Human Papillomavirus E6 Proteins Mediate Resistance to Interferon-Induced Growth Arrest through Inhibition of p53 Acetylation^▿

Cell culture.

Normal human foreskin keratinocytes (NHKs) were isolated from neonatal human foreskin epithelium as described previously and were cultured in keratinocyte basal medium supplemented with hydrocortisone, bovine pituitary extract, insulin, and epidermal growth factor (Lonza, Chicago, IL). Retroviral transductants were cultured with mitomycin C-treated NIH 3T3 J2 fibroblast feeders in E medium plus mouse epidermal growth factor (5 ng/ml; Collaborative Biomedical Products, Bedford, MA). Prior to the harvesting of cells for Western analyses, J2 3T3 fibroblast feeders were removed via treatment with 0.5 mM EDTA in phosphate-buffered saline (PBS).

Creation and mutagenesis of retroviral constructs.

The LXSN, 16E6, 16E7, and 16E6E7 retroviral constructs have been described previously (17). For mutagenesis of LXSN plasmids, the Stratagene (IDT, Coralville, IA) QuikChange XL kit was used with appropriate primers in accordance with the manufacturer's specifications.

Creation of retroviral cell lines.

PT67 cells (Clontech, Mountain View, CA) were transfected with previously described LXSN retroviral constructs by using FuGENE transfection reagent (Roche, Indianapolis, IN). Stable, virus-producing cells were selected by using 1,000 μg/ml G418 for 4 days, followed by an additional 4 days of selection with 500 μg/ml G418. Supernatants from cells were harvested, filtered, and used to infect NHKs for 6 h in the presence of 8 μg/ml Polybrene (Sigma, St. Louis, MO). Infected cells underwent antibiotic selection to generate stable cell lines as described previously (18).

Western blot analysis.

To isolate whole cell extracts, subconfluent cells were first treated with 0.5 mM EDTA in PBS to remove fibroblast feeders, trypsinized, and then pelleted by centrifugation at 1,200 rpm for 4 min. Pellets were washed with PBS, pelleted, and resuspended in 300 μl of RIPA buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 5 mM EDTA, 1% Triton X-100, protease inhibitors [Complete Mini; Roche], 100 μM phenylmethylsulfonyl fluoride). Samples were placed in ice for 15 min and then centrifuged at 16,100 × g for 5 min at 4°C. Supernatants were recovered and stored at −80°C. Protein was quantified against bovine serum albumin standards by using the Bradford method (Biotin Protein Assay Reagent; Bio-Rad, Hercules, CA) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described (18). Western analysis was conducted with anti-p53 (Oncogene, La Jolla, CA), anti-Rb (Santa Cruz, Santa Cruz, CA), anti-glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, MA), and anti-acetylated p53 (K305 or K382) (Biovision, Mountain View, CA) antibodies.

Interferon sensitivity assay.

Prior to plating for the interferon sensitivity assay, cells were trypsinized and stained with trypan blue and viable cells were counted with a hemocytometer. Fifty thousand viable cells were plated on J2 fibroblast feeders in each well of a six-well plate in E medium with three wells designated as controls and three designated for interferon treatment. On days 0, 2, and 4, the medium was changed to fresh with or without 1,000 IU/ml human recombinant beta interferon (EMD Biosciences, San Diego, CA). On the second day after plating, fresh J2 fibroblast feeders were added to each well. On day 5, cells were trypsinized after treatment with 0.5 mM EDTA in PBS and 2 aliquots from each well were trypan blue stained and counted on a hemocytometer, for a total of six counts each of untreated and interferon-treated cells. The highest and lowest values for each group were excluded, and the average number of interferon-treated cells was determined. For experiments extending to 12 days, 100,000 cells were initially plated in 10-cm dishes, fresh J2 cells were added, and the medium was replaced every 3 days.

Ras-induced senescence of IMR90 human fibroblasts.

