p53 promotes adenoviral replication and increases late viral gene expression


The tumor suppressor protein, p53, plays a critical role in viro-oncology. However, the role of p53 in adenoviral replication is still poorly understood. In this paper, we have explored further the effect of p53 on adenoviral replicative lysis. Using well-characterized cells expressing a functional p53 (A549, K1neo, RKO) and isogenic derivatives that do not (K1scx, RKOp53.13), we show that virus replication, late virus protein expression and both wtAd5 and ONYX-015 virus-induced cell death are impaired in cells deficient in functional p53. Conversely, by transfecting p53 into these and other cells (IIICF/c, HeLa), we increase late virus protein expression and virus yield. We also show, using reporter assays in IIICF/c, HeLa and K1scx cells, that p53 can cooperate with E1a to enhance transcription from the major late promoter of the virus. Late viral protein production is enhanced by exogenous p53. Taken together, our data suggest that functional p53 can promote the adenovirus (Ad) lytic cycle. These results have implications for the use of Ad mutants that are defective in p53 degradation, such as ONYX-015, as agents for the treatment of cancers.


The observation that adenovirus (Ad) E1b55kDa inactivates wild-type (wt) p53 function (Yew and Berk, 1992) was the basis for the development of the E1b55kDa deletion mutant ONYX-015 (originally designated dl1520 (Barker and Berk, 1987)), as a selective tumoricidal agent (Bischoff et al., 1996). It was hypothesized that viruses lacking the E1b55kDa would be prevented from replicating DNA due to p53 imposed cell stasis. In this way normal cells would be spared, but tumor cells with mutant p53 would not be able to protect themselves from viral infection and would eventually die from virus-induced cell lysis. Several studies have supported this theory, showing that ONYX-015 selectively killed tumor cells with a mutant p53 (Bischoff et al., 1996; Heise et al., 1997; Lee et al., 2000), but many other studies in vitro have suggested that ONYX-015 can replicate in, and kill, cells irrespective of their p53 status (e.g., Goodrum and Ornelles, 1998; Rothmann et al., 1998; Harada and Berk, 1999; Dix et al., 2000). To explain these differences, it was suggested that ONYX-015 might selectively target not just p53-deficient cells but also those defective in expression of the p14ARFgene (Ries et al., 2000) that functions as a positive regulator of p53. However, there is some doubt about this mechanism too (Edwards et al., 2002). Despite this, early clinical trials with ONYX-015 have looked promising, especially in combination with conventional pharmacological therapies (Nemunaitis et al., 2000, 2001; Rogulski et al., 2000), but the success is variable and the underlying mechanisms poorly understood. In particular, the precise role of p53 in Ad replication and tumor selectivity of ONYX-015 remains controversial. Factors that have been shown to impinge on this include tumor type, viral receptor expression and late viral export (Rauen et al., 2002; O'Shea et al., 2004).

Although we agree that ONYX-015 does not selectively target p53-deficient cells, we have reported that wt p53 might in fact promote virus-induced cell lysis (Hall et al., 1998; Dix et al., 2000) as we have often found that cells with wtp53 die more efficiently than those without. This is not to say that p53 is absolutely required for Ad to kill cells, but that it generally occurs more efficiently in cells with a functional p53 (Dix et al., 2000). A possible explanation for the apparent discrepancies is that not all cell lines genetically wt for p53 actually express a functionally wtp53 protein, due to defects in other cellular gene products as suggested above (Ries et al., 2000).

To test directly whether p53 can enhance Ad replication and cell death induction, we have carried out p53 transfection experiments and studied virus replication and death ability using a pair of well-characterized cell lines (K1neo and K1scx) derived from a thyroid papillary carcinoma (Wyllie et al., 1999). K1neo retains many features representative of these tumors, including a wt TP53 gene. The cells are nontumorigenic, contact inhibited and retain DNA damage-responsive G1/S and G2/M checkpoints, the former being p53 dependent. K1scx cells express the dominant-negative A143V human p53 mutant, but otherwise are an isogenic derivative. We show that Ad-induced cell death and virus replication occur more efficiently in the cells expressing a functional p53. We also show that p53 can increase virus yield and late virus gene expression, and, in conjunction with viral E1a products, stimulate the major late promoter (MLP).

The roles of E1b55kDa and p53 in regulating virus replication and virus-induced cell death are explored below.


Virus lacking E1b55kDa is attenuated for cell killing

Comparison of E1b55kDa defective viruses with wtAd5 shows them to be attenuated for cell death induction in most, although not all, cell types, despite expression of the principal apoptotic gene E1a (Bischoff et al., 1996; Goodrum and Ornelles, 1998 and reviewed in Dix et al., 2000). This is demonstrated in Figure 1a for the E1b55kDa defective virus, dl1520, which is unable to kill the wtp53-expressing A549 cells up to 4 days after infection, whereas wtAd5 reduces viability to about 10–20% of controls by this time, despite similar levels of the proapoptotic protein E1a as seen by Western blot (Figure 1b). The E1a defective virus dl312 is also unable to kill these cells, but complementation with dl1520 rescues the ability of both viruses to cause cell death. The levels of the Ad E2A DNA-Binding Protein (DBP), which promotes viral DNA replication, are comparable over the course of the infection studied, but with dl1520 slightly higher than wtAd5 at early times post-infection. Complementation of dl1520 with the E1a defective dl312 led to a similar expression of DBP over the 4 days. Collectively, these data suggest that E1b55kDa is likely to be important for efficient cell death induction, but is not essential for E1a early gene expression, nor for DBP expression.

