, quantified with a phosphoimager and expressed relative to the vehicle control (DMSO). Logarithmic inhibition curves and approximate IC50 values are shown for each compound. Error bars represent the standard deviation from five independent experiments.
Fig 5.
Tdp1 and PARP1 are essential cellular proteins for the initial amplification of the high-risk HPV genome.
U2OS cells were transfected with HPV18 wt minicircle and an sh_Tdp1 plasmid (A) or sh_PARP1 plasmid (B). Genomic DNA was extracted 3 and 4 days after the transfection, linearized and digested with DpnI. The HPV18 replication signal was detected with Southern blot analyses and quantified with a phosphoimager. C: U2OS cells were transfected with an HPV18 wt minicircle and grown for 3 and 4 days in the presence of different concentrations of the PARP1 inhibitor ABT-888. The HPV replication signal was detected with Southern blot analyses and quantified with a phosphoimager. D: U2OS cells were transfected with an HPV31, 33, 11 or 5 wt minicircle and the sh_Tdp1 or sh_PARP1 plasmid (2μg). Genomic DNA was extracted 3 days post-transfection, linearized and digested with DpnI The HPV replication signals were detected with Southern blot analyses and quantified with a phosphoimager. E and F: Western blot analyses showing the downregulation of the Tdp1 and PARP1 proteins by shRNA at the 3-day timepoint. Empty shRNA vector was used as a mock control for Tdp1 and PARP1 downregulation by sh_RNA plasmids. G: Error bars represent standard deviations from two to three independent experiments.
Fig 6.
Synergistic inhibitory effect between Camptothecin (CPT) and compounds identified in the HT screen on the initial amplification of the HPV18 genome.
U2OS-EBNA1 cells were transfected with HPV18 wt and oriP plasmids and grown in the presence of the indicated concentrations of compounds alone or together with 2 nM CPT for 5 days. Genomic DNA was extracted, linearized and digested with DpnI. Both HPV18 and oriP replication signals were detected with Southern blot and quantified using a phosphoimager. Compounds 9782, 88915, 109128 and 305831 showed modest synergistic inhibition together with CPT (panels A, C, D and E), whereas compound 82269 did not (panel B). Error bars represent standard deviations from at least three independent experiments. Lanes 15 and 16 are size markers for HPV18 and oriP.
Fig 7.
Identified compounds are specific high-risk HPV replication inhibitors.
U2OS cells were transfected with various types of HPV minicircle genomes and grown in the presence of the indicated concentrations of the compounds for 5 days. Genomic DNA was extracted, HPV DNA was linearized with the appropriate enzyme for each HPV type and bacterially produced input DNA was digested with DpnI. Replication signals were quantitated by qPCR (for HPV types 5, 11 and 16; panels A, B and C) or from Southern blots using a phosphoimager (for HPV types 31, 33 and 45; panels D, E and F) and are expressed relative to DMSO, as described in the Materials and Methods section. Panel G: CIN612E cells were grown in the presence of indicated concentrations of compounds for 6 days and HPV31 replication signal was quantitated from Southern blots using a phosphoimager and are expressed relative to DMSO. Error bars represent standard deviations from two to three independent experiments.
Fig 8.
Proposed model for the role of the Tdp1 and PARP1 proteins in high-risk (HR) HPV genome replication.
I: E1 and E2 dependent bi-directional replication is initiated from the HPV replication origin in the URR region. II: Topoisomerase I cleaving complex (Top1cc) will be entrapped in at least one site in the HR-HPV genome due to the specific DNA damage or replication fork collapse. III: Tdp1, which is activated through PARylation by the PARP1 protein, cleaves the phosphodiester bond between Top1cc and DNA, thereby releasing the complex. Replication fork collision will be repaired by homologous recombination-dependent DNA repair (using a homologous region from another HPV genome molecule), and HPV genome replication will proceed via unidirectional recombination-dependent replication.