Three mRNAs from the murine polyomavirus early region encode the three well-characterized tumor antigens. We report the existence of a fourth alternatively spliced mRNA which encodes a fourth tumor antigen, tiny T antigen, which comprises the amino-terminal domain common to all of the T antigens but is extended by six unique amino acid residues. The amount of tiny T antigen in infected cells is small because of its short half-life. Tiny T antigen stimulates the ATPase activity of Hsc70, most likely because of its DnaJ-like motif. The common amino-terminal domain may interface with chaperone complexes to assist the T antigens in carrying out their diverse functions of replication, transcription, and transformation in the appropriate cellular compartments. The small, middle, and large T antigens expressed by the murine polyomavirus (muPy) are formed of a common aminoterminal sequence juxtaposed to unique carboxy-terminal sequences (33, 65). Such parsimony in protein structures reflects the pattern of viral gene expression, for the T antigens are encoded by a single primary transcript containing two splice acceptor and two splice donor sites (76) (Fig. 1), creating the potential of four separate mRNAs. Three of these RNAs encode the small, middle, and large T antigens. The fourth mRNA, heretofore undetected, should encode a protein with the T antigen common amino-terminal domain extended by six residues. Here, we report the detection of this mRNA and the partial characterization of the protein it encodes (tiny T antigen). Genetic and biochemical evidence indicates the muPy T antigen common amino-terminal domain is required for cell transformation and promotes small and middle T antigen association with PP2A, c-Src, and perhaps other proteins (5, 26, 73). These cellular signal transducers strongly influence viral DNA replication, transcription, and translation; consequently, the T antigen common amino-terminal domain must contribute to virtually all aspects of the virus life cycle. Sequence homology between the T antigen amino-terminal domain and DnaJ proteins has been noted (36). DnaJ proteins modulate the ATPase activity of the hsp70 class of chaperones, and we observe that tiny T antigen expressed in Escherichia coli and purified to near homogeneity exhibits such a stimulatory activity in vitro. This activity within the T antigen amino-terminal domain very likely contributes to the functions of the T antigens by promoting their interactions with cellular chaperones. MATERIALS AND METHODS Cells and viruses. NIH 3T3, 3T6, and COS1 cells were obtained from the American Type Culture Collection. Polyomavirus transformed MOP-3T3 and MOP-3T6 cells were obtained from John Hassell (McMaster University, Ontario, Canada). Whole mouse embryo (WME) cells were prepared from outbred mice as described previously (24). All cells were grown in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal calf serum.