Our previous global analysis of the effects of Ad5 infection on expression of human genes in normal human fibroblasts identified changes in some 10% of the genes examined and in genes associated with a variety of specific cellular processes or programs (51
). For example, the great majority of E2F-responsive genes increased substantially in expression by 12 h after infection, and Ad5 infection reversed the quiescence program (12
) and induced the core serum response. In contrast to results obtained with other viruses, including several herpesviruses and retroviruses (see reference 39
), genes encoding proteins that participate in innate and adaptive immune defenses to viral infection were not significantly enriched in any of the clusters of genes defined on the basis of the kinetics of the changes in the corresponding RNA concentrations following Ad5 infection (51
). It is now clear from the data presented here that the expression of many such genes is repressed in Ad5-infected cells, by a mechanism that requires the E1B 55-kDa protein: consensus k-means clustering and GO term analysis established that the set of genes that were increased in expression only in cells infected by the E1B 55-kDa-protein-null mutant Hr6 were heavily enriched in those associated with innate and adaptive immune responses to viral infection (Fig. ). This set included four HLA class I heavy chain genes, interferon-inducible genes, and genes encoding interleukins and proteins for regulators of the NF-κB pathway (Fig. ; Table ). The reductions of expression of these cellular genes, some quite large (Table ), were observed 30 h after Hr6 infection, when infected, quiescent HFFs are just entering the late phase of infection (51
). This temporal pattern suggests that the reduced concentrations of transcripts of cellular genes associated with immune defenses cannot be ascribed to regulation of mRNA export by the E1B 55-kDa protein (see references 2
), which occurs only during the late phase of infection. The E1B 55-kDa protein therefore appears to fulfill a previously unrecognized function, repression of expression of host cell genes that encode proteins that induce or execute innate and adaptive antiviral defense mechanisms (Fig. ).
As summarized in the introduction, previous studies using in vitro transcription and transient expression assays have demonstrated that the E1B 55-kDa protein can act as a repressor of transcription by RNA polymerase II, an activity that correlates with transforming activity. The mechanism of such repression is not fully understood, but the viral protein is thought to act on the basal transcription machinery via an as-yet-unidentified cellular corepressor (see reference 2
). Alanine substitutions at two C-terminal sites of phosphorylation, Ser490 and Ser491, have been reported to impair both inhibition of p53-dependent transcription by the E1B 55-kDa protein and repression of transcription by a Gal4 DNA-binding domain-E1B protein fusion (80
). However, mutations that introduce these substitutions had no effect on repression of expression of the cellular genes identified in these experiments by the E1B 55-kDa protein: the concentrations of their transcripts were not altered significantly in cells infected by Ad5E1B-sub17 (Fig. ). This result indicates that the E1B 55-kDa protein represses expression of specific cellular genes by a mechanism distinct from that previously characterized in simplified experimental systems.
The E1B protein has been reported to interact with cellular proteins that repress transcription, the corepressor complex that contains Sin3A and histone deacetylase I (Hdac I) (61
), and the death domain-associated protein (Daxx) (95
). Although a central sequence of the E1B protein, between amino acids 156 and 261, binds directly to Hdac I in vitro, the viral protein also interacts strongly with Sin3A in transformed 293 and infected HeLa cells (61
). Binding to this corepressor complex has been reported to be required for the ability of the viral protein to block repression of transcription by p53 (62
). Binding of the E1B 55-kDa protein to Daxx, which also appears to be direct, was observed to inhibit the stimulation of p53-dependent transcription by Daxx in transient expression assays (95
). The repression of expression of endogenous genes by the E1B protein described here cannot be attributed to effects on p53. Of the cellular genes present in cluster 2 (Fig. ), only two have been identified as direct targets of transcriptional regulation by p53 (41
). Nor do the C-terminal phosphorylation site substitutions that impair repression of p53-dependent transcription by the E1B protein (81
) eliminate repression of expression of these genes following infection of HFFs (Fig. ). Finally, and perhaps most compelling, the p53 transcriptional program is inhibited as effectively in Hr6-infected HFFs as it is in Ad5-infected cells (Fig. ; Table ).
The Daxx protein has also been reported to block the activity of several other transcriptional activators, to interact with Hdac II, and to share sequences in two putative amphipathic α-helices with Sin3A, properties that suggest that this protein can also function as a corepressor (see reference 68
). It is therefore possible that the Sin3A- and Hdac I-containing corepressor and/or Daxx contributes to repression of expression cellular genes by the E1B 55-kDa protein in infected cells.
