Clodagh C. O'Shea

Heterochromatin silencing of p53 target genes by a small viral protein

The transcription factor p53 (also known as TP53) guards against tumour and virus replication and is inactivated in almost all cancers. p53-activated transcription of target genes is thought to be synonymous with the stabilization of p53 in response to oncogenes and DNA damage. During adenovirus replication, the degradation of p53 by E1B-55k is considered essential for p53 inactivation, and is the basis for p53-selective viral cancer therapies. Here we reveal a dominant epigenetic mechanism that silences p53-activated transcription, irrespective of p53 phosphorylation and stabilization. We show that another adenoviral protein, E4-ORF3, inactivates p53 independently of E1B-55k by forming a nuclear structure that induces de novo H3K9me3 heterochromatin formation at p53 target promoters, preventing p53–DNA binding. This suppressive nuclear web is highly selective in silencing p53 promoters and operates in the backdrop of global transcriptional changes that drive oncogenic replication. These findings are important for understanding how high levels of wild-type p53 might also be inactivated in cancer as well as the mechanisms that induce aberrant epigenetic silencing of tumour-suppressor loci. Our study changes the longstanding definition of how p53 is inactivated in adenovirus infection and provides key insights that could enable the development of true p53-selective oncolytic viral therapies.

Adenoviral proteins mimic nutrient/growth signals to activate the mTOR pathway for viral replication

Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra‐ and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor‐limiting conditions. E4‐ORF1 mimics growth factor signaling by activating PI3‐kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4‐ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4‐ORF4 protein phosphatase 2A‐binding domain. We also show that mTOR activation is required for efficient S‐phase entry, independently of E2F activation, in adenovirus‐infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.

Viruses : seeking and destroying the tumor program

DNA viruses have enormous utility in cancer research, both as tools for tumor target discovery as well as agents for lytic cancer therapies. This is because there is a profound functional overlap between the DNA viral and tumor cell programs. DNA viruses encode proteins that elicit growth deregulation in infected cells similar to that engendered by mutations in tumor cells. Evolution has refined viral proteins to target the critical cellular hubs that regulate growth. Thus, viral proteins are discriminating biochemical probes that can be used to identify and characterize novel tumor targets. Moreover, the overlap between the DNA viral and tumor programs can also be exploited for the development of lytic cancer therapies. Discovering whether tumor cells selectively complement the replication of viral mutants can reveal novel oncolytic viral therapies, as well as unexpected tumor properties. For example, altered RNA export was recently uncovered as a novel tumor cell property that underlies ONYX-015 replication, a promising oncolytic adenoviral therapy. A perspective is provided on how adenovirus could be systematically exploited to map the requisite role, or indeed the redundancy, of cellular pathways that act in an integrated program to elicit pathological replication. This knowledge has important applications for the rational design of the next generation of oncolytic viruses, as well as the discovery of efficacious combination cancer therapies.

Adenovirus Overrides Cellular Checkpoints for Protein Translation

mTOR is a critical regulator of protein translation, and plays an important role in controlling cellular replication. Recent studies indicate that nutrient and growth factor mediated activation of mTOR is deregulated in human cancer, and therefore represents an attractive tumor target. However, activation of mTOR is a complex process that is not yet fully understood. DNA viruses and tumor cells often perturb similar cellular pathways to facilitate their replication. In a recent study, we used adenovirus as a novel tool to probe the mechanisms underlying the inappropriate activation of mTOR in quiescent primary cells. These studies revealed that adenovirus encodes two viral proteins, E4-ORF1 and E4-ORF4, which activate mTOR, even in the absence of nutrient/growth factor signals, and which play a role in promoting viral replication. E4-ORF1 mimics growth factor signaling to mTOR by activating PI3-kinase, whereas E4-ORF4, which binds and relocalizes PP2A, can substitute for glucose mediated activation of mTOR. We discuss insights from this study, together with the similarities that may exist between viruses and tumor cells with respect to the mechanistic and functional requirements for mTOR activation in driving their aberrant DNA replication.

