Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four-to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.Simian virus 40 (SV40) encodes two proteins involved in tumorigenesis, the large and small tumor antigens. Large tumor antigen (T antigen) orchestrates many aspects of productive viral infection and is necessary and in many cases sufficient for tumorigenesis. T antigen is a 708-amino-acid multifunctional protein that elicits cellular transformation by acting on multiple targets, including members of the retinoblastoma tumor suppressor family (pRb, p107, and p130), members of the CBP family of transcriptional coactivators (CREB-binding protein [CBP], p300, and p400), and the tumor suppressor, p53. It is likely that additional T antigen targets important for transformation await discovery. T antigen sequences important for transformation map to two different regions of the molecule: the amino-terminal domain, which encompasses the first 125 amino acids, and a region located within the carboxyterminal half of the molecule (Fig. 1). Major questions that remain to be answered are the following. How does T antigen act on each of the cellular targets? How does the concerted action of T antigen on these multiple targets lead to tumorigenesis?Evidence that one or more independent transforming functions reside in the carboxy-terminal half o...

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity.Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Smail-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone Hi phosphorylated by protein kinase C. With the exception of histone HI, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone Hi, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone Hi as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-t antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.Neoplastic transformation by polyoma-, papilloma-, and adenoviruses involves complex formation between their transforming proteins and cellular proteins involved in regulating cell proliferation (20). For example, the transforming protein of simian virus 40 (SV40), large T antigen (large T), binds to and presumably inactivates the growth-suppressing proteins p53 (see reference 30 for review) and the product of the retinoblastoma gene (RB) (17,18). The transforming proteins of some human papillomaviruses also form complexes with p53 and the RB protein, suggesting a similar mechanism of transformation. On the other hand, the principal transforming protein of polyomavirus, medium-T antigen (medium T), does not bind to p53 or the RB protein.Instead, medium T associates with pp60csrc, the product of the c-src proto-oncogene (14-16), and strongly activates its protein-tyrosine kinase activity (4,9,14). Genetic evidence indicates that the activation of pp60csrc plays a role in transformation by polyomavirus (3,16). Medium T also binds to the pp62c-Yes (27) and pp59C-fy (10, 28) proteins, two pp60CcSrc_relat...

Cell-cycle progression is mediated by a coordinated interaction between cyclin-dependent kinases and their target proteins including the pRB and E2F͞DP-1 complexes. Immunoneutralization and antisense experiments have established that the abundance of cyclin D1, a regulatory subunit of the cyclin-dependent kinases, may be rate-limiting for G 1 phase progression of the cell cycle. Simian virus 40 (SV40) small tumor (t) antigen is capable of promoting G 1 phase progression and augments substantially the efficiency of SV40 transformation through several distinct domains. In these studies, small t antigen stimulated cyclin D1 promoter activity 7-fold, primarily through an AP-1 binding site at ؊954 with additional contributions from a CRE site at ؊57. The cyclin D1 AP-1 and CRE sites were sufficient for activation by small t antigen when linked to an heterologous promoter. Point mutations of small t antigen between residues 97-103 that reduced PP2A binding were partially defective in the induction of the cyclin D1 promoter. These mutations also reduced activation of MEK1 and two distinct members of the mitogen-activated protein kinase family, the ERKs (extracellular signal regulated kinases) and the SAPKs (stressactivated protein kinases), in transfected cells. Dominant negative mutants of either MEK1, ERK or SEK1, reduced small t-dependent induction of the cyclin D1 promoter. SV40 small t induction of the cyclin D1 promoter involves both the ERK and SAPK pathways that together may contribute to the proliferative and transformation enhancing activity of small t antigen.

The early region of simian virus 40 codes for at least two immunologically related polypeptides: large-T and small-t, with apparent molecular weights of 90,000-100,000 and 15,000-20,000, respectively. Because small-t shares methionine-containing tryptic peptides with large-T, the two polypeptides are probably coded, in part, by a common nucleotide sequence. To locate the coding sequences for large-T and small-t in the DNA, the production of these proteins was examined after infection of CV-1 cells with wild-type and deletion mttants of simian virus 40. We found that a deletion at the distal portion of the early region alters the structure of large-T but not of small-t; but deletions within the region between map coordinates 0.59 and 0.55 result in an alteration or absence of small-t and a normal large-T. These findings have been rationalized by a model that proposes the existence of two early mRNAs, one coding for large-T and the other for small-t. Both mRNAs span virtually the entire early region; but the mRNA coding for large-T lacks the nucleotide sequence between map coordinates 0.59 and 0.54. We suggest that small-t is translated from the larger of the two mRNAs, beginning at or near its 5' end and terminating at a termination codon at about map coordinate 0.54. Large-T, on the other hand, is translated from the shorter mRNA, beginning at the same initiator codon, and, because of the deletion of the terminator codon at 0.54, translation proceeds to the terminator codon at or near map position 0.18.The genome of simian virus 40 (SV40) has a defined molecular structure and a small number of genes, each of which is expressed during a particular interval in its multiplication cycle. Its ability to cause tumors in vivo and transform cells in vitro provides an opportunity for analyzing the mechanism of these phenomena as well (see refs. 1 and 2 for a review of the molecular biology of SV40). The oncogenic event and maintenance of the transformed phenotype require the expression of one or more viral genes. Because only those viral functions that occur prior to the onset of viral DNA replication in the lytic cycle (the early functions) are expressed in transformed cells, this narrows the search for the putative oncogenes to the early genes. One of the viral coded products required for transformation is the T antigen or T polypeptide (large-T), a protein that is synthesized early in the lytic infection and is coded by the early region of the DNA (the A cistron); the same protein is made in SV40-transformed or tumor cells (1, 2).The T antigen has an apparent polypeptide molecular weight of 90,000-100,000 (3); consequently, almost the entire nucleotide sequence of the early region would be needed to code for its primary structure. Therefore, it was not anticipated that a set of mutants with deletions in the early region between map coordinates 0.59 and 0.54 would produce a normal T polypeptide (4). The interpretation offered at that time (4) suggested that the nucleotide sequence between map positions 0.59 and...