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• :Molekulare Virologie
• :Arbeitsgebiete

Arbeitsgebiete

Adenovirus regulatory proteins

We use adenovirus type 5 (Ad5) as a model system to study the molecular interactions involved in viral oncogenesis, focusing particular attention on mechanisms regulated by the Ad5 early proteins: E1B-55K, E4orf6 and E4orf3. Our current studies are intended to determine the molecular mechanisms by which these proteins regulate viral replication, and how this regulation is related to their cellular growth transforming activities. Projects focus on genetic, molecular and biochemical analyses of viral E1B and E4 gene products, the identification of cellular interaction partners, and the evaluation of their oncogenic and mutagenic properties in tissue culture as well as animal models. Our basic studies should provide valuable information on molecular strategies used by adenovirus for viral replication and may reveal new principles of viral transformation relevant to human neoplasms. Moreover, with respect to the reported antitumoral effect of E1B-55K defective adenoviruses (e.g. ONYX-015/dl1520) in human malignant cells, detailed knowledge of the functions encoded by E1B-55K can provide a rational basis for the design of adenovirus-based cancer therapeutics.

Role of E1B-55K in productive infection and cell transformation

The 55-kDa product from early region 1B (E1B-55K) is a multifunctional regulator of adenovirus replication that controls several processes, including modulation of p53-induced apoptosis and growth arrest, selective late viral mRNA transport and shut-off of host cell protein synthesis. In addition, the adenovirus protein provides functions for complete oncogenic transformation of mammalian cells in cooperation with E1A oncoproteins. The transforming potential of E1B-55K correlates with its ability to act as a direct transcriptional repressor targeted to p53-responsive promoters by binding to the tumor suppressor protein. Considerable evidence suggests that these activities antagonize p53-induced apoptosis and/or cell cycle arrest. Although neutralizing p53 tumor suppressor functions is an integral part of the molecular mechanisms used by E1B-55K to contribute to complete transformation of primary cells, it is now becoming clear that the mode of action of E1B-55K during transformation may involve additional functions and other protein interactions.
We discovered that E1B-55K continuously shuttles between the nucleus and cytoplasm. Efficient nuclear import is mediated by conjugation with the small ubiquitin-related modifier protein 1 (SUMO1), whereas nuclear export is dependent on a leucine-rich nuclear export signal (NES) of the HIV-1 Rev-type. Our studies revealed that SUMOylation is absolutely required to enhance transformation in conjunction with E1A, and to inhibit p53-mediated transactivation. Recent data indicate that this posttranslational modification is required for efficient nuclear accumulation and intranuclear targeting of the adenovirus protein. This introduces a new function for Ad5 E1B-55K and suggests that regulation of nuclear targeting is an important key to how the adenovirus protein contributes to transformation. Work is in progress to determine whether continuous nuclear export of E1B-55K is required for transformation in combination with E1A and to investigate whether nucleo-cytoplasmic shuttling of the viral protein is essential for efficient viral replication.
We also found that the E1B-associated protein E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55K in lytically infected cells. Because expression of E1B-AP5 efficiently interferes with adenovirus E1A/E1B-mediated transformation of primary cells in culture it is possible that E1B-55K additionally exerts its oncogenic activities by perturbing post-transcriptional processes of the host cell. These observations would establish an exciting link between post-transcriptional regulation and transformation. An important aim of our current studies is, therefore, to determine the function of E1B-AP5 and to establish whether this protein represents a mediator for the oncogenic properties of E1B-55K.