Figure 1

Kinetics of cell death induced by wtAd5 and dl1520 in a human lung carcinoma cell line. A549 cells were infected with a total of 20 CPEU/cell of virus (in the case of dl1520 and dl312 complementation, 10 CPEU/cell of each virus was used). (a) Cell viabilities determined over time as described in Materials and methods. Viability experiments were performed at least three times. Error bars represent s.e.m. (b) Protein lysates were generated and analysed for E1a, DBP and β-actin expression levels using Western blotting.

The attenuated cell death that we observed in cells infected with dl1520 may be a direct effect of E1b55kDa as a death-inducing protein, or alternatively it could be because E1b55kDa promotes the virus lytic cycle. We therefore went on to investigate these two possibilities.

Ad-induced cell death is linked to the virus replication cycle

To determine whether E1b55kDa can function as a cell death-promoting protein, we transiently expressed E1b55kDa directly in various cell lines. We found no consistent evidence that E1b55kDa caused more cell death than vector alone (data not shown).

We then went on to test whether cell death is linked to virus replication using the temperature-sensitive (ts) virus ts125, which is unable to replicate its DNA at 39.5°C (Williams et al., 1974) due to a defect in the DBP (Kruijer et al., 1981), but is replication competent at the permissive temperature of 32.5°C. A549 cells were infected with wtAd5 or ts125 and incubated at either 39.5 or 32.5°C for 4 days and cell viability determined. Both viruses reduced cell viability to around 20% of control by 96 h after infection at the permissive temperature (Figure 2a). At the nonpermissive temperature (39.5°C) the ts125 was significantly impaired in its cytopathic effect (CPE), while cell viability was reduced to less than 20% of control in wtAd5-infected cells. Levels of viral DNA were determined by Southern blotting. At 39.5°C, little or no viral DNA was detected in ts125-infected cells, but at 32.5°C similar levels of viral DNA were detected to wtAd5 (data not shown), as shown previously (Williams et al., 1974; Braithwaite et al., 1981). Levels of E1a were not significantly different enough between wtAd5 and the ts mutant to explain the differences obtained in cell killing (Figure 2b). These data show that the attenuated cytopathogenicity of the ts mutant virus is not the consequence of a defect in expression of the proapoptotic E1a protein, but is instead linked to the impairment of viral DNA replication at the nonpermissive temperature. Thus, virus-induced cell death is linked to a successful viral replication cycle.

Figure 2

Cell death is linked to virus replication. A549 cells were infected with 20 CPEU/cell of either wtAd5 or ts125. After infection, cells were maintained at either 32.5 or 39.5°C and incubated for the indicated times. (a) Cells were harvested and cell viabilities determined. Error bars represent s.e.m. (b) Levels of E1a and β-actin protein expression were measured by Western blotting as in Figure 1. This experiment has been performed three times.

Ad cell lysis is impaired in the absence of p53

To investigate whether p53 influences the ability of Ad to cause cell lysis, dl1520 and wtAd5 were used to infect two isogenic pairs of cells at doses which infect nearly 100% of the cells (data not shown). Each pair of cells, RKO and RKOp53.13, and K1neo and K1scx, differs only in the expression of a dominant-negative mutant p53. At 20 CPEU/cell, dl1520 failed to cause appreciable cell death in RKO wtp53 cells, whereas wtAd5 caused 80% death at 4 days after infection (Figure 3). The E1a defective dl312 is also unable to kill these cells, but complementation with dl1520 rescues the ability of these viruses to cause cell death. In contrast, over the time course of the experiment, no virus or combination of viruses at 20 CPEU caused death of RKOp53.13 cells expressing a dominant-negative p53. Similar results were obtained for the thyroid cancer cell lines (K1neo and K1scx). These cells were infected with wtAd5 and dl1520 at 50 CPEU/cell, and viability determined over 10 days, as these cells are less permissive than A549 or RKO. Results (Figure 3 and Table 1) show that wtAd5 reduces the viability of K1neo to about 35% of control by 8–10 days post-infection, but dl1520 did not cause a significant decline in cell viability over the course of the experiment. dl1520 also has no effect on the viability of K1scx cells over the same time course and, in this case, wtAd5 also has little effect. Consistent with these data, approximately 20-fold less wtAd5 was harvested from K1scx cells compared to K1neo cells at 3 days post-infection (2.7 × 105 vs 1.5 × 104 CPEU/cell) (Table 1), and very little dl1520 was harvested from either cell type at this time (470 and 2.6 CPEU/cell for K1neo and K1scx, respectively). Interestingly, exogenous p53 is as effective as E1b55k in rescuing dl1520 in K1neo cells, but not in the presence of the dominant-negative mutant in K1scx (Table 1).

Figure 3

p53 promotes the Ad lytic cycle. The isogenic RKO pair (RKO and RKOp53.13) and the isogenic K1 pair (K1neo and K1scx) were infected and cell viabilities determined over time. The RKO pair were either mock infected (control), or infected with 20 CPEU/cell total virus of wtAd5, dl1520, dl312, or dl1520 and dl312 together (at 10 CPEU/cell per virus as in Figure 1). The K1 pair was either mock infected (control), or infected with wtAd5 or dl1520 at a dose of 50 CPEU/cell. Error bars represent s.e.m. These experiments have been performed many times with similar results.

Table 1 Viral recoveries (CPEU/cell) in K1neo and K1scx cells

These data suggest that wtp53 promotes Ad replication and cell death, as we have reported previously (Hall et al., 1998; Dix et al., 2000), and may function as a ‘permissivity’ factor to promote completion of the virus lifecycle and thereby enhance cell death. These data do not rule out the possibility that mutant p53 has some ‘gain of function’ that is inhibitory to viral replicative lysis. This possibility is explored later (Figure 7).

