Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60 c-src and in transformation.Polyomavirus T antigens function both in replication of the virus and in transformation of the host cell. Large T is central to virus production as the initiator of viral DNA replication (20). Middle T and small T also play important roles in different aspects of polyomavirus infection (21,23,55). Defects in viral DNA replication and transcription, as well as defects in viral assembly, have been observed in different mutants of middle T and small T (1,7,8,22,38). Each of the viral early proteins also contributes to regulation of host cell function. Large T is able to immortalize primary cells (44), to block differentiation (37), and to provoke apoptosis (18, 48). These activities are mediated via association with the retinoblastoma susceptibility (Rb) family of tumor suppressors. Middle T, the major transforming protein, works through activation of cellular signaling pathways that are regulated by src-family tyrosine phosphorylation (15). Small T is able to promote cell cycle progression via association with protein phosphatase 2A (PP2A) (39).All three T antigens are produced by differential splicing of common primary transcripts (56). As a result, they have the identical N-terminal sequence of 79 amino acids that encompasses a DnaJ domain. DnaJ domains, consisting of approximately 70 amino acids, have a helical structure in which a conserved HPD motif is found between helix 2 and helix 3 (2, 13, 32, 43, 54). DnaJ domains, found in a broad range of proteins, function to stimulate ...

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...