IMR90 human fibroblasts were first infected with retroviruses expressing wild-type or mutant E6 as described above to obtain stable cell lines. Ras-expressing retroviruses were obtained after transfection of PT67 cells with the vector control pBabe or H-rasV12 (pBabe-ras). Stable, virus-producing cells were selected, and supernatants were harvested after 72 h in 5 ml of medium. The virus-containing medium was filtered, supplemented with 8 μg/ml Polybrene, and used for infections. IMR90 cells expressing E6 and mutant forms of E6 were counted, plated at equal densities, and incubated overnight. For infections, the culture medium was replaced with the pBabe vector or Ras-containing supernatant and then incubated overnight at 37°C. Following infection, cells were expanded and selected for 2 days with 1 μg/ml puromycin. To screen for senescence-associated β-galactosidase activity at 5 days following selection, cells were washed three times with PBS, fixed with 3% formaldehyde, and then washed again with PBS. Cells were then incubated at 37°C with fresh SA-β-gal stain solution for 2 to 16 h. SA-β-gal solution (pH 6.0) contained 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal), 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄Fe(CN)₆, 2 mM MgCl₂, 150 mM NaCl, 40 mM citric acid, Na₂HPO₄ for pH, and PBS up to 20 ml (2, 33).

Cells expressing HPV type 16 (HPV-16) E7 exhibit increased sensitivity to interferon-induced growth inhibition.

In order to investigate how HPV oncoproteins modulate the cellular response to interferon, we generated a series of five independently derived matched sets of keratinocyte lines in which the HPV-16 E6 and E7 proteins were expressed either individually or in combination. NHKs were isolated from neonatal foreskins and infected with retroviruses expressing HPV-16 E6, E7, or both E6 and E7. Following selection, colonies were expanded and pooled. Cells were then treated with beta interferon for periods of up to 12 days. At various times after initiation of treatment, cells were harvested and stained with trypan blue to identify viable cells. A representative analysis of one matched set of cells expressing HPV proteins is shown in Fig. 1A and B. Similar effects were seen in all five sets of cells treated with interferon. We observed differences in the number of viable cells as early as day 3 of treatment with beta interferon, and by 5 days significant reductions in cell numbers, as well as growth rates, were observed (Fig. 1C). After 5 days, cells expressing E7 alone and treated with interferon exhibited little to no proliferation compared to E6- and E6-E7-expressing cells, which continued to divide (Fig. 1C). Untreated, E7-expressing cells showed growth rates similar to those of untreated cells expressing E6 alone but slightly reduced from those of cells expressing the combination. Similar effects were seen with all five sets of keratinocytes expressing HPV proteins. After 12 days of interferon treatment, cells expressing E7 alone were completely growth arrested while cells expressing E6 alone or both E6 and E7 continued to proliferate (Fig. 1A and B). Identical effects were obtained with cells infected with retroviruses that contained both E6 and E7 but had a translation termination mutation in E6 (data not shown).

The morphology of the cells expressing E7 alone changed following treatment with interferon, while no significant alterations were seen in LXSN vector-infected control cells or cells expressing E6 alone or in combination with E7 (Fig. 2A). By phase-contrast microscopy, we observed that vector control cells treated with interferon resembled untreated cells. Similarly, no dramatic changes in cell morphology were observed in interferon-treated cells expressing E6 alone or E6 together with E7. In contrast, cells expressing E7 alone formed colonies of large flat cells whose morphology resembled that observed in cells undergoing senescence. By 12 days of interferon treatment, cells expressing E7, but not E6 and E7, became positive for acidic β-galactosidase (SA-β-gal), indicating that the cells were undergoing senescence (Fig. 2B). No blebbing of cytoplasmic membranes or nuclear condensation was seen, indicating that the cells were not undergoing apoptosis (data not shown). Taken together, these results suggest that the presence of E7 leads to increased sensitivity to interferon-induced growth arrest and senescence. Importantly, the coexpression of E6 together with E7 abrogates these effects.

Inhibition of p53 pathway engagement correlates with sustained proliferation in interferon-treated cells.