Figure 7

p53 increases expression from the MLP. IIICF/c, HeLa and K1scx cells were transiently transfected with different combinations of CMVE1a, CMVhp53 and the MLPCAT reporter plasmid from Ad2. IIICF/c cells were transfected with 0–3.9 ng wtp53 (gray shading); HeLa cells were transfected with 0–240 pg wtp53, and K1scx cells were transfected with 0–3.8 pg wtp53. In the case of IIICF/c cells, CMVmp53 (a his273 expressing p53 mutant) was also transfected over the same concentration range as for wtp53 (0–3.9 ng) (black shading). Optimal concentration ranges were determined empirically (data not shown). After 48 h, cells were harvested and CAT activity determined as described in Materials and methods. Error bars represent s.e.m., where this is greater than 5% deviation. This experiment has been performed at least five times.

p53 is expressed in cells at times coincidental with expression of viral DBP

Since it is known that E1b55kDa and E4orf6 combine to destroy p53 during the adenoviral replicative cycle (Querido et al., 2001a), we now wished to determine if p53 expression could be detected at times relevant to its proposed function as a ‘permissivity’ factor. Using immunofluorescence, we showed that p53 is expressed in wtAd5-infected K1neo cells up to 6 days post-infection, coincidental with the viral DBP (Figure 4), and is substantially expressed in infected cells at 3 days PI.

Figure 4

p53 is present late into wtAd5 infection. K1neo cells were seeded onto glass coverslips and infected with 50 CPEU/cell of wtAd5 for the indicated times. Cells were fixed and stained for p53 and DBP expression, then visualized by immunofluorescence microscopy.

Exogenous expression of E1b55kDa restores dl1520 replication and p53 enhances the Ad yield

To further explore a role for p53, we asked what effect the addition of exogenous p53 or E1b55kDa has on virus production. K1neo and K1scx cells were transfected with plasmids expressing human p53 (CMVhp53) or E1b55kDa (CMVE1b55kDa), and then infected with dl1520 or wtAd5. Cells were incubated for 2–3 days, virus recovered and the amount of virus titrated on 293 cells. The results are shown in Table 1 and Figure 5a. Coexpression of E1b55kDa in K1scx cells infected with dl1520 48 h earlier enhanced the virus yield about 500-fold, but had little effect on viral yield from Ad5-infected cells. At 72 h post-infection, exogenous E1b55kDa also enhanced the virus yield over 500-fold and wtAd5 yield only about three-fold. Expression of exogenous p53 increased the viral yield of dl1520-infected cells about 40-fold at both 48 and 72 h post-infection. p53 also increased the yield of wtAd5-infected cells by about eight-fold at 72 h post-infection. The addition of exogenous wtp53 restored yields of wtAd5 from K1scx cells to levels similar to those seen in K1neo cells at 72 h PI (2.7 × 105 CPEU/K1neo cell vs 1.1 × 105 CPEU/K1scx cell plus exogenous wtp53 (Table 1)).

Figure 5

Reintroduction of p53 aids viral recovery. K1scx cells were transiently transfected with either CMVhp53, CMV55kDa or control vector (pUC19), then infected with 50 CPEU/cell of either wtAd5 or dl1520 as indicated. Cells were harvested at 48 and 72 h post-infection. (a) Virus was harvested and quantitated on 293 cells using a CPE assay as described in Materials and methods (see also Table 1). (b) Expression from CMV55kDa was confirmed on protein lysates using Western blotting. This experiment has been performed at least three times.

Western blot showed that the levels of exogenous E1b55kDa protein expression used to complement dl1520 infection were similar to the levels expressed in wtAd5-infected K1neo cells (Figure 5b). A similar experiment could not be performed for p53 as the dominant-negative mutant in K1scx is stabilized and expressed at high levels; however, since similar conditions were used for transfection of the E1b55kDa and the p53 expression plasmid, we anticipate that physiological levels of exogenous p53 expression were attained. Transfection efficiency was determined using FACS analysis and found to be approximately 50% (results not shown). These data strongly suggest that p53 can influence Ad replication and consequently its ability to cause cell death.

Expression of late viral proteins is delayed in the p53 defective K1scx cells and reduced in E1b55kDa-deleted virus

To determine which part of the virus replication cycle p53 is affecting, K1neo and K1scx cells were infected with wtAd5 and dl1520, and Southern blotting was carried out to measure the amount of viral DNA synthesized. A number of different time points were examined and the results for 2, 4 and 6 days post-infection are shown in Figure 6. No significant differences in the levels of virus DNA synthesized with either wtAd5 or dl1520 were observed at any time examined up to 6 days post-infection.

Figure 6

The level of hexon expression is reduced in p53-deficient cells, while Ad DNA replication is comparable. K1neo and K1scx cells were infected with 50 CPEU/cell of either wtAd5 or dl1520 and harvested at the indicated times. (a) DNA was extracted and the amount of virus DNA determined by Southern blotting using a [32P]Ad DNA probe. (b) Western blotting was carried out to assess hexon, penton, E2A DBP and β-actin protein expression.

Expression of penton and hexon proteins (late antigens) was measured by Western blotting at different times after infection. Both hexon and penton expressions were severely impaired in K1neo after infection with dl1520, compared to wtAd5-infected cells (Figure 6). These data show that E1b55kDa is needed for late viral protein expression as expected. There was also a considerable reduction in penton and hexon production after infection of K1scx cells compared with K1neo cells for wtAd5, also suggesting that p53 may facilitate this process.

Collectively, these data suggest that p53 may be exerting its influence on the Ad lytic cycle, possibly by affecting late viral gene expression.