Modulation of the ARF-p53 Pathway by the Small DNA Tumor Viruses

The small DNA tumor viruses encode proteins that subvert many of the pivotal growth regulatory pathways within the cell to facilitate their own replication. The cell responds to viral infection/proteins by activating the p53 tumor suppressor pathway. Activation of p53 could impair a productive viral infection at many levels, including the inhibition of viral DNA replication and/or the premature apoptosis of infected cells. Therefore, DNA viruses encode proteins that inactivate the p53 tumor suppressor pathway. Understanding how DNA viral proteins activate/inactivate the p53 pathway has provided invaluable insights into tumorigenesis. Recent studies with polyoma virus have identified a viral protein (PyST) that inhibits ARF-mediated activation of p53, and revealed a novel role for PP2A in the regulation of the ARF-p53 tumor suppressor pathway.

Critical Role for Arginine Methylation in Adenovirus-Infected Cells

ABSTRACT During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection.

The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication

One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system‐wide protein‐protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context‐dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein‐protein interactions and structures. WIREs Syst Biol Med 2011 3 48–73 DOI: 10.1002/wsbm.88

aMAGEing New Players Enter the RING to Promote Ubiquitylation

The MAGE proteins are best known as curious tumor-specific antigens. However, Doyle et al. (2010) reveal that MAGE proteins interact with RING proteins to promote ubiquitylation which provides important new insights into the physiological and pathological functions of this enigmatic family of proteins.

Viruses: tools for tumor target discovery, and agents for oncolytic therapies – an introduction

Viruses: tools for tumor target discovery, and agents for oncolytic therapies – an introduction

Adenovirus E4-ORF3 Targets PIAS3 and Together with E1B-55K Remodels SUMO Interactions in the Nucleus and at Virus Genome Replication Domains

ABSTRACT Adenovirus E4-ORF3 and E1B-55K converge in subverting critical overlapping cellular pathways to facilitate virus replication. Here, we show that E1B-55K and E4-ORF3 induce sumoylation and the assembly of SUMO2/3 viral genome replication domains. Using a conjugation-deficient SUMO2 construct, we demonstrate that SUMO2/3 is recruited to E2A viral genome replication domains through noncovalent interactions. E1B-55K and E4-ORF3 have critical functions in inactivating MRN and ATM to facilitate viral genome replication. We show that ATM kinase inhibitors rescue ΔE1B-55K/ΔE4-ORF3 viral genome replication and that the assembly of E2A domains recruits SUMO2/3 independently of E1B-55K and E4-ORF3. However, the morphology and organization of SUMO2/3-associated E2A domains is strikingly different from that in wild-type Ad5-infected cells. These data reveal that E1B-55K and E4-ORF3 specify the nuclear compartmentalization and structure of SUMO2/3-associated E2A domains, which could have important functions in viral replication. We show that E4-ORF3 specifically targets and sequesters the cellular E3 SUMO ligase PIAS3 but not PIAS1, PIAS2, or PIAS4. The assembly of E4-ORF3 into a multivalent nuclear matrix is required to target PIAS3. In contrast to MRN, PIAS3 is targeted by E4-ORF3 proteins from disparate adenovirus subgroups. Our studies reveal that PIAS3 is a novel and evolutionarily conserved target of E4-ORF3 in human adenovirus infections. Furthermore, we reveal that viral proteins not only disrupt but also usurp SUMO2/3 to transform the nucleus and assemble novel genomic domains that could facilitate pathological viral replication. IMPORTANCE SUMO is a key posttranslational modification that modulates the function, localization, and assembly of protein complexes. In the ever-escalating host-pathogen arms race, viruses have evolved strategies to subvert sumoylation. Adenovirus is a small DNA tumor virus that is a global human pathogen and key biomedical agent in basic research and therapy. We show that adenovirus infection induces global changes in SUMO localization and conjugation. Using virus and SUMO mutants, we demonstrate that E1B-55K and E4-ORF3 disrupt and usurp SUMO2/3 interactions to transform the nucleus and assemble highly structured and compartmentalized viral genome domains. We reveal that the cellular E3 SUMO ligase PIAS3 is a novel and conserved target of E4-ORF3 proteins from disparate adenovirus subgroups. The induction of sumoylation and SUMO2/3 viral replication domains by early viral proteins could play an important role in determining the outcome of viral infection.