Expression of high-risk E7 proteins in the absence of E6 results in increased levels of p53, as well as enhanced expression of downstream targets such as p21. As p53 has been implicated as a regulator of the type I interferon response (35), we next wanted to investigate whether it contributed to the response of HPV-positive cells to interferon. We therefore examined p53 protein levels in cells expressing E6, E7, or E6-E7 following treatment with beta interferon. As shown in Fig. 3A, cells expressing E7 alone contained levels of p53 higher than those observed in E6- or E6-E7-expressing cells or in vector controls. Following treatment with interferon, little or no change in p53 levels was observed in E6-, E7-, or E6-E7-expressing cells or in vector controls. Consistent with the known downstream targets of p53, the levels of the p53-regulated protein p21 were increased in E7-expressing cells following treatment with interferon but not when E6 was also present (Fig. 3B). Both hypo- and hyperphosphorylated forms of Rb were detected in both E7- and E6-E7-expressing cells following interferon treatment. The ratio of hyper- to hypophosphorylated forms changed slightly in E6-E7-positive cells following interferon treatment, while no such changes were seen in cells expressing E7 alone. Similar effects were seen in three independently derived cell lines. These observations are consistent with the hypothesis that high levels of p53 in E7-expressing cells may be important for the growth-repressive effects of beta interferon.

E6 binding of p53 and CBP/p300, but not p53 degradation, are essential to overcome interferon-mediated growth inhibition.

The studies described above suggested a correlation between increased levels of p53 and sensitivity of E7-expressing cells to interferon. This would be consistent with the increase in proliferation that was observed when E6 was coexpressed with E7 as it targets p53. We next investigated which activities of E6 were responsible for overcoming the growth inhibition of E7-expressing cells. For these studies, we analyzed a series of retrovirally transduced NHKs that expressed several previously characterized mutant E6 proteins in combination with wild-type E7 (Fig. 3C). We first analyzed two mutations that inhibit the ability of E6 to degrade p53. The Δ118-122 and I128T mutant E6 proteins block the increased turnover of p53 mediated through E6 binding to E6AP, resulting in higher steady-state levels of p53. NHKs were infected with retroviruses expressing these two mutant E6 proteins in combination with wild-type E7, and cell lines were isolated following drug selection. Surprisingly, no significant differences in growth were seen following interferon treatment in cells expressing either of the two mutant E6 proteins in combination with E7 compared to cells expressing wild-type E6-E7 (Fig. 3E). After 5 days of treatment, the number of wild-type E6-E7-positive cells was similar to that of cells expressing either the Δ118-122 or I128T mutant form of E6 in combination with E7. Examination of the p53 levels by Western blot analysis demonstrated elevated amounts in cells that expressed mutant E6 proteins compared to those seen in cells expressing wild-type E6 or E6-E7, although they did not reach the levels seen in cells expressing E7 alone (Fig. 3D). These results suggest that the absolute levels of p53 are not a major determinant of sensitivity to interferon.

A second activity of E6 that is directed against p53 function is the inhibition of acetylation through the binding of the histone acetyltransferases CBP/p300 (29, 37). We therefore examined the effects of E6-mediated interaction with these proteins in modulating the interferon-induced growth inhibition of E7-expressing cells (Fig. 3C and D). E6 forms a trimeric complex with p53 and p300/CBP. The abrogation of the binding of E6 to p300 or loss of E6 binding to p53 allows the acetylation of p53. The G134V mutation in HPV-16 E6 abrogates the ability to bind CBP/p300, thereby allowing p53 to be acetylated, but does not alter its ability to bind and degrade p53 (29, 37). Furthermore, the L37S mutation abolishes the ability of E6 to bind p53, thus blocking its increased rate of degradation, as well as the inhibition of acetylation (24). Cell lines were generated following infection with retroviruses expressing these mutant E6 proteins in combination with wild-type E7 and monitored for growth in the presence of interferon. Interestingly, neither the L37S nor the G134V mutant E6 protein was able to overcome growth arrest following treatment with interferon (Fig. 3F). After 5 days of treatment, the number of viable cells infected with either mutant was comparable to that of cells expressing E7 alone, suggesting that the ability of E6 to bind both p53 and CBP/p300 is necessary to overcome interferon-mediated growth inhibition. As expected, the levels of p53 in the G134V mutant were similar to those of E6-expressing cells while in the L37S cells the levels were similar to those seen in LXSN-infected control cells (Fig. 3D).

