Nuclear accumulation of the papillomavirus E1 helicase blocks S-phase progression and triggers an ATM-dependent DNA damage response

Abstract

Replication of the papillomavirus genome is initiated by the assembly of a complex between the viral E1 and E2 proteins at the origin. The E1 helicase is comprised of a C-terminal ATPase/helicase domain, a central domain that binds to the origin, and an N-terminal regulatory region that contains nuclear import and export signals mediating its nucleocytoplasmic shuttling. We previously reported that nuclear accumulation of E1 has a deleterious effect on cellular proliferation which can be prevented by its nuclear export. Here we have shown that nuclear accumulation of E1 from different papillomavirus types blocks cell cycle progression in early S phase and triggers the activation of a DNA damage response (DDR) and of the ATM pathway in a manner that requires both the origin-binding and ATPase activities of E1. Complex formation with E2 reduces the ability of E1 to induce a DDR but does not prevent cell cycle arrest. Transient viral DNA replication still occurs in S-phase-arrested cells but surprisingly is neither affected by nor dependent on induction of a DDR and of the ATM kinase. Finally, we provide evidence that a DDR is also induced in human papillomavirus type 31 (HPV31)-immortalized keratinocytes expressing a mutant E1 protein defective for nuclear export. We propose that nuclear export of E1 prevents cell cycle arrest and the induction of a DDR during the episomal maintenance phase of the viral life cycle and that complex formation with E2 further safeguards undifferentiated cells from undergoing a DDR when E1 is in the nucleus.

Fig. 1.

E1 proteins from different papillomavirus types inhibit cellular proliferation. (A) Colony formation assays (CFA). C33A cells were transfected with the indicated GFP-E1 expression vector (also containing a Zeocin resistance gene). Following 4-weeks' selection in bleomycin, colonies were stained with methylene blue. (B) Cell cycle analysis. C33A cells transiently expressing the indicated GFP-E1 or GFP were trypsinized 48 h posttransfection, and their DNA was stained with Hoechst stain and analyzed by flow cytometry. For each condition, the cells were either grown asynchronously (Asynchr.) or synchronized with mimosine for 24 h and then released in nocodazole for an additional 24 h (Released in Nocodazole). The cell cycle profile of each sample is shown as a histogram and quantified in Tables 1 and 2. (C) Graphical representation of the cell cycle distributions shown in panel B. ES, MS, and LS stand for early, mid-, and late S phase, respectively. (D) Western blot analysis of total protein extracts prepared from transfected C33A cells expressing the GFP-E1 proteins of the various papillomaviruses used in this study. E1 proteins were detected using an anti-GFP antibody, and tubulin (Tub.) was used as a loading control.

Fig. 2.

The OBD and ATPase/helicase domain of E1 are both required for its antiproliferative activity. (A) Schematic representation of HPV31 E1 truncations used in this analysis, highlighting the location of the p80-binding site (aa 10 to 40), the caspase-3/7 cleavage site, the shuttling module (aa 85 to 125), the origin-binding domain (OBD), and the ATPase/helicase domain. E1 fragments lacking the shuttling module were fused to the SV40 large T antigen nuclear localization signal (NLS). (B) Colony formation assay and cell cycle analysis. CFA and cell cycle analysis were done essentially as described in the legend for Fig. 1 with the exception that cells were selected in G418-containing medium for 3 weeks before staining the colonies with methylene blue. (C) Transient DNA replication activities of E1 truncations. The DNA replication activity of each E1 fragment was determined using 1.25, 2.5, and 5 ng of expression vector 72 h posttransfection. Replication activities are reported as a percentage of the Fluc/Rluc ratio obtained with the 5 ng of the 1–629 E1 expression vector. Error bars represent standard deviations. Cells transfected with EYFP or EYFP-NLS only were used as negative controls.

Fig. 3.

Amino acid substitutions in the origin-binding and ATPase domains of E1 abolish its antiproliferative activity. (A and B) CFAs (A) or cell cycle analysis (B) of cells transfected with EYFP alone or the indicated EYFP-E1 constructs carrying amino acid substitutions in the OBD, ATPase domain, and NES, as described in the text. For CFAs, cells were selected in G418-containing medium before staining. Cell cycle analyses were performed as described for Fig. 1. (C) Western blot analysis in which E1 proteins were detected using an anti-GFP antibody (α-GFP) and tubulin was used as a loading control (α-Tub.).