Although the E1B 55-kDa protein is necessary to prevent expression of specific genes by 30 h after infection of normal human cells, the data presented here establish unequivocally that it is not required to block the action of p53 in HFFs: the suppression of the p53 transcriptional program characteristic of Ad5-infected HFFs (Fig. ) (51
) was also complete in cells infected by Hr6 or Ad5E1B-sub17 (Fig. ; Table ). It has been reported previously that accumulation of p53 in infected human lung carcinoma (A549) cells resulting from an R239A substitution in the E1B protein was not accompanied by increased synthesis of two products of p53 target genes, Mdm-2 and p21. Rather, expression of both cellular genes was repressed as effectively as in Ad5-infected cells (36
). Similarly, infection of normal human small airway epithelial cells by Ad5 mutants that cannot direct synthesis of any of the proteins made from the E1B 55-kDa open reading frame (Onyx-015) or that encode an E1B 55-kDa protein that cannot bind to p53 (Onyx-053) led to accumulation of p53 but not activation of expression of seven genes transcriptionally activated by p53 (59
). Our global analysis extends such findings not only to a significantly larger set of genes that are transcriptionally activated by p53 but also to genes that are repressed by this cellular protein (Fig. ; Table ) and to a second type of normal host cell. Thus, there is a growing body of evidence indicating that the E1B 55-kDa protein is not necessary to block the transcriptional function of p53 in normal human cells infected by Ad5.
As p53 accumulates in various normal and transformed host cells infected by E1B 55-kDa-protein-null mutants or by viruses carrying mutations that block the interaction of the E1B protein with p53 or the E5 Orf6 protein (see the introduction), this conclusion implies that activation of p53 is prevented in Ad5-infected cells by one or more additional mechanisms. The activity of p53 is strictly regulated, and its activation requires various posttranslational modifications. One modification that is crucial is acetylation by the histone acetyltransferase p300/Cbp (79
), which is bound by the viral E1A proteins (see reference 2
). The E1A-p300 interaction has been reported to be required for accumulation of p53 (11
). However, p53-dependent transcription has not been observed to increase in cells infected by mutants producing E1A proteins with alteration that impair their interaction with p300 (36
). During its activation, p53 is also modified by phosphorylation of specific residues and removal of ubiquitin (see references 58
), raising the possibility that adenoviral proteins may inhibit one or both of these reactions. One possible candidate is the E4 Orf4 protein, which has been shown to interact with protein phosphatase 2A to induce dephosphorylation of viral E1A and cellular SR splicing proteins, as well as decreased activity of cellular transcriptional activators, including JunB and E2f (19
). It has been proposed that the modifications required to activate p53 take place in the nuclear structures termed Pml nuclear bodies (Pml oncogenic domains or nuclear domains 10): in response to various forms of stress, p53 associates with Pml nuclear bodies, which contain several enzymes that modify the protein, including Hausp (a p53 deubiquitinase), Hipk2 (a p53 kinase), and the acetyltransferase Cbp/p300 (reviewed in references 3
, and 60
). The disruption of Pml nuclear bodies and reorganization of their components induced by the viral E4 Orf3 protein (9
) could also block activation of p53 in Ad5-infected cells.
Cellular RNAs that specify proteins implicated in progression through the cell cycle and proliferation have been observed to increase substantially in concentration following Ad5 infection of quiescent human foreskin and lung fibroblasts and the diploid fibroblast line WI38 (51
). Comparison of the alterations in expression of such genes, particularly as exemplified by cyclin E1, induced by infection with Ad5 or the E1B mutant Adhz60 or dl1520 (Onyx-015), led to the conclusion that the E1B 55-kDa protein open reading frame is required to stimulate expression of cyclin E1 (66
). The former mutant lacks all E1B coding sequences, whereas the latter carries a stop codon in place of the second E1B 55-kDa protein codon and consequently cannot direct synthesis of the E1B 55-kDa protein or any of the smaller related proteins (Fig. ). In contrast, the Hr6 mutation affects only the E1B 55-kDa protein (87
). As we observed similar degrees of stimulation of expression of cyclin E1 and other genes associated with cell cycle progression in Ad5- and Hr6-infected cells, we conclude that the E1B 55-kDa protein is not required for this response to infection. Rather, this effect appears to be a function of one of the smaller, E1B 55-kDa-protein-related proteins, which also fail to be made in dl1520-infected cells. Little is known about the molecular properties and functions of these viral proteins. However, the 156R protein, which shares both N- and C-terminal sequences with the 55-kDa protein (78
), can transform rodent cells in cooperation with E1A gene products (73
). Furthermore, the rate of proliferation of such transformed cells correlated with the concentration of the E1B 156R protein produced (73
), suggesting that this E1B protein might stimulate expression of genes associated with cell cycle progression and proliferation.