Activation of NF-κB by human papillomavirus 16 E1 limits E1-dependent viral replication through degradation of E1

Abstract

NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop.

Importance: A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent genome replication of HPV16. These results suggest that NF-κB mediates a negative feedback loop to regulate HPV replication and that this feedback loop could be associated with control of the viral copy numbers. We could thus show for the first time that NF-κB activity is involved in the establishment and maintenance of persistent HPV infection.

FIG 1

Conditional induction of E1-dependent replication in immortalized human cervical keratinocytes containing HPV16 genome (HCK1T-HPV16). (A) Representative image of Southern blotting for HPV16 genomes in 10 μg of total genomic DNA isolated from cells with the indicated gene expression at 24 h after DOX (1-μg/ml) addition. The serially diluted, linearized full lengths of HPV16 genomes were included as copy number standards (lanes 1 to 3). The DNAs with BamHI digestion, which linearizes the HPV16 genome, and XhoI digestion, which does not cut the HPV16 genome, are shown in lanes 4 to 7 and lanes 8 to 11, respectively. Arrowheads indicate HPV16 DNAs which correspond to Form I, a supercoiled DNA, and Form II, a relaxed circular DNA. (B and C) qPCR results for the copy number of HPV16 genomes with the indicated gene expression at 24 h (B) and at the indicated time points following DOX incubation (C). (D) Indirect immunofluorescence analysis (IFA) for HA-E1 and E2. As a negative control, HCK1T-HPV16 with tetON E1+E2 incubated with a vehicle control is shown. (E) The number of cells with the indicated gene expression that stayed attached to tissue culture plates is shown. (F) Western blot analysis for the levels of E1 and the markers of apoptosis, PARP-1 and caspase 3, in HCK1T-HPV16 cells with the indicated gene expression following incubation with DOX (lanes 2, 4, 6, and 8) or vehicle as a control (lanes 1, 3, 5, and 7). Arrowheads indicate cleaved forms of PARP-1 and caspase 3. α-Tubulin was detected as a loading control. The averages of results from at least three independent experiments are shown. P values were evaluated by Student's t test.

FIG 2

Activation of NF-κB upon induction of E1 and/or E2 expression in HCK1T-HPV16 cells. (A) The status of NF-κB p65 (RELA) and its inhibitor, IκBα, was analyzed by Western blotting for HCK1T-HPV16 cells with the indicated gene expression at 24 h after DOX addition. α-Tubulin was included as a loading control. (B) Localization of RELA and HA-E1 and/or E2 was examined in HCK1T-HPV16 tetON E1+E2 by indirect IFA. A rabbit polyclonal antibody to RELA and a mouse monoclonal antibody to HA were simultaneously incubated, and secondary antibodies, Alexa 488-conjugated anti-rabbit IgG and Alexa 596-conjugated anti-mouse IgG, respectively, were used for detection. Nucleus was counterstained with DAPI. (C) Relative mRNA levels of indicated NF-κB target genes and viral transcripts containing E1^E4 spliced ORF were measured by RT-qPCR. The mRNAs of the indicated genes were normalized to β-actin mRNA. Fold increases of the indicated mRNAs in DOX-treated samples over those in vehicle-treated samples are shown. The averages of at least three independent experiments are shown.

FIG 3

Activation of NF-κB upon induction of E1 and/or E2 expression in HCK1T in the absence of an episomal HPV16 genome. (A) The level of IκBα and expression of E1 and E2 were analyzed in parental HCK1T cells that do not contain an HPV16 genome by Western blotting. HCK1T-tetON cells were included as a negative control. Cell lysates were prepared at 24 h following incubation with DOX (lanes 2, 4, 6, and 8) or a vehicle (lanes 1, 3, 5, and 7). Vinculin was detected as a loading control. (B) qRT-PCR analysis for mRNA levels of the NF-κB target genes in HCK1T-tetON E1+E2 or E1 alone. The mRNAs of the indicated genes were normalized to β-actin mRNA. Fold increases of the indicated mRNAs in DOX-treated samples over those in vehicle-treated samples are shown. The averages of at least three independent experiments are shown. (C) RT-PCR analysis for mRNA levels of the NF-κB target genes in HCK1T, HCK1T-HPV16, and CIN612-9E cells. As a negative control, an RNA isolated from HCK1T not subjected to RT reaction was used as a template for PCRs (lane 1).

