Ahmed F. Yousef

Intrinsic Structural Disorder in Adenovirus E1A: a Viral Molecular Hub Linking Multiple Diverse Processes

Viruses are obligate intracellular parasites. Their genomes are not large enough to encode all the functions required to independently produce progeny; hence, viruses are absolutely dependent on host cell functions. Mechanistically, these host cell processes in eukaryotes are founded on an exquisitely complex series of molecular interactions. In particular, the execution of complex biological processes requires the precise interaction and regulation of thousands of proteins. The definition of cellular interactomes by systematic analysis of protein-protein interactions has revealed complex molecular networks (39, 82, 111, 122). Most cellular proteins interact with only one or two other proteins, making only one or two connections. However, the minority of proteins interact with tens, or even hundreds, of other proteins to form network hubs. Hub proteins play key roles in regulating and orchestrating the activity of the proteins they interact with, effectively creating functional modules within the cellular interactome (35, 48, 62). The central role served by cellular hub proteins in regulating cell functions makes them ideal targets during a viral infection. By targeting a single cellular hub, a viral regulatory protein can effectively gain control over an entire module, potentially comprised of hundreds of proteins. By targeting multiple cellular hubs, a virally encoded hub can transform the architecture of the cellular protein interaction network, reprogramming virtually all aspects of cell function and behavior. The viral oncogenes of the small DNA tumor viruses encode some of the most versatile and potent viral hub proteins. Among these, adenovirus E1A is one of the best characterized and is the subject of this review.

Roles for APIS and the 20S proteasome in adenovirus E1A-dependent transcription

We have determined distinct roles for different proteasome complexes in adenovirus (Ad) E1A‐dependent transcription. We show that the 19S ATPase, S8, as a component of 19S ATPase proteins independent of 20S (APIS), binds specifically to the E1A transactivation domain, conserved region 3 (CR3). Recruitment of APIS to CR3 enhances the ability of E1A to stimulate transcription from viral early gene promoters during Ad infection of human cells. The ability of CR3 to stimulate transcription in yeast is similarly dependent on the functional integrity of yeast APIS components, Sug1 and Sug2. The 20S proteasome is also recruited to CR3 independently of APIS and the 26S proteasome. Chromatin immunoprecipitation reveals that E1A, S8 and the 20S proteasome are recruited to both Ad early region gene promoters and early region gene sequences during Ad infection, suggesting their requirement in both transcriptional initiation and elongation. We also demonstrate that E1A CR3 transactivation and degradation sequences functionally overlap and that proteasome inhibitors repress E1A transcription. Taken together, these data demonstrate distinct roles for APIS and the 20S proteasome in E1A‐dependent transactivation.

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation

Hub proteins have central roles in regulating cellular processes. By targeting a single cellular hub, a viral oncogene may gain control over an entire module in the cellular interaction network that is potentially comprised of hundreds of proteins. The adenovirus E1A oncoprotein is a viral hub that interacts with many cellular hub proteins by short linear motifs/molecular recognition features (MoRFs). These interactions transform the architecture of the cellular protein interaction network and virtually reprogram the cell. To identify additional MoRFs within E1A, we screened portions of E1A for their ability to activate yeast pseudohyphal growth or differentiation. This identified a novel functional region within E1A conserved region 2 comprised of the sequence EVIDLT. This MoRF is necessary and sufficient to bind the N-terminal region of the SUMO conjugase UBC9, which also interacts with SUMO noncovalently and is involved in polySUMOylation. Our results suggest that E1A interferes with polySUMOylation, but not with monoSUMOylation. These data provide the first insight into the consequences of the interaction of E1A with UBC9, which was initially described in 1996. We further demonstrate that polySUMOylation regulates pseudohyphal growth and promyelocytic leukemia body reorganization by E1A. In conclusion, the interaction of the E1A oncogene with UBC9 mimics the normal binding between SUMO and UBC9 and represents a novel mechanism to modulate polySUMOylation.

Adenovirus E1A Directly Targets the E2F/DP-1 Complex

ABSTRACT Deregulation of the cell cycle is of paramount importance during adenovirus infection. Adenovirus normally infects quiescent cells and must initiate the cell cycle in order to propagate itself. The pRb family of proteins controls entry into the cell cycle by interacting with and repressing transcriptional activation by the E2F transcription factors. The viral E1A proteins indirectly activate E2F-dependent transcription and cell cycle entry, in part, by interacting with pRb and family members to free the E2Fs. We report here that an E1A 13S isoform can unexpectedly activate E2F-responsive gene expression independently of binding to the pRb family of proteins. We demonstrate that E1A binds to E2F/DP-1 complexes through a direct interaction with DP-1. E1A appears to utilize this binding to recruit itself to E2F-regulated promoters, and this allows the E1A 13S protein, but not the E1A 12S protein, to activate transcription independently of interaction with pRb. Importantly, expression of E1A 13S, but not E1A 12S, led to significant enhancement of E2F4 occupancy of E2F sites of two E2F-regulated promoters. These observations identify a novel mechanism by which adenovirus deregulates the cell cycle and suggest that E1A 13S may selectively activate a subset of E2F-regulated cellular genes during infection.

