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Interaction of the Human Papillomavirus E1 Helicase With UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes

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Interaction of the Human Papillomavirus E1 Helicase With UAF1-USP1 Promotes Unidirectional Theta Replication of Viral Genomes

Marit Orav et al. mBio.

Abstract

Human papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCE Human papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.

Keywords: DNA replication; FA pathway; human papillomavirus.

Figures

FIG 1
FIG 1
E1-UAF1-USP1 interaction is necessary for efficient replication of the HPV11 genome in U2OS cells. A Southern blot analysis of the transient replication of the HPV11 WT genome and of three UBSmut genomes was performed (top), and a bar graph representing the replication efficiencies of the UBSmut genomes relative to the replication efficiency of the WT genome is shown (bottom). Total cellular DNA was extracted from HPV11-transfected U2OS cells 72 h posttransfection and digested prior to analysis with the single-cutter restriction enzyme HindIII and the methylation-sensitive restriction enzyme DpnI. The bar graph data represent quantitated Southern blot signals from two independent experiments; error bars represent standard deviations. Statistical significance was determined using ordinary one-way ANOVA followed by Dunnett’s multiple-comparison test. (B and C) Southern blot analysis of the transient replication of HPV11 WT (B) or VE (C) genomes in the presence of increasing concentrations of ML323 (top); bar graph representing the effect of ML323 on the replication efficiency of the HPV11 WT (B) or VE (C) genome (bottom). ML323 (Millipore Sigma) or DMSO as a vehicle was added to the cell culture medium 24 h posttransfection. The DMSO concentration was constant for all ML323 dilutions and for the no-ML323 control. Total cellular DNA was extracted from HPV11-transfected U2OS cells 48 h posttransfection and linearized prior to analysis. Bar graphs represent quantitated Southern blot signals from four independent experiments; error bars represent standard deviations. Statistical significance was determined using ordinary one-way ANOVA followed by Dunnett’s multiple-comparison test.
FIG 2
FIG 2
Disrupting the E1-UAF1-USP1 interaction specifically inhibits the unidirectional replication of the HPV11 genome. (A and B) 2D N/N AGE analysis of single-cutter restriction enzyme-digested RIs generated from the HPV11 WT genome (A) or VE genome (B). Low-molecular-weight DNA was extracted from HPV11-transfected U2OS cells 72 h posttransfection and digested with FspI. The results are representative of 4 experiments. Experiments were normalized to the number of transfected cells. White arrows mark uni-RIs, black arrows mark theta-RIs, and thin black arrows mark late theta-RIs. (D and E) 2D N/N AGE analysis of single-cutter restriction enzyme-digested RIs generated from the HPV11 WT genome in the presence of DMSO vehicle (D) or 20 µM ML323 (E). Low-molecular-weight DNA was extracted from HPV11-transfected U2OS cells 72 h posttransfection and digested with FspI. The results are representative of 3 experiments. Experiments were normalized to the number of transfected cells. White arrows mark uni-RIs, black arrows mark theta-RIs, and thin black arrows mark late theta-RIs. (C and F) Scatter graph depicting the ratio of signals representing theta-RIs and uni-RIs. Scatter graph data represent quantitated Southern blot signals; the areas used for quantitation are marked with boxes in panels A, B, D, and E. Each dot represents a separate experiment. The scatter graph represents quantitated signals from 4 (C) or 3 (F) separate experiments. Statistical significance was determined using unpaired Student's t tests; error bars represent the standard deviations. P = 0.0013, t = 5.701 (C); P = 0.0010, t = 8.679 (F). (G) Schematic overview of DNA molecules produced by FspI-digestion of RIs generated by bidirectional theta replication of HPV11 genomes, and their expected migration patterns during 2D N/N AGE (64).
FIG 3
FIG 3
HPV11 RIs produced in the HaCaT cell line are identical to those generated in U2OS cells. 2D N/N AGE was performed with single-cutter restriction enzyme-digested RIs generated from the HPV11 WT and E8mut genomes in HaCaT cells. Low-molecular-weight DNA was extracted from HPV11-transfected HaCaT cells 72 h posttransfection and digested with FspI. Experiments were normalized to the number of transfected cells. White arrows mark uni-RIs, black arrows mark theta-RIs, and thin black arrows mark late theta-RIs.