FIG 4

Effects of overexpression of IκBα on E1-dependent replication of HPV16 and cell proliferation of subclones of HCK1T-HPV16 cells with tetON E1+E2. (A) Shown are results of Western blot analysis (left) for E1 and E2 levels and the steady-state levels of IκBα in representative clones and the results of qPCR for HPV16 genomes (right) following incubation with DOX or vehicle for 24 h. Images with short and long exposures with the anti-HA antibody were included to indicate expression of HA-E2. α-Tubulin is shown as a loading control for the top three panels, and vinculin is shown for the HA panel comparing clones to the parental population. (B) Western blot analysis to verify overexpression of IκBα in clone B2 transduced with retroviruses carrying an empty vector designated MCS (lanes 3 and 4) or the vector expressing IκBα^S32/36A mutant (IκBαMT) (lanes 5 and 6) or wild-type IκBα (IκBαWT) (lanes 7 and 8). Parental HCK1T-HPV16 treated with TNF-α for 15 min was included as a positive control for degradation of IκBα (lanes 1 and 2). Vinculin was detected as a loading control. (C) RT-qPCR analysis of NF-κB target genes and viral early transcripts containing spliced E1^E4 ORF. Shown are levels of the indicated mRNAs relative to MCS control without DOX treatment. Two asterisks indicate a P value of <0.01 for the comparison of the mRNAs in MCS with those in IκBαWT- or IκBαMT-expressing cells in the absence of DOX. Differences for all of the mRNA levels between MCS and IκBαWT- or IκBαMT-expressing cells with DOX treatment were statistically significant (P < 0.01). The highlighting of statistical significance was omitted for DOX-treated samples to avoid making graphs too busy. (D) The copy number of HPV16 genomes was measured by qPCR with indicated gene expression. An asterisk indicates a P value of <0.05. (E) The number of cells staying attached to tissue culture plates is shown. An asterisk indicates a P value of <0.05. The averages of at least three independent experiments are shown.

FIG 5

Effects of inhibitors of DDR on activation of NF-κB, E1-dependent replication of HPV16 genomes, and cell proliferation. (A) Western blot analysis for activation of ATM- and ATR-mediated DDR pathways. The left panel (lanes 1 to 8) shows the results of original cell populations of HCK1T-HPV16 with the indicated gene expression. The right panel (lanes 9 to 18) shows the results for representative clones of HCK1T-HPV16 tetON E1+E2 cells. Vinculin or α-tubulin indicates a loading control in each panel. White arrowheads indicate a slower migration pattern of NBS1 and CHK2. (B) Time course experiment for DDR activation and the level of IκBα in the presence of DMSO as a control, 10 μM wortmannin, or 2.5 μM KU55933. (C) Transcriptional activation of NF-κB target genes and the viral early transcripts was examined by RT-qPCR in the presence or absence of the inhibitors incubated with or without DOX for 24 h. An asterisk indicates a P value of <0.05 for the comparison of the mRNAs between DMSO-treated and wortmannin-treated cells in the absence of DOX. The mRNAs of NF-κB target genes but not the viral early transcripts in wortmannin- or KU55933-treated cells compared to those in DMSO-treated cells in the presence of DOX were significantly reduced (P < 0.05). The highlighting of P values less than 0.05 was omitted for DOX-treated samples. (D) The copy number of HPV16 genomes was measured by qPCR at 24 h after DOX addition in the presence of the indicated inhibitors. (E) The number of cells staying attached to tissue culture plates in the presence or absence of inhibitors was counted. An asterisk indicates a P value of <0.05. (F) Relative mRNA levels of HA-E1 were analyzed by RT-qPCR in the presence or absence of wortmannin following incubation with DOX or vehicle for 24 h. (G) Western blot analysis for solubility of E1 in the presence of wortmannin or vehicle. S and I indicate soluble (lanes 1, 3, and 5) and insoluble (lanes 2, 4, and 6) fractions, respectively. The averages or representative images of at least three independent experiments are shown.

