Large E1B proteins of adenovirus types 5 and 12 have different effects on p53 and distinct roles in cell transformation.

The formation of complexes between oncoproteins of DNA tumor viruses and the cellular protein p53 is thought to result in inactivation of the growth suppressor function of p53. In cells transformed by nononcogenic human adenovirus type 5 (Ad5), the 55-kDa protein encoded by E1B forms a stable complex with p53 and sequesters it in the cytoplasm. However, the homologous 54-kDa protein of highly oncogenic Ad12 does not detectably associate with p53. Yet in Ad12-transformed cells, p53 is metabolically stable, is present at high levels in the nucleus, and contributes to the oncogenicity of the cells. Such properties have previously been described for mutant forms of p53. Here, we show that stable p53 in Ad12-transformed cells is wild type rather than mutant and that stabilization of p53 is a direct consequence of the expression of the Ad12 E1B protein. We also compared the effects of the E1B proteins on transformation of rodent cells by different combinations of oncogenes. A synergistic interaction was observed for the gene encoding the 54-kDa E1B protein of Ad12 with myc plus ras oncogenes, resembling the effect of mutant p53 on myc plus ras. In contrast, the Ad5 55-kDa E1B protein strongly inhibited transformation by myc plus ras but stimulated transformation by E1A plus ras. The data are explained in terms of different interactions of the two E1B proteins with endogenous p53. The results suggest that in cultured rat cells, endogenous wild-type p53 plays an essential role in cell proliferation, even in the presence of myc plus ras. The dependence on p53 is lost, however, when the adenovirus E1A oncogene is present.     

Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis

The adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated transactivation and apoptosis. The proposed mechanisms include tethering the E1B repression domain to p53-responsive promoters via direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the 55-kDa protein would impose additional inhibition on p53-mediated effects. To investigate further the role of cytoplasmic sequestration of p53 in its inhibition by the E1B 55-kDa protein we systematically examined domains in both the Ad12 55-kDa protein and p53 that underpin their colocalization in the cytoplasmic body and show that the N-terminal transactivation domain (TAD) of p53 is essential for retaining p53 in the cytoplasmic body. Deletion of amino acids 11 to 27 or even point mutation L22Q/W23S abolished the localization of p53 to the cytoplasmic body, whereas other parts of TAD and the C-terminal domain of p53 are dispensable. This cytoplasmic body is distinct from aggresome associated with overexpression of some proteins, since it neither altered vimentin intermediate filaments nor associated with centrosome or ubiquitin. Formation of this structure is sensitive to mutation of the Ad12 55-kDa protein. Strikingly, mutation S476/477A near the C terminus of the Ad12 55-kDa protein eliminated the formation of the cytoplasmic body. The equivalent residues in the Ad5 55-kDa protein were shown to be critical for its ability to inhibit p53. Indeed, Ad12 55-kDa mutants that cannot form a cytoplasmic body can no longer inhibit p53-mediated effects. Conversely, the Ad12 55-kDa protein does not suppress p53 mutant L22Q/W23S-mediated apoptosis. Finally, we show that E1B can still sequester p53 that contains the mitochondrial import sequence, thereby potentially preventing the localization of p53 to mitochondria. Thus, cytoplasmic sequestration of p53 by the E1B 55-kDa protein plays an important role in restricting p53 activities.     

Association between the cellular p53 and the adenovirus 5 E1B-55kd proteins reduces the oncogenicity of Ad-transformed cells.

The association of the cellular p53 protein with the E1B-55kd protein of adenovirus 5 (Ad5) is thought to result in inactivation of the p53 recessive oncogene product. Here we show that Ad5 E1-transformed 3Y1 rat cells which express low levels of the 55 kd E1B protein do not contain the p53-E1B 55kd complex. These cells have nuclearly located p53 and are highly oncogenic in nude mice. In 3Y1 cells expressing the E1B protein at a sufficiently high level, association between p53 and E1B-55kd occurs, resulting in an almost complete trapping of p53 into a discrete cytoplasmic body. These cells only form tumors after a very long latency period and in the tumors that eventually appear selection has occurred in favor of cells lacking the complex and containing free nuclear p53. Comparable results were found when highly oncogenic Ad12-transformed cells were supertransfected with the Ad5 E1B region. In none of the Ad-transformed mouse, rat and human cell lines examined, could we detect p53 of abnormal molecular weight or association with hsc70, neither could we immunoprecipitate p53 by the mutant specific antibody PAb240. These data suggest that a high level of nuclear p53 with a wild-type conformation contributes to the oncogenicity of Ad transformed cells.     