Fig. 4.

E1 inhibits cellular proliferation in p53-positive and -negative cell lines. CFAs and cell cycle distributions of EYFP- and EYFP-E1-transfected C33A, HeLa, or U2OS cells. CFA were performed in G418-containing medium. Cell cycle distributions were determined 24 h posttransfection using cells stained with propidium iodide. Results are represented as histograms here and are quantified in Table 3.

Fig. 5.

E1 arrests cell cycle progression in early S phase. (A) Cellular localization of RFP-PCNA in transfected cells expressing either the wild type or an origin-binding-defective E1 protein. Twenty-four hours posttransfection, cells were fixed, mounted, and visualized by ×40 fluorescence confocal microscopy. Nuclei (DNA) were stained with DAPI. Cells containing large PCNA foci (such as the one indicated by the white arrow) were quantified and are reported on the right side of the panel as a percentage of the total number of RFP-PCNA-expressing cells. (B) BrdU incorporation. Twenty-four, forty-eight, and seventy-two hours posttransfection, cells expressing EYFP or EYFP-E1 wild-type or NES/OBD or NES/ATPase mutant proteins were pulsed for 1 h with 10 μM BrdU. The percentage of cells that incorporated BrdU was then determined by flow cytometry and is reported in the histogram. (C) BrdU incorporation throughout the cell cycle. Cells expressing EYFP or EYFP-E1 wild type or the indicated mutant proteins were pulsed with BrdU 24 h posttransfection and analyzed as described for panel B. In addition, cells were stained with 7-AAD to measure their DNA content. For each sample, BrdU incorporation is represented as a function of DNA content (cell cycle distribution) in a scatter plot. Boxes indicate the populations of cells in G₁, S, and G₂. EYFP-transfected cells treated with hydroxyurea (HU) and aphidicolin (Aph) were used as controls.

Fig. 6.

Nuclear accumulation of enzymatically active E1 induces a DNA damage response. (A) The indicated EYFP and EYFP-E1 wild-type or mutant proteins were transiently expressed in C33A cells. Twenty-four hours posttransfection, cells were fixed and stained for phospho-serine 139 of γH2AX. Mounted cells were then visualized by ×20 confocal fluorescence microscopy. Mock-transfected cells and cells treated for 24 h with 50 μM etoposide (Eto.) were used as negative and positive controls, respectively. (B) Analysis similar to that for panel A but using the E1 fragments described in the legend for Fig. 2.

Fig. 7.

The ability of E1 to induce a DDR is a conserved feature of papillomavirus E1 and involves activation of the ATM pathway. (A) The ability of E1 to induce a DDR is conserved in HPV31, -16, and -11 and BPV E1. The activation of γH2AX was analyzed in C33A cells expressing the indicated GFP or GPF-E1 proteins. To analyze γH2AX activation, cells were fixed 24 h posttransfection, stained for phosphoserine 139 of γH2AX, mounted, and visualized by ×20 fluorescence confocal microscopy. (B) Expression of E1 over time correlates with activation of γH2AX. The EYFP-E1 wild type was transiently expressed in C33A cells. Six and twelve hours posttransfection, cells were analyzed as described for panel A. (C to E) E1 activates components of the ATM pathway. The indicated EYFP or EYFP-E1 wild-type or mutant proteins were transiently expressed in C33A cells. Twenty-four hours posttransfection, cells were fixed and stained for phosphoserine 1981 of ATM (pATM) (C), phosphothreonine 68 of Chk2 (pChk2) (D), or phosphoserine 345 of Chk1 (pChk1) (E). Mock-transfected cells were used as negative controls, and cells treated for 24 h with 50 μM etoposide (Eto.) or 2 mM hydroxyurea (HU) were used as positive controls.

Fig. 8.

