Journal: Journal of Virology

Article Title: Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis

doi: 10.1128/jvi.00830-22

Figure Lengend Snippet: Plasmid retention/segregation by E2 in U2OS cells. (A) Our luciferase-based segregation/retention assay, as summarized in the text. (B) Luciferase activity detected in U2OS cells. Day 3 luciferase activity for both E2-WT and E2-S23A was statistically identical . For ptk6E2-luc, at day 6, E2-WT retained around 85% of day 3 activity, while E2-S23A retained around 15%. At day 9, E2-WT retained around 15% of day 3 activity, while most activity was lost with E2-S23A. For pSV40-luc, activity was lost rapidly in both E2-WT and E2-S23A cells. Asterisk (*) indicates a significant difference between this sample and the others on days 6 and 9, P < 0.05. Standard error bars are too small to appear on the log scale; results represent the summary of three independent experiments.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay: one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in the manuscript), which has no E2 DNA-binding sites; the other with the HSV1 tk promoter driving expression of luciferase with 6 E2 target sites upstream (see results for details).

Techniques: Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis

doi: 10.1128/jvi.00830-22

Figure Lengend Snippet: Average luciferase activity on the indicated days from three independent experiments (used to generate Fig. 3B ) ^a

Article Snippet: Two luciferase reporter plasmids were used for our novel assay: one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in the manuscript), which has no E2 DNA-binding sites; the other with the HSV1 tk promoter driving expression of luciferase with 6 E2 target sites upstream (see results for details).

Techniques: Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis

doi: 10.1128/jvi.00830-22

Figure Lengend Snippet: Fold change relative to E2-WT at day 3 = 1 from the novel quantitative luciferase-based assay (represented in Fig. 3B ) ^a

Article Snippet: Two luciferase reporter plasmids were used for our novel assay: one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in the manuscript), which has no E2 DNA-binding sites; the other with the HSV1 tk promoter driving expression of luciferase with 6 E2 target sites upstream (see results for details).

Techniques: Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis

doi: 10.1128/jvi.00830-22

Figure Lengend Snippet: E2 plasmid segregation/retention function is dependent on TopBP1 in U2OS cells. (A) Small interfering RNA (siRNA) knockdown of TopBP1 (bottom panels) removes the interaction of E2-WT with mitotic chromatin. (B) U2OS cells were treated with the indicated siRNAs and their growth measured over the indicated time period. (C) siRNA was added to cells on day 3 of our luciferase based plasmid retention/segregation assay, as described in . Protein was prepared from cells 6 and 9 days following transfection, and Western blotting was performed for the indicated proteins. TopBP1 siRNA knocked down TopBP1 expression, which persisted from day 6 until day 9. The knockdown was quantitated by averaging out the TopBP1 levels in the three different siRNA treatment sets (bottom panel). (D) Knockdown of TopBP1 expression abolishes the ability of E2-WT to retain ptk6E2-luc. Asterisk (*) indicates a significant difference between this sample and the others on days 6 and 9, P < 0.05. Standard error bars are too small to show up on the log scale. (E and F) An alternative TopBP1 siRNA was used to those shown in in panels C and D, and these duplicate experiments demonstrate that the effects of the TopBP1 siRNA were not due to off-target effects.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay: one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in the manuscript), which has no E2 DNA-binding sites; the other with the HSV1 tk promoter driving expression of luciferase with 6 E2 target sites upstream (see results for details).

Techniques: Plasmid Preparation, Small Interfering RNA, Knockdown, Luciferase, Transfection, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis

doi: 10.1128/jvi.00830-22

Figure Lengend Snippet: Knockdown of CK2 components disrupts E2 segregation/retention function in U2OS cells. (A and B) Indicated siRNA was added to cells on day 3 of our luciferase-based plasmid retention/segregation assay, as described in . Protein was then prepared from cells 6 and 9 days following transfection, and Western blotting was carried out for the indicated proteins. The knockdown was quantitated by averaging out the TopBP1 levels in the three different siRNA treatment sets (bottom panels). (C) Knockdown of CK2 components compromises the ability of E2-WT to retain ptk6E2-luc. Asterisk (*) indicates a significant difference between this sample and the others on days 6 and 9, P < 0.05. Standard error bars are too small to show up on the log scale; results represent the summary of three independent experiments.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay: one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in the manuscript), which has no E2 DNA-binding sites; the other with the HSV1 tk promoter driving expression of luciferase with 6 E2 target sites upstream (see results for details).