Exogenous p53 increases expression of late viral genes

Given that p53 is a transcription factor, as is E1a, the principal activator of virus gene transcription, we asked whether both proteins might cooperate to enhance late gene transcription. To do this, a reporter plasmid containing the Ad MLP was used in p53 null cells (IIICF/c), cells with HPV E6-mediated degradation of p53 (HeLa) and in cells with a dominant-negative mtp53 (K1scx). The MLP is the principal promoter regulating expression of late virus proteins (Shenk, 1996). For this experiment, cells were cotransfected with a fixed amount of CMVE1a (0.05 μg/dish) and differing amounts of CMVhp53 depending on cell type (see legends), along with a chloramphenicol acetyl transferase (CAT) reporter plasmid linked to the MLP from Ad2 (Weyer and Doerfler, 1985). After 48 h, cells were harvested and CAT activity was determined. Results (Figure 7) showed that E1a alone gave low expression as reported previously (Parks and Shenk, 1997), but that E1a in conjunction with p53 caused a dose-dependent increase in MLP CAT activity, which was up to 300-fold higher than E1a alone. The level of p53 enhancement varied between cell lines, with HeLa giving the greatest enhancement of 300-fold at 15 pg of CMVhp53 per dish of 2 × 105 cells. p53 had no effect on the MLP promoter in the absence of E1a, except at high concentrations (>0.5 μg plasmid/2 × 105 cells) at which it repressed promoter activity (data not shown). Wtp53 enhanced MLP expression at levels of 0.06–15.2 pg of transfected plasmid, whereas mtp53 (CMVmp53 R273H) required 100–250 pg plasmid to get some enhancement in IIICF/cs (Figure 7). This shows that the inhibition of replicative lysis by mtp53, as seen in Figure 3 and Table 1, is unlikely to be due to a dominant ‘gain of function’ effect of mtp53, but more likely to be due to loss of wtp53 function. Western blotting showed that hexon expression was increased by addition of exogenous p53 to wtAd5-infected HeLa cells, particularly at lower levels (0.06 pg plasmid/2 × 105 cells) of p53 expression (Figure 8a).

Figure 8

p53 increases expression of the adenoviral hexon protein. (a) HeLa cells were transiently transfected with different combinations of CMVhp53 and pUC19, then infected with 1 CPEU/cell of wtAd5. After 48 h, cells were harvested and assayed for expression of hexon, p53 and β-actin protein levels. (b) HeLa cells were cotransfected with wtAd2 DNA and CMVhp53. At 24 h after transfection, cells were labelled with [S35]methionine, extracts prepared and immunoprecipitations carried out using normal rabbit serum (control), an antibody to E1b55kDa (55 kDa), an antibody to hexon protein (hexon) and protein A Sepharose beads. Immunoprecipitates were analysed by SDS–PAGE and fluorography. This experiment has been performed twice and also once in other cells.

To determine directly whether p53 is able to increase expression of late viral proteins, HeLa cells were transfected with wtAd2 DNA, and then transfected with CMVhp53. HeLa cells were used as they are very permissive for Ad, essentially p53 deficient due to the degradative influence of the papillomavirus E6 protein present in these cells (Scheffner et al., 1991) and gave the best results in the in vitro CAT assay. At 24 h after transfection, cells were metabolically labeled with [S35]methionine, lysates prepared and assayed for virus protein expression by immunoprecipitation with hexon- and E1b55kDa-specific antibodies and protein A sepharose beads. Results (Figure 8b) show that, although no hexon or E1b55kDa proteins were detected after transfection of Ad2 DNA alone, hexon was clearly detectable if p53 was present, although E1b55kDa was not. These data show that p53 can selectively increase expression of late viral proteins and are consistent with the observations in previous sections.


The precise impact of E1b55kDa and p53 on Ad replicative lysis is poorly understood. Our results show that both E1b55kDa and p53 are important for induction of cell death. The mechanisms behind these observations are explored further.

Although ONYX-015 (dl1520) was originally reported to replicate in tumor cells deficient in the p53 pathway, the bulk of more recent evidence does not support this (reviewed in Dix et al., 2001). Many reports suggest that the replication of both wtAd5 and dl1520 occurs independently of the p53 status of cells (Goodrum and Ornelles, 1998; Rothmann et al., 1998; Harada and Berk, 1999). The work of Harada and Berk (1999) is particularly clear on this point. Despite this, in many experiments we have observed a replicative bias towards cells having a wtp53. These studies encompass some 20 human cell lines. However, we too find exceptions to the trend. For example, we find very efficient replication and cell death with both wtAd5 and dl1520 in the mutant p53 C33A cervical cancer cell line (Harada and Berk, 1999; Dix et al., 2000). Nonetheless, the tendency of Ads to kill cells with wtp53 more efficiently still stands. Some other studies have also observed a similar bias (e.g., Geoerger et al., 2002), and one report noted that recombinant Ads expressing wtp53 replicated and spread to other cells more efficiently than control viruses (Sauthoff et al., 2002). In addition, a recent report suggested that one ‘gain-of-function’ p53 mutant (R248W) could partially rescue the deficiency in dl1520 replication (Hann and Balmain, 2003). Evidence was provided that this and other mutants had lost cell cycle arrest ability, but no direct evidence of ‘gain of function’ was provided other than virus rescue. Another interpretation is that mutant R248W still retains a number of wtp53 functions. If so, these data would be consistent with our own. This may also explain why the C33A cells are so permissive for Ads. These cells have a R273C p53 mutant which does retain some residual functional activity (Ory et al., 1994).

A possible explanation for the equivocal results concerning a role for p53 in Ad replication is that not all cell lines genetically wt for TP53 actually express a functional p53 protein, due to defects in other cellular gene products. This thinking led to the hypothesis that p14ARF might be a key determiner of ONYX-015 replication (Ries et al., 2000) as p14ARF is an important positive regulator of p53 (Pomerantz et al., 1998) and defects in p14ARF expression are commonplace in many tumor cells containing a wt TP53 gene (Stott et al., 1998; Ries et al., 2000). However, this explanation alone may not be the whole story (Edwards et al., 2002).

