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

The early region 1B (E1B) of adenovirus type 5 (Ad5) encodes overlapping functions essential for virus mediated cellular transformation in cooperation with E1A by suppressing p53-mediated apoptosis or a G1 growth arrest (52, 62). The E1A proteins are potent transactivators that relieve cellular growth suppression and induce quiescent cells to enter S phase by binding members of the retinoblastoma protein family and transcription factors such as p300 (reviewed in reference 17). This action of E1A results in the accumulation of p53. p53 is a cellular growth suppressor that acts as a G1 checkpoint control (reviewed in references 37 and 74). In response to viral challenge, p53 may induce a G1 growth arrest by inducing genes such as the cyclin-dependent kinase inhibitor p21/WAF1/Cip1 gene (16, 69) or apoptosis by inducing genes such as bax1 (43). Either response by p53 is expected to severely hinder Ad replication and transformation (38). The E1B 55-kDa and 19-kDa proteins are required to fully transform cells (4, 19, 23, 56, 64). The E1B 19-kDa protein is a functional homologue of the proto-oncogene-encoded Bcl-2 and prevents apoptosis by similar mechanisms (12, 52). The E1B 55-kDa proteins of Ad5 and Ad12 have been hypothesized to permit E1A-induced DNA synthesis and transactivation by preventing a p53-mediated G1 growth arrest (51, 60, 61). The 55-kDa protein complexes with the amino-terminal end of p53 and inhibits its activity as a transcription factor (33, 72, 73). This inhibition of p53-mediated transactivation by the large E1B protein is required for transformation by both the weakly oncogenic group C and highly oncogenic group A Ads (33, 72, 75, 76). E1B 55-kDa protein-mediated inactivation of p53 has been hypothesized to be required for viral replication in the lytic infection (7); however, this has not been demonstrated. The Ad E4orf6 protein was recently shown to bind p53 and block transcriptional activation mediated by p53 (14, 46). Subsequently, the E4orf6 protein was shown to cooperate with the E1A and E1B proteins to transform baby rat kidney cells (46), to convert the nontumorigenic 293 human cell line (24) into a tumorigenic cell line in nude mice, and to block p53-dependent apoptosis (44). In both transformed and productively infected cells, coexpression of the E1B 55-kDa and the E4orf6 proteins decreased the stability of p53 (44, 46, 50). These findings suggest that the E4orf6 protein may encode some overlapping and redundant functions with the E1B 55-kDa protein with regard to transformation. At late times in the lytic infection, the E1B 55-kDa protein facilitates the transport of viral late mRNA while inhibiting the transport of most cellular mRNA in association with the E4orf6 protein (3, 8, 26, 36, 49). Ad mutants that fail to express the E1B 55-kDa protein are defective for expression of late viral proteins and replicate poorly. The functional interaction between the E1B 55-kDa and E4orf6 proteins may be mediated by primate-specific cellular factors (22, 47). The transport of several cellular messages, including the heat shock protein 70, β-tubulin, and interferon-inducible Mx-A and 6-16 mRNAs, requires the E1B 55-kDa protein late in Ad infection. This effect correlates with activation of their transcription during the late phase (45, 71). In addition to selectively blocking transport of most cellular mRNA, the E1B 55-kDa protein further modulates host cell shutoff by inhibiting host protein synthesis by mechanisms unrelated to the inhibition of mRNA transport (2). We have recently demonstrated that the E1B 55-kDa protein functions in promoting Ad replication independently of the cell cycle. E1B 55-kDa mutant Ads produce virus most efficiently when cells are infected during S phase and are restricted from replication in cells infected during G1 (20). These findings suggest that the E1B 55-kDa protein plays a role in deregulating the cell cycle to the advantage of the lytic infection. Perhaps this function represents a link between the functions of the E1B 55-kDa protein in the lytic infection and transformation. Because the E1B 55-kDa protein inhibits the function of p53, it has been hypothesized that an E1B 55-kDa mutant Ad can replicate only in cells lacking a functional p53. Evidence supporting this hypothesis includes the finding that the E1B 55-kDa mutant Ad dl1520 failed to lyse U2OS cells which contain wild-type p53. Furthermore, the E1B 55-kDa mutant virus induced more severe cytopathic effect in the RKO human colon cancer cell line transfected with a dominant negative p53 gene than in the parental cell line containing a wild-type p53 gene (7). However, p53 null tumor cells were not more susceptible to lysis by the E1B 55-kDa mutant virus than some cancer cells containing wild-type p53 (28). Furthermore, the relative ability of the E1B 55-kDa mutant virus to suppress tumor growth compared to the wild-type Ad was unaffected by the status of p53 (7). By contrast, Ridgway et al. (54) suggest that the interaction between p53 and the E1B 55-kDa protein is necessary for efficient Ad replication and that neither the wild-type nor E1B 55-kDa mutant virus replicated in p53-mutant cell lines. These authors reported that the wild-type Ad failed to efficiently shut off α-actin synthesis, synthesize viral DNA, and induce cytopathic effect in the p53 mutant cell lines T98G and 143B. They suggest that p53 may mediate the interaction between the E1B 55-kDa and E4orf6 proteins in the lytic infection (54). However, Rubenwolf et al. (55) demonstrated that p53 is not required for coimmunoprecipitation of the E1B 55-kDa and E4orf6 proteins although the two proteins have been shown to bind p53 independently (14, 55, 57). The work presented here demonstrates that the inability of the E1B mutant virus to replicate efficiently and produce virus in all infected cells is not strictly due to the failure to abrogate p53 function. Among a variety of cell lines analyzed, the capacity of the E1B 55-kDa mutant virus to replicate, synthesize viral DNA, and produce virus in all infected cells did not correlate with the status of p53. However, the ability of the E1B 55-kDa mutant virus to replicate differed among the cell types studied. Furthermore, the ability of the E1B 55-kDa mutant virus to induce cell killing correlated with permissivity to virus growth and not the status of p53. In a cell line expressing a temperature-sensitive p53 allele, active p53 only moderately affected the replication of the E1B 55-kDa mutant virus. The defect in E1B 55-kDa mutant virus replication due to reduced temperature was as much as 10-fold greater than the defect due to p53 function. Indeed, the E1B 55-kDa mutant virus produced equivalent yields of virus and synthesized equivalent levels of late viral proteins compared to the wild-type virus in cells maintained at 39°C but not at 32°C. The cell cycle restriction of the E1B 55-kDa mutant virus was also partially overcome at 39°C, and progeny virus were produced in a larger fraction of infected cells. By contrast, the apparent cell cycle restriction was exacerbated at 32°C. Since both the cell cycle restriction and the defect in late gene expression exhibited a cold-sensitive phenotype, we speculate that the functions of the E1B 55-kDa protein in promoting mRNA transport and cell cycle-independent viral replication may be linked.