Techniques: Knockdown, Luciferase, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Interaction with TopBP1 Is Required for Human Papillomavirus 16 E2 Plasmid Segregation/Retention Function during Mitosis

doi: 10.1128/jvi.00830-22

Figure Lengend Snippet: TopBP1 interaction regulates E2 plasmid segregation/retention function and mitotic expression in human keratinocyte cells. (A) Luciferase activity was detected in the indicated N/Tert-1 cells. Day 3 luciferase activity for both E2-WT and E2-S23A was statistically identical . For ptk6E2-luc, on day 6, E2-WT retained around 90% of day 3 activity while E2-S23A retained around 15%. On day 9, E2-WT retained around 20% of day 3 activity while most activity was lost with E2-S23A. For pSV40-luc, activity was lost rapidly in both E2-WT and E2-S23A cells. Asterisk (*) indicates a significant difference between this sample and the others at days 6 and 9, P < 0.05. Standard error bars are too small to show up on the log scale; results represent the summary of three independent experiments. (B) N/Tert-1 were double thymidine blocked and released at the indicated time points. Harvested proteins were then Western blotted for the indicated proteins, and the results demonstrate a peak of E2 and TopBP1 expression at the 16-h release time point. This corresponds with the peak in expression of the mitotic marker Cyclin B1, indicating that, 16 h following double thymidine block release, there is a significant enrichment of mitotic cells. Results are from two different Western blots from the same extracts, and two GAPDH (glyceraldehyde 3-phosphate dehydrogenase) controls are therefore included. Separate blots are indicated by the brackets to the right-hand side of the figure. (C) N/Tert-1 cells were double thymidine blocked and released for 16 h when the cells are in mitosis (B). Harvested proteins were then Western blotted for the indicated proteins, and the results demonstrate increased expression of E2-WT and TopBP1 during mitosis, but neither E2-S23A nor TopBP1 in the E2-S23A cells. This was repeated and quantitated, demonstrating statistically significant increases in both E2 and TopBP1 in the E2-WT cells compared with that in E2-S23A. (D) Human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16) were double thymidine blocked and released at the indicated time points. Protein extracts were prepared and the indicated Western blottings carried out. Cyclin B indicates G 2 /M phase. Results demonstrate significant increases in both E2 and TopBP1 corresponding with the peak in Cyclin B1 expression (19 h), demonstrating that E2 and TopBP1 levels increase during mitosis in HPV16-positive cells. The experiment was repeated and the duplicates at 19 h post-double thymidine block release demonstrate significant increases in E2 and TopBP1 during mitosis. (E) As reported previously, E2-WT, but not E2-S23A, protein levels are increased in mitotic U2OS cells . We harvested RNA from repeat experiments at the indicated time points (lower panel) and demonstrated that there was no significant difference between E2-WT and E2-S23A RNA levels during mitosis. Therefore, any changes in E2 protein levels are due to post-transcriptional events.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay: one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in the manuscript), which has no E2 DNA-binding sites; the other with the HSV1 tk promoter driving expression of luciferase with 6 E2 target sites upstream (see results for details).

Techniques: Plasmid Preparation, Expressing, Luciferase, Activity Assay, Western Blot, Marker, Blocking Assay

Journal: bioRxiv

Article Title: Interaction with TopBP1 mediates human papillomavirus 16 E2 plasmid segregation/retention function and stability during the viral life cycle

doi: 10.1101/2022.01.28.478274

Figure Lengend Snippet: A. This figure summarizes our luciferase-based segregation/retention assay summarized in the text. B. Luciferase activity detected in U2OS cells. The day 3 luciferase activity for both E2-WT and E2-S23A was statistically identical . For ptk6E2-luc, at day 6, E2-WT retained around 85% of day 3 activity while E2-S23A retained around 15%. At day 9, E2-WT retained around 15% of day 3 activity while most activity was lost with E2-S23A. For pSV40-luc, activity was lost rapidly in both E2-WT and E2-S23A cells. * indicates a significant difference between this sample and the others at days 6 and 9, p-value<0.05. The standard error bars are too small to show up on the log scale, the experiments represent the summary of three independent experiments.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay; one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in this manuscript) which has no E2 DNA binding sites, the other with the HSV1 tk promoter driving expression of luciferase with 6-E2 target sites upstream; see results for details.

