Activation of CREB/ATF Sites by Polyomavirus Large T Antigen

Abstract

Polyomavirus large T antigen (LT) has a direct role in viral replication and a profound effect on cell phenotype. It promotes cell cycle progression, immortalizes primary cells, blocks differentiation, and causes apoptosis. While much of large T function is related to its effects on tumor suppressors of the retinoblastoma susceptibility (Rb) gene family, we have previously shown that activation of the cyclin A promoter can occur through a non-Rb-dependent mechanism. Here we show that activation occurs via an ATF/CREB site. Investigation of the mechanism indicates that large T can synergize with CREB family members to activate transcription. Experiments with Gal4-CREB constructs show that synergy is independent of CREB phosphorylation by protein kinase A. Examination of synergy with Gal4-CREB deletion constructs indicates that large T acts on the constitutive activation domain of CREB. Large T can bind to CREB in vivo. Genetic analysis shows that the DNA-binding domain (residues 264 to 420) is sufficient to activate transcription when it is localized to the nucleus. Further analysis of the DNA-binding domain shows that while site-specific DNA binding is not required, non-site-specific DNA binding is important for the activation. Thus, CREB binding and DNA binding are both important for large T activation of CREB/ATF sites. In contrast to previous models where large T transactivation occurred indirectly, these results also suggest that large T can act directly at promoters to activate transcription.Polyomavirus large T antigens have a dual role, acting directly in viral DNA replication and transcription but also functioning to alter host cell signaling. The role of large T antigens in viral DNA replication has been extensively studied. Simian virus 40 (SV40) has provided the major model for establishing the mechanisms of cellular DNA replication, and its replication is still the best understood (8, 70). During a productive infection, murine polyomavirus large T initiates viral DNA replication (23). The interaction of large T with AP-1 enhances origin unwinding (29). In addition, association with histone acetyltransferases is involved in large T-mediated DNA replication (87). By analogy to SV40 (73, 85), large T is also likely to participate in the elongation phase of DNA synthesis. Its role in replication is important in other contexts as well. In transformation it is responsible for integration (16) and excision (4) of the viral genome and can also promote recombination (75, 76).Large T antigens have broad effects on the host cell. Large T, the major transforming protein of SV40, can participate in the transformation of human cells (30,31). Polyomavirus large T does not transform by itself, and viruses that make only large T do not cause tumors. Much of the difference in phenotype comes from the obvious interaction of SV40 large T with p53 (41, 44). Although murine polyomavirus large T can interact with p53 phosphorylated on serine 18 (17), polyomavirus tumors show no evidence of a block in p53 function seen in S...

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