In this paper, we have carried out studies in isogenic pairs of cells in which the normal functional status of p53 has been verified, together with a variant in which p53 activity has been inhibited with a dominant-negative mutant. The purpose of this approach was to avoid the confounding genetic variables that are inherent in the use of panels of cell lines. In the case of the K1neo cells, the verification that p53 is functional is particularly clear (Wyllie et al., 1999). Results from these studies show that wtAd5 and dl1520 kill cells expressing a functional p53 protein in preference to the mutant derivative, and that virus lacking E1b55kDa is attenuated (Figure 3). Furthermore, we show that cell death is in fact linked to the viral replication cycle (Figure 2), thus ruling out direct killing due to early events or to early gene expression alone. In fact, we tested the possibility that E1b55kDa could function directly as a cell death-promoting protein, but we could find no evidence for this (results not shown). We did however show (Figure 6) that cell killing is associated with a successful production of the late viral proteins, but not with the amount of viral genome replicated. E1b55kDa promotes the virus replication cycle due to its host shut-off capacity and promotion of the selective translation of late viral genes. Thus, it appears as though p53 may be acting analogously to E1b55kDa to advance the virus replication cycle by promoting expression of late viral genes. This would be consistent with the ability of some p53 mutants to rescue the defect in dl1520 replication (Hann and Balmain, 2003). To test the hypothesis that p53 can promote viral replication and late gene expression, we transfected Ad-infected cells with a p53 expression construct. Exogenous p53 increased expression of hexon protein, particularly at low levels (Figure 8), and increased virus yield (Figure 5). To explain this, we asked whether p53 might increase expression of the MLP of Ad, which regulates expression of late viral proteins. In reporter assays, we found that p53 did not increase activity of an MLPCAT reporter by itself. However, in a range of cells, cotransfection of p53 along with E1a resulted in significantly higher MLPCAT activity than E1a alone (Figure 7). Thus, physiological levels of p53 cooperate with E1a to increase activity of the promoter that controls expression of late Ad mRNAs such as hexon. This would lead to increased availability of late proteins for assembly into virus particles, thus increasing the virus yield and enhancing cell death. Small increases in late protein expression have the potential to cause amplified effects on virion production due to the mass action nature of the process. It has been reported that a two- to four-fold increase in expression of late viral RNAs could have a 25-fold effect on the number of infectious particles (O'Shea et al., 2004); thus, an increase in the efficiency of translation from the MLP could be expected to have a similar amplifying effect on virus yield and concomitant cell lysis.

E1a induces p53 expression in cells (Braithwaite et al., 1990); however, it has been suggested widely that E1b55kDa and E4orf6 co-operate in promoting ubiquitination and degradation of p53, thus removing it and rendering virally infected cells effectively null for p53 (Querido et al., 2001a, 2001b). The question arises therefore, ‘How can p53 aid late gene expression if the protein has been degraded?’. In order to answer this, we have shown that p53 is present in the nucleus of cells at the same time as the viral DBP (Figure 4), and at a time therefore when it could aid MLP function. Thus, we would argue that the p53 in virally infected cells is not simply degraded, but is ‘highjacked’ by the virus for its own purpose, before being destroyed. Our data suggest that, after infection, p53 is induced and remains stable in the cell, possibly in a modified state, for a large part of the infectious cycle, without antagonizing viral replication. These results are in agreement with others who have shown that p53 activity does not impair Ad replication (Koch et al., 2001; Hobom and Dobbelstein, 2004), but we have now gone further by showing that p53 augments late stages of viral replication. The possibility exists that E1b55k and E4orf6 only degrade a subset of cellular p53 molecules, identified either by their location or by post-translational modification, leaving the residual p53 for MLP function.

The mechanism of cooperation between E1a and p53 is not clear. It is known however that E1a cannot bind directly to DNA, but requires binding partners to exert its transactivation function. The possibility exists that p53 could act as a chaperone or intermediary, bringing together components of the transcription machinery at the required DNA sites. The MLP contains binding sites for several transcription factors, including the CCAAT-box binding factor, the TATA-box binding protein (reviewed in Shenk, 1996) and the transcription factor Sp1 (Parks and Shenk, 1997), all of which have been reported to interact with both p53 (reviewed in Prives and Hall, 1999) and E1a (reviewed in Shenk, 1996). It could be that p53 helps to stabilize an interaction between E1a and essential transcription factors on the MLP, thereby enhancing transcription. The interaction with Sp1 would have to be a strong candidate, given its marked ability to cooperate with E1a (Parks and Shenk, 1997). In this respect, it is interesting to note that we have found transfection of exogenous E1a into dl1520-infected K1scx cells increases virus yield approximately 10-fold, with little effect on wtAd5 (results not shown). Thus, E1b55kDa, E1a, and p53 can have a major effect on viral replication by increasing the efficiency of MLP function, with the latter two being particularly significant for the E1b55kDa-deficient viruses for which late viral RNA function is a limiting factor (O'Shea et al., 2004).

A very different kind of explanation involves YB1, a member of the highly conserved family of cold shock proteins. YB1 is reported to have a number of different functions, among them being transcriptional regulation (Kohno et al., 2003). YB1 is mostly located in the cytoplasm, but various stimuli cause YB1 to translocate to the nucleus, where it is able to transcriptionally regulate different genes that contain CCAAT boxes as YB1 binds to an inverted CCAAT site (Y-box). WtAd5 stimulates nuclear YB1 translocation, which requires E1b55kDa, and this results in the transactivation of the AdE2 promoter that is also important for regulating viral replication and late gene expression (Holm et al., 2002). Moreover, an E1 defective virus that constitutively expresses YB1 is able to replicate with modest efficiency, and several fold better than an E1-deleted virus without YB1 (Holm et al., 2002). YB1 may therefore be an important permissivity factor for Ad. We have recently reported that p53 is required for, or improves the efficiency of, YB1 nuclear translocation (Zhang et al., 2003). Thus, p53 may increase virus replication as shown in the present paper, by stimulating YB1 nuclear translocation and the consequent activation of late viral promoters.