Variants of adenovirus type 5 with deletions in early genes: ability for selective replication in p53-defective human tumor cells

Site-directed mutagenesis was used to construct human adenovirus serotype 5 (Ad5) variants defective in E1A or E1B. Mutant Adel3 with deletion from E1A was markedly attenuated in permissive cell cultures regardless of the p53 status, and replicated efficiently only in cells of the complementing 293 line. Mutant Adel2 with deletion from E1B55K infected the 293 line cells and p53-deficient human tumor cells (A431, SW480, HEp2) with efficiencies similar to those of Ad5, whereas its replication in normal p53-positive cells was substantially limited. Thus, Adel2 proved to be capable of selective infection and lysis of p53-deficient human tumor cells in vitro. On intratumor injection, Adel2 dramatically suppressed the growth of human epidermoid carcinoma (A431) in nude mice. Adel2 is thus a promising model for designing therapeutic agents against p53-deficient human tumors.     

Adenovirus E1A oncogene expression in tumor cells enhances killing by TNF-related apoptosis-inducing ligand (TRAIL)

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-alpha and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-alpha and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-alpha- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.     

Adenovirus Serotype 5 Variants Defective in Early Genes: Selective Replication in p53-Deficient Human Tumor Cells

Site-directed mutagenesis was used to construct human adenovirus serotype 5 (Ad5) variants defective in E1A or E1B. Mutant Adel3 with deletion from E1A was markedly attenuated in permissive cell cultures regardless of the p53 status, and replicated efficiently only in cells of the complementing 293 line. Mutant Adel2 with deletion from E1B55K infected the 293 line cells and p53-deficient human tumor cells (A431, SW480, HEp2) with efficiencies similar to those of Ad5, whereas its replication in normal p53-positive cells was substantially limited. Thus, Adel2 proved to be capable of selective infection and lysis of p53-deficient human tumor cells in vitro. On intratumor injection, Adel2 dramatically suppressed the growth of human epidermoid carcinoma (A431) in nude mice. Adel2 is thus a promising model for designing therapeutic agents against p53-deficient human tumors.     

Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells.

The adenovirus type 5 243R E1A protein induces p53-dependent apoptosis in the absence of the 19- and 55-kDa E1B polypeptides. This effect appears to result from an accumulation of p53 protein and is unrelated to expression of E1B products. We now report that in the presence of the E1B 55-kDa polypeptide, the 289R E1A protein does not induce such p53 accumulation and, in fact, is able to block that induced by E1A 243R. This inhibition also requires the 289R-dependent transactivation of E4orf6 expression. E4orf6 is known to form complexes with the E1B 55-kDa protein and to function both in the transport and stabilization of viral mRNA and in shutoff of host cell protein synthesis. We demonstrated that the block in p53 accumulation is not due to the generalized shutoff of host cell metabolism. Rather, it appears to result from a mechanism targeted specifically to p53, most likely involving a decrease in the stability of p53 protein. The E1B 55-kDa protein is known to interact with both E4orf6 and p53, and as demonstrated recently by others, we showed that E4orf6 also binds directly to p53. Thus, multiple interactions between all three proteins may regulate p53 stability, resulting in the maintenance of low levels of p53 following virus infection.     

A cell cycle G0-ts mutant, tsJT60, becomes lethal at the nonpermissive temperature after transformation with adenovirus 12 E1B 19K mutant

tsJT60, a temperature-sensitive (ts) cell-cycle mutant of Fischer rats, is viable at both the permissive (34 degrees C) and nonpermissive (40 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with serum from G0 phase they enter S phase at 34 degrees C but not at 40 degrees C. tsJT60 cells transformed with human adenovirus (Ad) 12 dl205, which lacks the E1B 19-kDa polypeptide gene, were lethal at 40 degrees C, whereas tsJT60 cells transformed with Ad12 wt, dl207, which lacks E1B 58-kDa protein gene, or in206B, which produces 19- to 58- kDa fused protein, were viable. Degradation of cell DNA occurred in dl205-transformed tsJT60 cultured at both 34 degrees C and 40 degrees C. Neither cytocidal phenotype nor degradation of DNA occurred in 3Y1 cells (a parental line of tsJT60) transformed with dl205. These results suggest that the lethal phenotype and degradation of DNA are related to the ts mutation in tsJT60 and also to the lack of Ad12 E1B 19kDa polypeptide.     

Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells.

In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.     

p53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection

The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G₁. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39°C. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.     

Adenovirus type 5 E1A immortalizes primary rat cells expressing wild-type p53

Adenovirus (Ad) E1A induces apoptosis in cells expressing wild-type p53, and stable transformation by Ad E1A requires the co-introduction of an anti-apoptotic gene such as Ad E1B 19K. Thus, cells immortalized by Ad E1A alone might have lost functional p53. In order to analyze the p53 in rat cells expressing Ad E1A, we established rat cell lines by transfecting primary rat embryo fibroblast (REF) and baby rat kidney (BRK) cells with cloned Ad5 E1A. By using a yeast functional assay, we analyzed p53 in six primary REF and three BRK cell lines immortalized by Ad5 E1A as well as five spontaneously immortalized rat cell lines (REF52, NRK, WFB, Rat-1 and 3Y1). The yeast functional assay revealed that all of the spontaneously and Ad5 ElA-immortalized rat cell lines except for 3Y1 expressed wild-type p53. All of the Ad5 E1A-immortalized rat cell lines contained p53 detectable by immunoprecipitation. Recombinant adenovirus expressing rat p53 cloned from a REF cell line immortalized by Ad5 E1A, as well as that expressing murine wild-type p53, induced apoptosis in p53-null cells in collaboration with E1A. Thus, it is suggested that the mutation of p53 appears to be not frequent in the spontaneous immortalization of primary rat cells, and that the functional loss of wild-type p53 is not a prerequisite of E1A-mediated immortalization.     

Adenovirus serotype determines association and localization of the large E1B tumor antigen with cellular tumor antigen p53 in transformed cells.

The distribution and stability of the cellular tumor antigen p53 were studied in baby rat kidney cells transformed by region E1 sequences of nononcogenic adenovirus (Ad) type 5 (Ad5) or oncogenic type 12 (Ad12). In transformed cells expressing the large E1B T antigen of Ad5, p53 was associated with this T antigen. The complexed proteins were concentrated in a cytoplasmic body, which has been shown to consist of a cluster of 8-nm filaments (A. Zantema et al., Virology 142:44-58, 1985). In transformed cells expressing the E1B region of Ad12, however, no association between the viral large T antigen and p53 was detectable. In the latter case, both proteins were found almost exclusively in the nucleus. The stability of p53 in both Ad5- and Ad12-transformed cells was increased relative to that in primary cells or cells immortalized by the E1A region only. Thus, the increased stability of p53 in Ad-transformed cells is not caused by association with a viral T antigen, but it correlates with expression of E1B and with morphological transformation.     

Inhibition of p53-Dependent,but Not p53-Independent,Cell Death by U19 Protein from Human Herpesvirus 6B

Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.     

Induction of the cell cycle in baby rat kidney cells by adenovirus type 5 E1A in the absence of E1B and a possible influence of p53.

From previous studies on the induction of DNA synthesis in quiescent primary baby rat kidney cells by adenovirus type 5 (Ad5) E1A deletion mutants, we concluded that induction is prevented only when cellular proteins p300 and pRb are both uncomplexed with E1A (J.A. Howe, J.S. Mymryk, C. Egan, P.E. Branton, and S.T. Bayley, Proc. Natl. Acad. Sci. USA 87:5883-5887, 1990). We have now examined induction by these same mutants in virus lacking the E1B region, so that cellular p53 was no longer complexed to the E1B 55-kDa protein. E1A mutants that fail to bind pRb induced DNA synthesis at a significantly lower level in Ad5 lacking E1B than in Ad5 containing E1B. Apparently, therefore, uncomplexed p53 can partially replace p300 in cooperating with pRb to suppress DNA synthesis in baby rat kidney cells.