Although no structural information about the multifunctional species C adenovirus E1B 55-kDa protein is available, a considerable body of information about sequence motifs, sites of posttranslational modification, and sequences required for interaction with various viral or cellular proteins has been collected (reviewed in reference 58
). None of the substitution mutations in the E1B protein-coding sequence that altered such previously characterized features, including the nuclear export signal, RNP motif, and C-terminal sites of phosphorylation, examined in these experiments specifically impaired viral replication in normal human cells exposed to IFN (). In conjunction with our previous observation that mutations that prevent assembly of the virus-specific E3 ubiquitin ligase in infected cells also fail to render viral replication sensitive to exogenous IFN (46
), these data suggest that the E1B 55-kDa protein blocks the antiviral action of IFN by a mechanism that does not depend on previously described properties of the protein. Several Ala substitutions in segments of the E1B 55-kDa protein predicted to be surface exposed and not previously studied, such as the alterations in the N-terminal segment that also encodes the C terminus of the E1B 19-kDa protein in a different reading frame introduced by the AdEasyE1Sub1, AdEasyE1Sub2, and AdEasyE1Sub3 mutations, were also without effect (). In fact, only the replacement with Ala of residues 443 to 448, a run of 6 charged or hydrophilic residues, in the E1B Sub19 mutations increased the sensitivity of viral replication to IFN () independently of any reductions in accumulation of the altered protein (). As mentioned previously, estimation of the impact of the Sub19 mutations on viral yield in IFN-treated cells was complicated by the very small plaque phenotype exhibited by AdEasyE1Sub19 (see Results), which suggests that this altered E1B 55-kDa protein acts as a dominant negative. This interpretation is consistent with both the larger quantities of the E1B Sub19 protein than of the endogenous wild-type protein likely produced in AdEasyE1Sub19-infected 293 cells () and the ability of the E1B 55-kDa protein to self-associate when synthesized in the absence of other viral proteins (71
). Regardless, measurement of the increase in the concentration of viral genomes over the value determined soon after entry (2 h p.i.), a parameter that is independent of any differences in the numbers of infecting particles or genomes, established that the E1B Sub19 mutations result in a severe defect in viral DNA synthesis specifically in IFN-treated cells ().
We have previously reported that expression of some 130 IFN-inducible genes is increased significantly when the E1B 55-kDa protein is not present in infected cells, as are the concentrations of several pre-mRNAs synthesized from such genes (44
). The large increases in accumulation of IFN-inducible mRNAs in untreated or IFN-treated cells infected with AdEasyE1Sub19 over the levels measured in AdEasyE1-infected cells () suggested that protection of viral replication and DNA synthesis against IFN-induced inhibition depends on transcriptional repression by the E1B protein: insertion of 4 amino acids at R443, the first residue replaced by Ala in the E1B Sub19 protein (), impairs both repression of p53-dependent transcription and the ability of an E1B 55-kDa protein–Gal4 DNA-binding domain fusion protein to act as a direct repressor of transcription in transient expression assays (35
). The increases in concentrations of newly synthesized, IFN-inducible pre-mRNAs observed in AdEasyE1Sub19-infected cells compared to AdEasyE1-infected cells () provide direct experimental support for the conclusion that the E1B 55-kDa protein represses transcription of specific genes in infected normal human cells.
Although the mechanism of such repression is not yet known (see below), it can be distinguished in several respects from that proposed for inhibition of transcription of p53-regulated genes (2
), which has been implicated in the ability of the E1B 55-kDa protein to cooperate with viral E1A proteins in transformation of rodent cells (see Introduction). Substitutions of the C-terminal sites of phosphorylation of the Ad5 or Ad12 E1B protein reduced its ability to inhibit p53-dependent transcription or to act as a repressor when fused to a heterologous DNA-binding domain (37
). However, such mutations neither induced increased expression of E1B 55-kDa protein-repressed genes in infected HFFs (44
) nor increased the sensitivity of viral replication () or DNA synthesis (data not shown) to IFN. The E1B protein alone is sufficient to repress p53-dependent transcription in cells transiently or stably synthesizing the viral protein (35
). The E1B 55-kDa protein fully competent to rescue the defects in genome replication of the null mutant AdEasyE1Δ2347 when stably produced in HFFs () also inhibited accumulation of mRNAs encoded by the p53-regulated BAX and MDM2 genes (). In contrast, it had no effect whatsoever on activation of expression of IFN-inducible genes by exogenous IFN ( and ). It therefore appears that the E1B 55-kDa protein can inhibit transcription of cellular genes by multiple mechanisms, depending on whether it is synthesized in the absence of other viral proteins or in the context of infected cells.