Growth inhibition corresponds to p53 acetylation at lysine 382.

We next wanted to investigate the acetylation status of p53 in cells expressing the various HPV oncoproteins. We first examined keratinocytes expressing wild-type E6, E7, and E6-E7 for the levels of p53 acetylated at two sites targeted by CBP/p300. For these analyses, we used antibodies specific for acetylation of p53 at lysine 305 or lysine 382 (Fig. 4A). No differences in the levels of p53 acetylated at lysine 305 were found in cells expressing any of combination of the viral proteins compared to LXSN-infected controls. Furthermore, levels of p53 acetylated at lysine 305 were not altered by treatment with interferon in either control or HPV-expressing cells. In contrast, high levels of acetylation of p53 at lysine 382 were observed in cells expressing E7 alone but not in control cells or cells expressing E6 or E6-E7. This acetylated form of p53 was seen in E7-expressing cells in the absence of interferon, and the levels were not altered following addition of interferon.

Similar analyses of the levels of p53 acetylation at lysine 382 were next performed by using extracts of cells expressing mutant forms of E6 in the presence of wild-type E7 (Fig. 4B). In cells expressing either the Δ118-122 deletion or the I128T mutant form, the levels of p53 acetylation at lysine 382 were similar to those seen in cells expressing wild-type E6 or E6-E7. In contrast, cells containing the L37S or the G134V mutant form of E6 exhibited levels of p53 acetylation at lysine 382 significantly higher than those seen in wild-type E6-E7-expressing cells and nearly identical to levels observed in cells expressing E7 alone. Our results indicate that inhibition of p53 acetylation at lysine 382 closely correlates with the ability of E6 to overcome interferon-mediated growth inhibition in the presence of the E7 protein.

To investigate if acetylation of p53 at lysine 382 was necessary for the induction of other forms of growth arrest leading to senescence, we used a well-characterized method by which growth arrest, and eventually senescence, is induced through the overexpression of Ras in human fibroblasts (2, 33). The ability of E6 to overcome Ras-mediated senescence of human fibroblasts has previously been reported, but the mechanisms responsible have not been characterized (25, 26). To determine which activities of E6 were important in this system, we first infected IMR90 human embryonic lung fibroblasts with control retroviruses or E6-expressing retroviruses and selected stable cell lines. These cell lines were then superinfected with Ras-expressing retroviruses and after 5 days, screened for the number of viable cells. In addition, we examined the cells for expression of SA-β-gal to assay for senescence. Consistent with previous studies, we observed that high-level expression of Ras in IMR90 cells inhibited cell growth, caused flattening of cells, and induced expression of SA-β-gal (Fig. 5A). In contrast, cells expressing both wild-type E6 and Ras continued to proliferate, exhibited a small-cell phenotype, and were not positive for β-galactosidase (Fig. 5A). We next repeated these experiments with the panel of mutant forms of E6 described above and observed that cells expressing the mutant forms of E6 that were unable to degrade p53 were not able to overcome Ras-induced growth arrest (Fig. 5B). In addition, the mutant forms of E6 that failed to block acetylation of p53 (G134V) in keratinocytes were still able to overcome Ras-mediated growth arrest, although at a reduced rate compared to wild-type E6 (Fig. 5B). The L37S mutant that does not bind p53 was also not able to overcome Ras-mediated arrest. We confirmed the presence of E6 in these cells by the ability to reduce p53 levels (data not shown). Our data suggest that blocking the ability to bind p53 or inhibiting its rapid turnover is important for overcoming Ras-induced growth arrest and senescence in fibroblasts. In contrast, E6-mediated effects on p53 acetylation were not essential for blocking senescence induced in this system. Consistent with this observation, we failed to observe acetylation of p53 in Ras-expressing IMR90 cells with or without the presence of E6 or mutant forms of E6 (Fig. 5D). We conclude that the ability of E6 to block p53 acetylation through p300/CBP binding is important in overcoming interferon-induced senescence but not other types of senescence.