Transactivation of Murine Cyclin A by Polyomavirus Large and Small T Antigens

Abstract

Polyomavirus large and small T antigens cooperate in the induction of S phase in serum-deprived Swiss 3T3 cells. While the large T antigen is able to induce S phase-specific enzymes, we have recently shown that both T antigens contribute to the production of the cyclins E and A and that the small T antigen is essential for the induction of cyclin A-dependent cdk2 activity (S. Schüchner and E. Wintersberger, J. Virol. 73:9266-9273, 1999). Here we present our attempts to elucidate the mechanisms by which the large and the small T antigens transactivate the murine cyclin A gene. Using Swiss 3T3 cells carrying the T antigens and various mutants thereof under the hormone-inducible mouse mammary tumor virus promoter, as well as transient-cotransfection experiments with the T antigens and cyclin A promoter-luciferase reporter constructs, we found the following. The large T antigen activates the cyclin A promoter via two transcription factor binding sites, a cyclic AMP responsive element (CRE), and the major negative regulatory site called CDE-CHR. While an intact binding site for pocket proteins is required for the function of this T antigen at the CDE-CHR, its activity at the CRE is largely independent thereof. In contrast, an intact J domain and an intact zinc finger are required at both sites. The small T antigen also appears to have an influence on the cyclin A promoter through the CRE as well as the CDE-CHR. For this an interaction with protein phosphatase 2A is essential; mutation of the J domain does not totally eliminate but greatly reduces the transactivating ability.The induction of S phase of the cell cycle is a complex reaction usually initiated at the cell surface through binding of ligands to receptors. Signal transduction pathways lead to the synthesis of S phase-specific enzymes and regulators, including the cyclins E and A. DNA tumor viruses require cells in S phase because they heavily depend on the cellular DNA synthesis machinery for the replication of their own DNA. Since they frequently infect differentiated cells, they have to interfere with the cellular mechanisms of growth regulation in order to drive cells out of the G 0 phase and into the S phase (reviewed in reference 19). This is accomplished by viral proteins which interact with various intermediates of the signal transduction pathway downstream of events taking place at the cell surface. One class of target for such viral proteins are the pocket proteins: pRB and its relatives p107 and p130. They negatively regulate members of the transcription factor family E2F (reviewed in reference 8). Adenovirus E1A, human papillomavirus (HPV) E7 protein, and the large T (LT) antigens of simian virus 40 (SV40), and polyomavirus (Py) bind to pocket proteins and cause a dissociation of the repressive complexes, which results in the transactivation of E2F responsive genes (19). Another region of the pleiotropic T antigens which is essential for this reaction is the J domain (reviewed in references 4 and 16), a sequence present in many chaperones capable ...

Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56γ Knockdown

“…Thus, in the presence of LT and activated Ras signaling, the NH 2 -terminal domain of ST appears to be sufficient to drive cells into S phase. Consistent with this hypothesis, mutations in the DnaJ domain of polyoma ST has been shown to strongly inhibit activation of the cyclin A promoter (50).…”

Transactivation of E2F-Regulated Genes by Polyomavirus Large T Antigen: Evidence for a Two-Step Mechanism

“…For growth arrest, asynchronously growing cells were seeded at 5 ϫ 10 5 cells per 100-mm-diameter petri dish; the next day, the serum concentration was reduced to 0.2% for 72 h. The cells were then either reinduced by addition of fresh medium containing 20% FCS or treated with dexamethasone at a concentration of 10 Ϫ6 mol/liter. Cell lines expressing the wild-type Py LT antigen (LTwt) and the E145D, P43A, and C452S mutant forms were described previously (31). Cell lines conditionally expressing LT antigens with other point mutations in the C-terminal region (Y442S and P670S) were produced by Polybrene-assisted transfections as described previously (24,31).…”

“…Mutations in either one of these sites abolish the transactivation of S phase-specific genes by LT antigen. In our previous work on Py LT antigen-mediated transactivation of the genes coding for thymidine kinase (25) and cyclin A (31), we observed that, in addition to the N terminus, regions within the C-terminal part of the LT protein play an important role (31). A mutation of the zinc finger present within this domain (C452S) interferes with transactivation.…”

Polyomavirus Small T Antigen Controls Viral Chromatin Modifications through Effects on Kinetics of Virus Growth and Cell Cycle Progression

“…SV40 sT regulates cell cycle progression at least in part through induction of cyclins D1 and A (49,56). Polyomavirus sT has been shown to promote cell cycle progression (34) and to synergize with large T for S-phase induction in Swiss 3T3 mouse fibroblasts (47,48). sT/PP2A may also affect rates and efficiency of encapsidation through phosphorylation of VP1 (18).…”