Whatever the precise mechanism, data such as we have provided offers an explanation for the killing bias we have observed by Ad for cells with wtp53, for the ability of p53 to enhance virus replication ((Sauthoff et al., 2002); our own data), and possibly for the ability of the R248W p53 mutant to rescue the defect in ONYX-015 replication (Hann and Balmain, 2003). Moreover, our results suggest that the limiting function of E1b55kDa is in selective translation of viral late message, as suggested by Harada and Berk (1999) and O'Shea et al. (2004), not in the degradation of p53.

Mutant p53 is stabilized and maintained at high levels in many tumour types. Since we have shown that at high levels, mutant p53 (R175H) can also co-operate with E1a at the MLP (Figure 7), the deficiency in dl1520 may even be rescued by certain p53 mutants in some cancers. Other researchers have shown similar effects for the R248W p53 mutant (Hann and Balmain, 2003). The collective data suggest that if viruses such as ONYX-015 are to be used as tumoricidal agents, the functional status of p53 and the nature of the p53 mutant may well be important.

In conclusion, we have presented data that refine the current concept that successful Ad replication requires the inactivation and destruction of p53. We show that the reverse can be true and that Ad actually benefits from p53. The effect is more significant for the defective dl1520/ONYX-015 virus. These findings have important implications for our understanding of infection by DNA viruses and for their use in treatment of diseases such as cancer.

Materials and methods

Cell lines

Isogenic human thyroid carcinoma cell lines: K1neo with wtp53 and K1scx with the A143V dominant-negative p53 mutant (Wyllie et al., 1999); RKO and RKOp53.p13 are colorectal carcinoma cell lines, respectively, with wtp53 and the A135V dominant-negative mouse p53 mutant (Slichenmyer et al., 1993). Other human cell lines used were: A549 (lung carcinoma; Lehman et al., 1991) containing wtp53; HeLa cervical carcinoma cells that are essentially p53 null due to the papillomavirus E6 protein (Scheffner et al., 1991), and p53 null fibroblasts from a LiFraumeni patient (IIICF/c; Rogan et al., 1995). Cell lines were grown in DMEM (Life Technologies, Inc.) supplemented with 10% FCS, L-glutamine, and the antibiotics penicillin and streptomycin. Cells were routinely cultured at 37°C and 10% CO2.


CMVE1b55kDa expresses the Ad5 E1b55kDa gene (Goodrum et al., 1996); CMVneo, used as a vector control, expresses the gene conferring resistance to the neomycin/kanamycin family of antibiotics (Braithwaite et al., 1987); CMVE1a expresses the E1a gene products from Ad2 (Morris and Mathews, 1991); CMVhp53 expresses human wtp53; CMVmp53 expresses a his273 p53 mutant (Jackson et al., 1994); MLPCAT contains the MLP promoter from Ad2 linked to the bacterial CAT gene (Weyer and Doerfler, 1985), and pUC19 used as a vector control.

Antibodies and Western blotting

Cells were seeded and infected as below. Cells were harvested at 24, 48 and 72 h post-infection and lysates prepared in SDS sample buffer. Samples equivalent to 2 × 105 cells per lane were loaded and run on 10% SDS–PAGE prior to transfer to PVDF membrane. Proteins were detected using specific antibodies with the chemiluminescent method (Supersignal West Pico substrate from Pierce, Rockford, IL, USA). Actin was used as a loading control. Antibodies: E1a was 13S-5 (Santa Cruz); p53 was DO-1 (Santa Cruz); E1b55kDa was the 2A6 monoclonal (Sarnow et al., 1982); hexon was a rabbit polyclonal anti-hexon antibody (gifted from Professor Peter van der Vliet, University Medical Center, Utrecht); actin was SC1615 (Santa Cruz).

Immunofluorescence analysis

K1neo cells were seeded at 2–4 × 104 cells per glass coverslip in a 24-well culture plate. Cells were infected 4–5 h after seeding with 50 CPEU/cell of wtAd5. Cells were then harvested at the indicated times post-infection by washing with PBS and fixation in −20°C methanol for 5 min, followed by air-drying. p53 (1:500) and Ad5 E2A DBP (1:2000) were visualized using AlexaFluor™-conjugated secondary antibodies (Molecular Probes) on a ZEISS Axioplan fluorescence microscope. Images were captured using a mounted SPOT camera (Diagnostic Instruments Inc.) and processed using the SPOT® 4.1 and Adobe Photoshop® 5.0 (Adobe) software packages.


Virus stocks were grown in human 293 cells and titered on 293 monolayers using a CPE assay as described previously (O'Carroll et al., 2000). WtAd5 and E1b55kDa deletion mutant ONYX-015 (dl1520; Barker and Berk, 1987) were used in this paper, as well as the E1a deletion mutant dl312 (Jones and Shenk, 1978, 1979). Ad mutant ts125 containing a ts defect (Williams et al., 1974) in the viral DBP was also used. In general, cells were infected and incubated at 37°C or, for ts125, at 32.5°C and 39.5°C, for the indicated times.