FIG 6

ATR, but not DNA-PKcs and p38MAPK, is involved in the E1-dependent reduction of IκBα. (A) Activation of p38MAPK was examined in HCK1T-HPV16 populations with the indicated gene expression. (B) Effects of wortmannin and KU55933 on activation of p38MAPK and DNA-PKcs in clone B2. Cell lysates were prepared at 0 and 24 h after DOX addition following preincubation with the inhibitors for 2 h. Vinculin was detected as a loading control. (C) Western blot analysis for the levels of IκBα and E1 at 24 h after incubation with DOX in the presence of indicated inhibitors. A bottom graph shows intensities of E1 with inhibitors relative to that with a vehicle control. The intensities of E1 were normalized to that of vinculin. Concentrations of inhibitors used were 10 μM wortmannin, 0.5 nM VE-822 (ATR inhibitor), 20 μM Nu7026 (DNA-PKcs inhibitor), 2.5 μM KU55933, and 10 μM SB203580 (p38MAPK inhibitor). (D) Western blot analysis for a time course experiment in the presence of wortmannin (lanes 5 to 8), VE-822 (lanes 9 to 12), or a vehicle control (lanes 1 to 4). (E) The copy number of HPV16 genomes was measured by qPCR. (F) The number of cells staying attached was examined after incubation with DOX or vehicle in the presence of DMSO or VE-822 for 24 h. An asterisk indicates a P value of <0.05. (G) Western blot analysis for the stability of E2 in the presence of indicated inhibitors as described in the Fig. 5 legend. The averages or representative images of at least three independent experiments are shown.

FIG 7

Activation of NF-κB mediates destabilization of E1 and promotes polyubiquitination. (A) Time course experiments for clone B2 transduced with MCS (lanes 1 to 4), IκBαWT (lanes 9 to 12), or IκBαMT (lanes 5 to 8) following incubation with DOX. A white arrowhead indicates a slower-migrating pattern of NBS1. Vinculin was detected as a loading control. (B) RT-qPCR analysis for HA-E1 in the indicated cells upon DOX incubation. (C) Western blot analysis for the levels of E1 and IκBα in clone B2 incubated with vehicle (lanes 1 and 2) or 10 nM epoxomicin (lanes 3 and 4) for 12 h. (D) A ubiquitin conjugation assay was performed as described in Materials and Methods. The left panel (lanes 8 to 13) shows results of Western blot (WB) analysis for input cell lysates subjected to immunoprecipitation (IP). The right panel (lanes 14 to 19) shows results of IP-WB to detect His-Ub conjugation to HA-E1. The cell lysates from 293FT cells transfected without a His-Ub expression vector (lanes 1 to 6) were also included. Wor and TNF indicate treatment with 10 μM wortmannin and 20 ng/ml of TNF-α, respectively, from 20 h posttransfection. IkB indicates cotransfection with an expression vector of IκBαMT.

FIG 8

E1 and E2 expression induces accumulation of cells in S and G₂ phases. Clone B2 was collected at indicated time points after a vehicle (above) or DOX (bottom) addition, and cell cycle profiles were analyzed by flow cytometry. Representative profiles are shown. The graphs indicate the percentages of cells in the cell cycle phases.

FIG 9

Effect of constitutive activation of NF-κB in maintenance replication of HPV genome. (A) The copy number of HPV16 genomes in HCK1T-HPV16 transduced with retrovirus carrying an empty (MCS) or IκBαMT-expressing vector was measured at 5 days after drug selection was completed. (B) The copy number of HPV16 or HPV31 was measured at 5 days after incubation of TNF-α at indicated concentrations (black bars). The cell proliferation levels were also compared (white bars). (C) Schematic presentation for a negative feedback loop between E1 and NF-κB. E1 induces activation of NF-κB most likely through degradation of IκBα as a consequence of DDR activation. NF-κB activation in turn limits E1-dependent replication and disrupts the stability of E1 through facilitating proteasomal degradation.