γH2AX staining is punctuated in cells that accumulate low levels of E1 in their nucleus. The activation of γH2AX in cells transiently expressing EYFP-E1, either wild type (WT) or a cyclin-binding motif mutant derivative (CBM), was analyzed by immunofluorescence confocal microscopy 24 h posttransfection. Where indicated, the E1 CBM-expressing cells were treated with 7.5 ng/ml of leptomycin B (LMB) for 4 h. Detection of γH2AX was performed at a magnification of ×20 (A) or ×63 (B).

Fig. 9.

E2 and the viral origin attenuate the E1-induced DDR. γH2AX levels in cells transiently expressing wild-type EYFP-E1 or the NES/ATPase mutant derivative, with or without E2, and containing or lacking the viral origin, are indicated. γH2AX levels were quantified by flow cytometry. For each sample, γH2AX levels were determined as a function of EYFP-E1 expression (low-, medium-, or high-EYFP-E1-expressing cells; shaded histograms), and the non-EYFP-expressing cells of the same cell population were used as an internal control (open histograms). For each condition, the fold increase in γH2AX activation is indicated in the upper right corner and was obtained by dividing the average value of activation measured for the EYFP-E1-positive cells by that of the controls cells.

Fig. 10.

E2 and the viral origin modulate the E1-induced cell cycle arrest. BrdU incorporation in EYFP-E1-expressing cells throughout the cell cycle. (A) Scatter plots obtained with cells transfected with a wild-type E1 expression vector, either alone or together with two different amounts (1× and 8×) of wild-type or E39Q E2 expression vector and the origin, as indicated. BrdU incorporation and DNA content were determined by flow cytometry. (B) Proportions of cells in G₁, S, and G₂ were quantified from the samples shown in panel A and from those transfected with a plasmid expressing the EYFP-E1 NES/ATPase mutant protein as a control (data not shown). Gates showed for the mock-transfected sample were used to quantity the cell cycle distribution of each sample, and the results were reported in a histogram. Results obtained with cells expressing EYFP-E1 are shown in the upper panel, while those obtained with cells expressing the NES/ATPase mutant E1 are shown in the lower panel. The blue and red arrows highlight the concomitant changes in the proportions of cells in G₁ and S phase, respectively, brought about by increasing amounts of E2 with or without the origin. (C) CFA performed with 3F-HPV31 E2 or with 3F-HPV31 E1 expression vector. Cells were selected in G418-containing medium before staining.

Fig. 11.

Effect of increasing amounts of E2 and the origin on E1-catalyzed DNA replication. (A) Activation of γH2AX in cells transfected with an EYFP-E1 expression plasmid and two different amounts (1× and 8×) of wild-type E2 expression vector plus the origin. Twenty-four hours posttransfection, E1-expressing cells were fixed, stained for γH2AX, mounted, and visualized by ×40 fluorescence confocal microscopy. (B) Transient DNA replication activity of E1 in the presence of increasing amounts of E2 and the origin. DNA replication activity of EYFP-E1 was determined 24, 48, and 72 h posttransfection. Cells were transfected with 5 ng of EYFP-E1 expression plasmid and increasing amounts of wild-type E2 expression vector plus the origin (E2 WT), as indicated (1×, 2×, 4×, and 8×). Background signal was measured in the absence of E2 (5 ng of E1 and increasing amounts of ori-plasmid) (No E2) or in the absence of E1 (No E1). Replication activities are reported as Fluc/Rluc ratios, and error bars represent standard deviations. (C) Effect of the ATM kinase inhibitor KU-55933 on viral DNA replication. Transient HPV DNA replication assays were performed under standard conditions (1×) or in the presence of an excess of E2 and the origin (8×). KU-55933 (KU) was tested at increasing concentrations ranging from 0.156 to 10 μM. Gemcitabine (Gemci., from 1.56 to 100 nM) and 0.1% dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. DNA replication levels (Fluc/Rluc ratios) from which background signals were subtracted are reported as percentages of the activity measured in the presence of DMSO, which was set at 100%.

Fig. 12.

γH2AX activation in keratinocytes immortalized with a wild-type or E1 NES mutant genome. Early-passage keratinocytes immortalized with the indicated HPV31 genome were fixed and stained with an antibody against γH2AX. Nuclei (DNA) were stained with DAPI.