Cell viability

Cells were seeded at 2 × 105 per well in six-well tissue culture plates and grown overnight before infection with 1–50 CPEU/cell depending on the ease of infectivity of the cell line. Optimal doses of virus for each cell line giving near 100% infectivity were used. These were empirically determined in advance of each experiment (data not shown). Floating and trypsinized cells were harvested at indicated times post infection. Both viable and nonviable cells were counted using trypan blue exclusion and a hemocytometer. All counts were performed at least in duplicate.

Virus complementation assays

These experiments were carried out using A549 and RKO cells and either wtAd5 or a combination of the E1b55kDa mutant dl1520 (ONYX-015) and the E1a defective mutant dl312. Cells were infected with a total of 20 CPEU/cell of virus. Thus, for a single infection 20 CPEU/cell was used, and for complementation 10 CPEU/cell of each virus was used. Cells were infected in the usual way and cells harvested and viability determined as above.

Plasmid complementation assays

Cells were seeded at a density of 2 × 105 cells per well in six-well culture plates, grown overnight, then transfected with 1 μg of plasmid DNA per well using FuGENE6™ (Roche) according to the manufacturer's instructions. (1) Cell viability: At 5 h post-transfection, cells were infected. Cells were harvested and live and dead cells counted at intervals up to 6 days post-infection as above. (2) Virus rescue and CPE assay: Cells were transfected with either CMVhp53 or CMVE1b55kDa, followed by infection with 50 CPEU/cell of virus as described above. Intracellular virus was harvested at the indicated times post-infection as described (Edwards et al., 2002) and virus titer assayed on 293 cells using a CPE assay (O'Carroll et al., 2000).

ts mutant analysis

A549 cells were seeded at a density of 1 × 106 cells per 175 cm2 culture vessel and first incubated at 37°C for 3–4 h, before being equilibrated at 32.5 or 39.5°C for 2–3 h. Monolayers were infected with 10 CPEU/cell of either wtAd5 or ts125 at both temperatures. At the indicated times post-infection, cells were harvested for viability studies and for Western blotting as described.


Approximately 1 × 106 HeLa cells were transfected with 1 μg of Ad2 DNA or 1 μg of Ad2 DNA and 0.5 μg of CMVhp53. At 24 h after transfection, cells were incubated for 1 h with methionine/cysteine-free medium and then incubated with 250 μCi of [35S] translabel/well for 1–2 h. Cells were then lysed (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 and 0.1% SDS), centrifuged to remove debris, and the supernatant preimmune cleared with protein A sepharose beads and normal rabbit serum. Beads were then removed by centrifugation and lysates immunoprecipitated overnight with specific antibodies. Proteins were separated on SDS–PAGE and visualized by fluorography.

Promoter/reporter assays

Cells (2–3 × 105) were transfected with the Ad2 MLP reporter, together with different combinations of CMVE1a and CMVhp53 plasmids (a total of 2 μg). Many different combinations were tried and those shown in Figure 5 are optimal. About 48 h later, cells were lysed (0.25 M Tris-HCl, pH 7.5) by repeated freeze–thaw cycles, and cell debris removed by centrifugation. Supernatants were normalized for protein concentration using the BCA protein assay kit (Pierce) and CAT activity determined as described previously (Sleigh, 1986).

Southern blotting

K1neo cells were seeded (2–3 × 105 cell per well) and infected at 50 CPEU/cell as described above. At different times after infection, cells were lysed, replicates pooled, and total DNA extracted, restricted with XbaI, blotted on Hybond N+ (Amersham) and probed with 32P Ad2 DNA.