FIG 4
FIG 4
Eliminating the expression of the E8^E2 repressor protein further decreases the replication efficiency of HPV11 genomes unable to bind UAF1. (A) Southern blot analysis of total DNA extracted from U2OS cells transfected with HPV11 WT, UBSmut, E8mut, and E8mutUBSmut genomes. Total cellular DNA was extracted from HPV11-transfected U2OS cells 72 h posttransfection and digested prior to analysis with the single-cutter restriction enzyme HindIII and the methylation-sensitive restriction enzyme DpnI. The results are representative of 2 experiments. (B) Bar graph representing the replication efficiencies of different HPV11 genomes relative to the replication efficiency of the WT genome. Data represent quantitated Southern blot signals from 2 separate experiments; statistical significance was determined using ordinary one-way ANOVA followed by Dunnett’s multiple-comparison test. Error bars represent the standard deviations.
FIG 5
FIG 5
RIs generated by bidirectional theta replication and unidirectional replication arise from HPV11 genomes in different topological forms. (A and B) 2D N/N AGE analysis of uncut RIs generated from the HPV11 WT (A) or VE (B) genomes. Low-molecular-weight DNA was extracted from HPV11-transfected U2OS cells 72 h posttransfection and digested prior to analysis with the methylation-sensitive restriction enzyme DpnI to remove input HPV11 genomes. The results are representative of 2 experiments. Experiments were normalized to the number of transfected cells. White arrow marks uni-RIs, and black arrows mark theta-RIs. (C) Schemes depicting the expected migration patterns of circular plasmids in different topological conformations (left) and uncut theta and rolling-circle RIs (right).
FIG 6
FIG 6
HPV11 E1 protein interacts with BLM helicase. (A) Yeast two-hybrid screen for E1 interaction partners. A diagram of HPV11 E1 indicating the positions of the N-terminal regulatory region, DNA-binding domain (BD), and C-terminal helicase domain is shown. The minimal region required for E1 oligomerization in yeast cells is marked with “O.” The amino acid sequence boundaries of each bait are indicated on the left; levels of measured β-galactosidase activity are indicated on the right. The ability of different baits to interact with E1 and E2 is indicated as a control. AD, activation domain. (B) GST pull-down assay demonstrating in vitro interaction between HPV11 E1 and BLM. Two different GST-tagged E1 fragments were used in the experiment: one fragment spanning E1 amino acid sequences from position 191 to position 649 and the other fragment spanning sequences from position 353 to position 649. An EtBr control was included to check for DNA contamination. E2 was used as a positive control and luciferase enzyme as a negative control for in vitro interaction.
FIG 7
FIG 7
BLM helicase stimulates the generation of uni-RIs. (A and D) 1D AGE analysis of the overall replication efficiency of the HPV11 WT genome (A) or VE genome (D) in the absence (control) or presence (+BLMwt) of wild-type BLM overexpression. Low-molecular-weight DNA was extracted from HPV11-transfected U2OS cells 72 h posttransfection and normalized to cell count. The number of cells per sample is indicated. Samples were digested with the single-cutter endonuclease HindIII and the methylation-sensitive restriction enzyme DpnI. The results are representative of 2 experiments. (B and E) 2D N/N AGE analysis of RIs generated from the HPV11 WT genome (B) or VE genome (E) in the absence (control) or presence (+BLMwt) of wild-type BLM overexpression. Low-molecular-weight DNA was extracted from HPV11-transfected U2OS cells 72 h posttransfection and digested prior to analysis with the single-cutter endonuclease FspI and the methylation-sensitive restriction enzyme DpnI. The results are representative of 3 experiments. Experiments were normalized to the number of transfected cells. White arrows mark uni-RIs, black arrows mark theta-RIs, and thin black arrows mark late theta-RIs. (C and F) Scatter graph depicting the ratio of signals representing uni-RIs and theta-RIs. Scatter graph data represent quantitated Southern blot signals from 3 separate experiments for each data set; areas used for quantitation are marked with boxes in panels B and E. Each dot represents a separate experiment. Statistical significance was determined using an unpaired Student's t test, and error bars represent the standard deviations. P = 0.0100, t = 4.605 (C); P = 0.6102, t = 0.5522 (F).
FIG 8
FIG 8
The proposed involvement of the UAF1-USP1 complex and BLM helicase in HPV11 replication.

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