The amino-terminal domain of SV40 large tumor antigen (TAg) is required for efficient viral DNA replication. However, the biochemical activity associated with this domain has remained obscure. We show here that the amino-terminal domain of TAg shares functional homology with the J-domain of DnaJ/hsp40 molecular chaperones. DnaJ proteins function as cofactors by regulating the activity of a member of the 70-kD heat shock protein family. Genetic analyses demonstrated that amino-terminal sequences of TAg comprise a novel J-domain that mediates a specific interaction with the constitutively expressed hsc70 and show that the J-domain is also required for efficient viral DNA replication in vivo. Furthermore, we demonstrated that the J-domain of two human DnaJ homologs, HSJ1 or DNAJ2, could substitute functionally for the amino-terminus of TAg in promoting viral DNA replication. Together, our findings suggest that TAg uses its J-domain to support SV40 DNA replication in a manner that is strikingly similar to the use of Escherichia coli DnaJ by bacteriophage g in DNA replication. However, TAg has evolved a more efficient strategy of DNA replication through an intrinsic J-domain to associate directly with a partner chaperone protein. Our observations provide evidence of a role for chaperone proteins in the process of eukaryotic DNA replication.

Polyomavirus causes a broad spectrum of tumors as the result of the action of its early proteins. This work compares signaling from middle T antigen (MT), the major transforming protein, to that from small T antigen (ST). The abilities of MT mutants to promote cell cycle progression in serum-starved NIH 3T3 cells were compared. Transformation-defective mutants lacking association with SHC or with phosphatidylinositol 3-kinase (PI3-K) retained the ability to induce DNA synthesis as measured by bromodeoxyuridine incorporation. Only when both interactions were lost in the Y250F/Y315F double mutant was MT inactive. ST promoted cell cycle progression in a manner dependent on its binding of protein phosphatase 2A (PP2A). Since the Y250F/ Y315F MT mutant was wild type for PP2A binding yet unable to promote cell cycle progression, while ST was capable of promoting cell cycle progression, these experiments revealed a functional difference in MT and ST signaling via PP2A. Assays testing the abilities of MT and ST to induce the c-fos promoter and to activate c-jun kinase led to the same conclusion. ST, but not Y250F/Y315F MT, was able to activate the c-fos promoter through its interaction with PP2A. In contrast, MT, but not ST, was able to activate c-jun kinase by virtue of its interaction with PP2A.Polyomaviruses have proven to be valuable models for studying growth regulation. The viruses require the apparatus of cellular DNA synthesis for their own replication. To meet this need, these viruses have evolved many different ways to intervene in cellular growth regulation, which can cause a broad range of tumors in different types of cells (26, 31). Examining how these viruses work has provided repeated leads that can be applied to understanding normal and abnormal cell behavior. Tyrosine phosphorylation (30) and phosphatidylinositol 3-kinase (PI3-K) (99) signaling are examples of two avenues of investigation driven by studies of the polyomavirus middle T antigen (MT).MT is the most important of the early gene products for transformation. MT is necessary (14, 87) and in many cases sufficient (89) for transformation in vitro. The importance of MT in tumor induction is also evident (3, 33). MT is associated with membranes and underlying cytoskeletal elements (1, 47,77,80). Its ability to transform depends upon those associations (14).MT functions as a kind of adaptor on which a collection of cellular signaling proteins are assembled (Fig. 1). MT, like polyomavirus small T antigen (ST), binds the A and C subunits of protein phosphatase 2A (PP2A) (70, 92). PP2A is a heterotrimeric serine-threonine phosphatase present in most cell types that has been implicated in the regulation of cell cycle progression, transcription, and DNA replication and translation (60, 61, 79). The B subunit, which is replaced by MT or ST, confers substrate specificity (79) and localization (85). The viral proteins should provide useful insight into PP2A regulation. It is becoming clear that binding of cell proteins can also modulate PP2A activity (41, 5...

Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.

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