Transforming potential of Ad5 E4orf3 and E4orf6 oncoproteins

Although expression of E1A and E1B oncogenes from Ad5 is sufficient for full transformation of primary cells in culture, it has long been discussed whether gene products encoded in early region 4 (E4) are also implicated in the process of virus-mediated oncogenesis. Evidence for involvement of the E4 region in transformation events stems from several earlier studies indicating that some E4 proteins may have partial transforming activities, stimulating E1A/E1B transformation and contributing to the transformed cell phenotype when present.
Consistent with this interpretation we found that two gene products from this early transcription unit, E4orf6 and E4orf3, substantially enhance transformation of primary rat cells in cooperation with Ad5 E1A/E1B gene products. Transformed cells stably expressing E4orf6 and/or E4orf3 exhibit multiple properties commonly associated with advanced oncogenic transformation and, in the case of E4orf6, dramatically accelerated tumor growth in nude mice. Studies in this laboratory and others suggest that the transforming and oncogenic activities of both E4 gene products are based on novel molecular mechanisms mediated by interactions with key cellular regulators involved in transcription, apoptosis, cell cycle control, DNA repair and proteolytic degradation.
We discovered that E4orf6, like E1B-55K, physically interacts with p53 and blocks p53 transcriptional functions. In addition, the E4 protein antagonizes the E1A-induced accumulation of p53 by targeting the tumor suppressor protein into the ubiquitin-dependent proteasomal degradation pathway in combination with E1B-55K and a novel SCF-like E3 ubiquitin ligase complex. In light of these observations, we are currently investigating the molecular basis underlying the E4orf6/E1B-55K-induced degradation of p53, and determining whether this activity plays a role in the E4orf6/E1B-55K-dependent stimulation of viral late mRNA export in productively infected cells.
Our studies also revealed that the ability of E4orf3 to promote oncogenic cell growth is probably due to combinatorial effects that involve the binding to E1B-55K, and the reorganization of dot-like matrix-associated multiprotein complexes known as nuclear bodies (NBs) or PML oncogenic domains (PODs). The later activity fits well in a model in which the modulation of NB-associated cellular factors by viral oncoproteins may trigger a cascade of processes that cause uncontrolled cell proliferation and neoplastic growth. We are now testing the model, and attempting to elucidate the mechanisms by which E4orf3 disrupts the integrity of NBs using genetic and biochemical approaches.

"Hit-and-run" transformation

Despite some functional similarities between E1B-55K and both E4 proteins, E1A/E4-mediated transformation differs fundamentally from the classical E1A/E1B pathway. While in the later the viral genes persist in the transformed cells, the majority of E1A/E4orf6 or E1A/E4orf3 transformants lack E4- and E1A-specific DNA sequences indicating that E4 proteins are necessary to initiate but not to maintain cellular transformation. The fact that transient expression of E1A plus E4orf6 or E4orf3 proved to be mutagenic suggests that the viral genes mediate such "hit-and-run" transformation by inducing oncogenic mutations in cellular genes.
Although not yet understood at the molecular level, there is good reason to believe that accumulation of mutations is probably triggered by the cooperation of various factors, including the unscheduled induction of cellular DNA synthesis by E1A as well as the modulation of p53 and NBs by E4orf6 and E4orf3, respectively. The later possibility is intriguing because other viral oncoproteins similarly implicated in "hit-and-run" transformation, such as SV40 Tag and HCMV IE1/IE2, also modulate the function of p53 as well as the integrity of NBs. Perhaps more significant, the reported inhibition of DNA double-strand break repair by E4orf3 and E4orf6 through binding to DNA-PK and/or modulation of the Mre11/Rad50/NBS1 complex could potentially contribute to the induction of mutations and genetic instability, thus forming the basis for "hit-and-run" transformation as observed with both E4 gene products. An important goal of our current studies is, therefore, to test whether the mutagenic activities of both E4 proteins correlate with their ability to interact with and to modulate the function of cellular components involved in DNA double-strand break repair.

Adenovirus-based cancer therapeutics

In cancer gene therapy the failure to achieve enough tumor transduction to cause complete remission has presently led to the revival of replication-competent viruses. Among those conditional replicative adenoviruses, such as the E1B-55K-deleted virus ONYX-015 (dl1520), have become the vector of choice in many preclinical studies and clinical cancer therapy protocols. However, the widespread use of these viruses as potential anti-cancer agents is still hampered by several problems, most notably their low degree in tumor-selective replication. Several observations indicate that this limitation can be overcome, at least in part, by regulation of viral genes with tumor-specific promoters and/or deletions of viral functions dispensable for replication in cancer cells.
On the basis of these findings we are investigating whether adenovirus replication and cell lysis can be restricted to tumor cells by introducing specific mutations in viral key regulatory genes. Our approach involves the generation of Ad5 mutants carrying amino acid changes in functional domains of the E1B-55K, E4orf6 and/or E4orf3 proteins. Genetic engineering of these virus mutants is done using an efficient bacimd cloning system which was established in our laboratory. Current projects focus on the phenotypic characterization of these mutant viruses in tissue culture cells, and the evaluation of their antitumoral efficacy following intratumoral administration to nude mouse-human tumor xenografts.