Identification of a second independent binding site for the pCAF acetyltransferase in adenovirus E1A

The conserved region 3 (CR3) portion of the human adenovirus (HAdV) 5 E1A protein functions as a potent transcriptional activator that induces expression of viral early genes during infection. Expression of HAdV-5 CR3 in the yeast Saccharomyces cerevisiae inhibits growth, as do the corresponding regions of the HAdV-3, 4, 9, 12 and 40 E1A proteins, which represent the remaining five HAdV subgroups. Growth inhibition is alleviated by disruption of the SAGA transcriptional regulatory complex, suggesting that CR3 targets the yeast SAGA complex. In yeast, transcriptional activation by several, but not all, of the CR3 regions requires the Gcn5 acetyltransferase component of SAGA. The CR3 regions of HAdV-3, 5, 9 and 40, but not HAdV-4 and 12 interact with the pCAF acetyltransferase, a mammalian ortholog of yeast Gcn5. Disruption of the previously described N-terminal pCAF binding site abrogates binding by the HAdV-5 243R E1A protein, but not the larger 289R E1A protein, which is otherwise identical except for the presence of CR3. RNA interference directed against pCAF decreased HAdV-5 CR3 dependent transcriptional activation in mammalian cells. Our results identify a second independent binding site for pCAF in E1A and suggest that it contributes to CR3 dependent transcriptional activation.

Coactivator requirements for p53-dependent transcription in the yeast Saccharomyces cerevisiae

p53 is a sequence‐specific DNA‐binding transcription factor and key regulator of cell cycle arrest and apoptosis. p53 is mutated in most human cancers and these mutations generally impair its ability to activate transcription. When expressed in Saccharomyces cerevisiae, p53 acts as a strong transcriptional activator allowing yeast to be used as a model system to study the effects of p53 mutations on activity. However, little is known about the exact mechanisms by which p53 functions in yeast. Using 76 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, INO80, ISW1, Mediator, RSC, SAGA, SAS, SLIK, SWI/SNF, and SWR1 transcriptional regulatory complexes on p53‐dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on p53 activation. Overall, our analysis indicates that there are several remarkable similarities between p53‐dependent transcription in yeast and mammalian cells, suggesting that yeast can serve as a valid model system for at least some aspects of p53 function.

Dissection of the C-Terminal Region of E1A Redefines the Roles of CtBP and Other Cellular Targets in Oncogenic Transformation

ABSTRACT Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A.

Comparison of E1A CR3-Dependent Transcriptional Activation across Six Different Human Adenovirus Subgroups

ABSTRACT The largest E1A isoform of human adenovirus (Ad) includes a C-4 zinc finger domain within conserved region 3 (CR3) that is largely responsible for activating transcription of the early viral genes. CR3 interacts with multiple cellular factors, but its mechanism of action is modeled primarily on the basis of the mechanism for the prototype E1A protein of human Ad type 5. We expanded this model to include a representative member from each of the six human Ad subgroups. All CR3 domains tested were capable of transactivation. However, there were dramatic differences in their levels of transcriptional activation. Despite these functional variations, the interactions of these representative CR3s with known cellular transcriptional regulators revealed only modest differences. Four common cellular targets of all representative CR3s were identified: the proteasome component human Sug1 (hSug1)/S8, the acetyltransferases p300/CREB binding protein (CBP), the mediator component mediator complex subunit 23 (MED23) protein, and TATA binding protein (TBP). The first three factors appear to be critical for CR3 function. RNA interference against human TBP showed no significant reduction in transactivation by any CR3 tested. These results indicate that the cellular factors previously shown to be important for transactivation by Ad5 CR3 are similarly bound by the E1A proteins of other types. This was confirmed experimentally using a transcriptional squelching assay, which demonstrated that the CR3 regions of each Ad type could compete with Ad5 CR3 for limiting factors. Interestingly, a mutant of Ad5 CR3 (V147L) was capable of squelching wild-type Ad5 CR3, despite its failure to bind TBP, MED23, p300/CBP-associated factor (pCAF), or p300/CBP, suggestive of the possibility that an additional as yet unidentified cellular factor is required for transactivation by E1A CR3.

Stigmasterol and Cholesterol Regulate the Expression of Elicitin Genes in Phytophthora sojae

Sterol acquisition by soilborne plant pathogens of the genus Phytophthora is presumed to involve extracellular proteins belonging to class-I elicitins. However, little is known about the relationship between sterol availability and elicitin secretion. The objective of this study was to determine the expression of class-I elicitin genes in Phytophthora sojae when grown in a medium containing stigmasterol or cholesterol. P. sojae growth was stimulated by nanomolar concentrations of stigmasterol and cholesterol, which also resulted in the down-regulation of its elicitin genes over time when expression profiles were monitored using real time Reverse Transcription Polymerase Chain Reaction (RT-PCR). The down-regulation of elicitin genes in response to the two sterols also coincided with a reduction in the amount of elicitins detected in spent filtrates. Our study is the first to show the influence of sterols on elicitin gene expression in Phytophthora, which is important with respect to the ecology of elicitin secretion as sterol carrier proteins in the environment.

Requirements for E1A dependent transcription in the yeast Saccharomyces cerevisiae

BackgroundThe human adenovirus type 5 early region 1A (E1A) gene encodes proteins that are potent regulators of transcription. E1A does not bind DNA directly, but is recruited to target promoters by the interaction with sequence specific DNA binding proteins. In mammalian systems, E1A has been shown to contain two regions that can independently induce transcription when fused to a heterologous DNA binding domain. When expressed in Saccharomyces cerevisiae, each of these regions of E1A also acts as a strong transcriptional activator. This allows yeast to be used as a model system to study mechanisms by which E1A stimulates transcription.ResultsUsing 81 mutant yeast strains, we have evaluated the effect of deleting components of the ADA, COMPASS, CSR, INO80, ISW1, NuA3, NuA4, Mediator, PAF, RSC, SAGA, SAS, SLIK, SWI/SNF and SWR1 transcriptional regulatory complexes on E1A dependent transcription. In addition, we examined the role of histone H2B ubiquitylation by Rad6/Bre1 on transcriptional activation.ConclusionOur analysis indicates that the two activation domains of E1A function via distinct mechanisms, identify new factors regulating E1A dependent transcription and suggest that yeast can serve as a valid model system for at least some aspects of E1A function.