The impaired repression of transcription of IFN-inducible genes in cells infected by AdEasyE1Δ2347 or AdEasyE1Sub19 could not be attributed to alterations in the kinetics or degree of activation of Stat1 () or its nuclear localization (data not shown), suggesting that the E1B 55-kDa protein does not target the signal transduction pathway responsible for activation of transcription of IFN-inducible genes. However, it is not clear whether this protein acts directly to repress transcription of IFN-inducible genes as it can in in vitro
or transient expression assays. The proximity of the E1B Sub19 substitutions to the R443 insertion in the previously described repression domain is consistent with such a mechanism, and the well-characterized E1B protein-containing E3 ubiquitin ligase is dispensable for protection against IFN-induced inhibition of viral replication (47
). On the other hand, mutations that prevent binding by the only motif in the protein implicated in interaction with nucleic acids, the RNP motif (79
), did not reduce the resistance of viral replication to IFN (), nor did analysis of the clusters of genes differentially expressed in cells infected by Ad5 and an E1B 55-kDa protein null mutant (44
) by using FIRE (Finding Informative Regulatory Elements) (80
) identify any sequence motif(s) common to or overrepresented among the promoters of E1B-repressed genes (data not shown). Furthermore, the E1B 55-kDa protein has more recently been demonstrated to function as a Sumol E3 ligase (33
) and could therefore regulate transcription indirectly via this activity.
The failure of the E1B protein to block activation of expression of IFN-inducible genes in response to exogenous IFN when synthesized in uninfected HFFs () implies that either one or more additional viral gene products or, perhaps less likely, alterations in the host cell environment induced by infection are also required. Analysis of proteins associated with the E1B 55-kDa protein by mass spectrometry identified the viral IVa2
and L4 100-kDa proteins (4
). The E1B-IVa2
protein interaction was confirmed by coimmunoprecipitation (4
) but seems unlikely to contribute to inhibition of expression of IFN-inducible genes: although this protein possesses sequence-specific DNA-binding activity, it contributes to activation of transcription from the viral major late promoter (81
). Furthermore, only 15 cellular genes were found to carry sequences corresponding to the sequence recognized by the IVa2
protein between positions −1000 and +500, and none were repressed by the E1B 55-kDa protein (44
). The small RNA VA-RNAI was the first of several adenovirus-encoded inhibitors of the antiviral effects of IFN to be identified (84
). However, this RNA acts downstream of expression of IFN-inducible genes by blocking activation of a specific effector of the IFN response, interferon-induced double-stranded RNA activated protein kinase E1 (E1F2AK2, or Pkr) (85
). The viral E1A proteins prevent inhibition of replication of vesicular stomatitis virus by exogenous IFN (60
) and inhibit the activation of transcription of IFN-inducible genes (86
). Such inhibition is the result of suppression of the Jak-Stat signaling pathway that leads to assembly in the nucleus of the crucial activator Isgf3 (89
) and also sequestration of the coactivator p300 (93
). Although the E1A proteins are potent repressors of expression of IFN-inducible genes when synthesized in the absence of any other viral proteins (86
), they are incapable of maintaining suppression of expression of such cellular genes (), even when produced in large quantities in AdEasyE1Sub19-infected cells ( and ).
Like the E1B 55-kDa protein () (46
), the viral E4 Orf3 protein prevents inhibition of viral genome replication in cells exposed to exogenous IFN (61
). This protective function of the E4 Orf3 protein, which has long been known to reorganize components of Pml nuclear bodies, including the Pml protein, into track-like structures (94
), becomes dispensable when production of Pml or Daxx is impaired by RNA interference (97
). Although the relocalization of Pml and association with the E4 Orf3 protein are indistinguishable in IFN-treated (or untreated) HFFs infected by wild-type and E1B 55-kDa protein null mutant viruses (46
), it is possible that the E1B protein functions downstream of E4 Orf3-dependent Pml body disruption. Indeed, IFN-treated cells infected by E1B 55-kDa protein null or E4 Orf3 protein null mutants display a strikingly similar failure in the formation of viral replication centers (46
). Furthermore, the E1B protein has been reported to interact with both E4 Orf3 (96
) and Daxx (98
) and to induce proteasomal degradation of the latter by a mechanism that does not require the Ad E3 ubiquitin ligase (99
). The species C human adenovirus E4 orf3 protein also sequesters components of the Mre11-Rad50-Nbs1 (MRN) double-strand break repair complex into the track-like structures (8
), a function that is necessary for efficient viral DNA synthesis when these cellular proteins cannot be targeted for proteasomal degradation by the virus-specific E3 ubiquitin ligase (19
). Replication of the genome of a double mutant virus null for production of both the E1B 55-kDa and E4 Orf3 proteins proved to be so defective in HFFs that it was not possible to compare the sensitivity of its replication to exogenous IFN to that of the single mutant parents (data not shown). It will therefore be of considerable interest to determine whether the E1A or E4 Orf3 proteins allow repression of IFN-inducible genes when also made in HFFs stably producing the E1B 55-kDa protein.