Techniques: Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Interaction with TopBP1 mediates human papillomavirus 16 E2 plasmid segregation/retention function and stability during the viral life cycle

doi: 10.1101/2022.01.28.478274

Figure Lengend Snippet: A. siRNA knockdown of TopBP1 (bottom panels) removes the interaction of E2-WT with mitotic chromatin. B. siRNA was added to cells at day-3 of our luciferase-based plasmid retention/segregation assay described in . Protein was then prepared from cells 6 days and 9 days following transfection and western blotting for the indicated proteins carried out. The TopBP1 siRNA knocked down TopBP1 expression that persisted from day 6 until day 9. C. The knockdown of TopBP1 expression abolishes the ability of E2-WT to retain ptk6E2-luc. * indicates a significant difference between this sample and the others at days 6 and 9, p-value<0.05. The standard error bars are too small to show up on the log scale, the experiments represent the summary of three independent experiments.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay; one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in this manuscript) which has no E2 DNA binding sites, the other with the HSV1 tk promoter driving expression of luciferase with 6-E2 target sites upstream; see results for details.

Techniques: Knockdown, Luciferase, Plasmid Preparation, Transfection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Interaction with TopBP1 mediates human papillomavirus 16 E2 plasmid segregation/retention function and stability during the viral life cycle

doi: 10.1101/2022.01.28.478274

Figure Lengend Snippet: A and B. Indicated siRNA was added to cells at day-3 of our luciferase-based plasmid retention/segregation assay described in . Protein was then prepared from cells 6 days and 9 days following transfection and western blotting for the indicated proteins carried out. C. The knockdown of CK2 components compromises the ability of E2-WT to retain ptk6E2-luc. * indicates a significant difference between this sample and the others at days 6 and 9, p-value<0.05. The standard error bars are too small to show up on the log scale, the experiments represent the summary of three independent experiments.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay; one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in this manuscript) which has no E2 DNA binding sites, the other with the HSV1 tk promoter driving expression of luciferase with 6-E2 target sites upstream; see results for details.

Techniques: Luciferase, Plasmid Preparation, Transfection, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Interaction with TopBP1 mediates human papillomavirus 16 E2 plasmid segregation/retention function and stability during the viral life cycle

doi: 10.1101/2022.01.28.478274

Figure Lengend Snippet: A. Luciferase activity detected in N/Tert-1 cells. The day 3 luciferase activity for both E2-WT and E2-S23A was statistically identical . For ptk6E2-luc, at day 6, E2-WT retained around 90% of day 3 activity while E2-S23A retained around 15%. At day 9, E2-WT retained around 20% of day 3 activity while most activity was lost with E2-S23A. For pSV40-luc, activity was lost rapidly in both E2-WT and E2-S23A cells. B. N/Tert-1 (top panel) or U2OS (lower panel) were double thymidine blocked and released for the indicated time points where cells have progressed into mitosis . Harvested proteins were then western blotted for the indicated proteins and the results demonstrate an increase in E2-WT, but not E2-S23A, protein expression in mitosis. In A, * indicates a significant difference between this sample and the others at days 6 and 9, p-value<0.05. The standard error bars are too small to show up on the log scale, the experiments represent the summary of three independent experiments.

Article Snippet: Two luciferase reporter plasmids were used for our novel assay; one containing the SV40 promoter and enhancer (pGL3 Control, Promega, described as pSV40-luc in this manuscript) which has no E2 DNA binding sites, the other with the HSV1 tk promoter driving expression of luciferase with 6-E2 target sites upstream; see results for details.

Techniques: Luciferase, Activity Assay, Western Blot, Expressing