  1. Barker DD, Berk AJ . (1987). Virology 156: 107–121.

  2. Bischoff JR, Kirn DH, Williams A, Heise C, Horn S, Muna M et al. (1996). Science 274: 373–376.

  3. Braithwaite A, Nelson C, Skulimowski A, McGovern J, Pigott D, Jenkins J . (1990). Virology 177: 595–605.

  4. Braithwaite AW, Murray JD, Bellett AJ . (1981). J Virol 39: 331–340.

  5. Braithwaite AW, Sturzbecher HW, Addison C, Palmer C, Rudge K, Jenkins JR . (1987). Nature 329: 458–460.

  6. Dix BR, Edwards SJ, Braithwaite AW . (2001). J Virol 75: 5443–5447.

  7. Dix BR, O'Carroll SJ, Myers CJ, Edwards SJ, Braithwaite AW . (2000). Cancer Res 60: 2666–2672.

  8. Edwards SJ, Dix BR, Myers CJ, Dobson-Le D, Huschtscha L, Hibma M et al. (2002). J Virol 76: 12483–12490.

  9. Geoerger B, Grill J, Opolon P, Morizet J, Aubert G, Terrier-Lacombe MJ et al. (2002). Cancer Res 62: 764–772.

  10. Goodrum FD, Ornelles DA . (1998). J Virol 72: 9479–9490.

  11. Goodrum FD, Shenk T, Ornelles DA . (1996). J Virol 70: 6323–6335.

  12. Hall AR, Dix BR, O'Carroll SJ, Braithwaite AW . (1998). Nat Med 4: 1068–1072.

  13. Hann B, Balmain A . (2003). J Virol 77: 11588–11595.

  14. Harada JN, Berk AJ . (1999). J Virol 73: 5333–5344.

  15. Heise C, Sampson-Johannes A, Williams A, McCormick F, Von Hoff DD, Kirn DH . (1997). Nat Med 3: 639–645.

  16. Hobom U, Dobbelstein M . (2004). J Virol 78: 7685–7697.

  17. Holm PS, Bergmann S, Jurchott K, Lage H, Brand K, Ladhoff A et al. (2002). J Biol Chem 277: 10427–10434.

  18. Jackson P, Ridgway P, Rayner J, Noble J, Braithwaite A . (1994). Biochem Biophys Res Commun 203: 133–140.

  19. Jones N, Shenk T . (1978). Cell 13: 181–188.

  20. Jones N, Shenk T . (1979). Proc Natl Acad Sci USA 76: 3665–3669.

  21. Koch P, Gatfield J, Lober C, Hobom U, Lenz-Stoppler C, Roth J et al. (2001). Cancer Res 61: 5941–5947.

  22. Kohno K, Izumi H, Uchiumi T, Ashizuka M, Kuwano M . (2003). BioEssays 25: 691–698.

  23. Kruijer W, van Schaik FM, Sussenbach JS . (1981). Nucleic Acids Res 9: 4439–4457.

  24. Lee H, Kim J, Lee B, Chang JW, Ahn J, Park JO et al. (2000). Int J Cancer 88: 454–463.

  25. Lehman TA, Bennett WP, Metcalf RA, Welsh JA, Ecker J, Modali RV et al. (1991). Cancer Res 51: 4090–4096.

  26. Morris GF, Mathews MB . (1991). J Virol 65: 6397–6406.

  27. Nemunaitis J, Ganly I, Khuri F, Arseneau J, Kuhn J, McCarty T et al. (2000). Cancer Res 60: 6359–6366.

  28. Nemunaitis J, Khuri F, Ganly I, Arseneau J, Posner M, Vokes E et al. (2001). J Clin Oncol 19: 289–298.

  29. O'Carroll SJ, Hall AR, Myers CJ, Braithwaite AW, Dix BR . (2000). Biotechniques 28: 408–410.

  30. O'Shea CC, Johnson L, Bagus B, Choi S, Nicholas C, Shen A et al. (2004). Cancer Cell 6: 611–623.

  31. Ory K, Legros Y, Auguin C, Soussi T . (1994). EMBO J 13: 3496–3504.

  32. Parks CL, Shenk T . (1997). J Virol 71: 9600–9607.

  33. Pomerantz J, Schreiber-Agus N, Liegeois NJ, Silverman A, Alland L, Chin L et al. (1998). Cell 92: 713–723.

  34. Prives C, Hall PA . (1999). J Pathol 187: 112–126.

  35. Querido E, Blanchette P, Yan Q, Kamura T, Morrison M, Boivin D et al. (2001a). Genes Dev 15: 3104–3117.

  36. Querido E, Morrison MR, Chu-Pham-Dang H, Thirlwell SW, Boivin D, Branton PE . (2001b). J Virol 75: 699–709.

  37. Rauen KA, Sudilovsky D, Le JL, Chew KL, Hann B, Weinberg V et al. (2002). Cancer Res 62: 3812–3818.

  38. Ries SJ, Brandts CH, Chung AS, Biederer CH, Hann BC, Lipner EM et al. (2000). Nat Med 6: 1128–1133.

  39. Rogan EM, Bryan TM, Hukku B, Maclean K, Chang AC, Moy EL et al. (1995). Mol Cell Biol 15: 4745–4753.

  40. Rogulski KR, Freytag SO, Zhang K, Gilbert JD, Paielli DL, Kim JH et al. (2000). Cancer Res 60: 1193–1196.

  41. Rothmann T, Hengstermann A, Whitaker NJ, Scheffner M, Zur Hausen H . (1998). J Virol 72: 9470–9478.

  42. Sarnow P, Sullivan CA, Levine AJ . (1982). Virology 120: 510–517.

  43. Sauthoff H, Pipiya T, Heitner S, Chen S, Norman R, Rom W et al. (2002). Hum Gene Ther 13: 1859–1871.

  44. Scheffner M, Munger K, Byrne J, Howley P . (1991). Proc Natl Acad Sci USA 88: 5523–5527.

  45. Shenk T . (1996) In: Fields B, Knipe D, Howley P, Chanock R, Melnick J, Monath T, Roizman B, Strauss S (eds). Virology vol. 2. Lippincott-Raven: Philadelphia. pp 2111–2148.

  46. Sleigh MJ . (1986). Anal Biochem 156: 251–256.

  47. Slichenmyer WJ, Nelson WG, Slebos RJ, Kastan MB . (1993). Cancer Res 53: 4164–4168.

  48. Stott FJ, Bates S, James MC, McConnell BB, Starborg M, Brookes S et al. (1998). EMBO J 17: 5001–5014.

  49. Weyer U, Doerfler W . (1985). EMBO J 4: 3015–3019.

  50. Williams J, Young C, Austin P . (1974). Cold Spring Harbor Sympos Quant Biol 39: 427–437.

  51. Wyllie FS, Haughton MF, Rowson JM, Wynford-Thomas D . (1999). Br J Cancer 79: 1111–1120.

  52. Yew PR, Berk AJ . (1992). Nature 357: 82–85.

  53. Zhang YF, Homer C, Edwards SJ, Hananeia L, Lasham A, Royds J et al. (2003). Oncogene 22: 2782–2794.

Download references


We thank Mike Kastan (Johns Hopkins, Baltimore) for the RKO pair of cells, Roger Reddel (CMRI, Sydney) for the IIICF/c p53 null human fibroblast cell line, Walter Doerfler (Institut fur Genetik, Cologne) for the MLPCAT plasmid, and Peter van der Vliet (University Medical Center, Utrecht) for the DBP antibody. We wish to thank Nicky Real, Deidre Dobson-Le, Craig Homer and Rhodri Harfoot for technical assistance. This work was supported by grants from the Royal Society and the Health Research Council of New Zealand.

Author information

Correspondence to J A Royds.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Royds, J., Hibma, M., Dix, B. et al. p53 promotes adenoviral replication and increases late viral gene expression. Oncogene 25, 1509–1520 (2006). https://doi.org/10.1038/sj.onc.1209185

Download citation


  • p53
  • adenovirus
  • E1b55kDa
  • oncology

Further reading