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...

Simian virus 40 gene A has previously been shown to promote the replication of viral DNA and the transcription of late viral RNA in productive infection and to maintain the growth characteristics of some transformed cells. The present study examines the effect of the A function on proteins synthesized during productive and transforming infections. Under restrictive conditions, temperature-sensitive A mutants induce the overproduction of a 100,000-dalton protein both in productively infected monkey cells and in transformed rabbit cells. Immunoprecipitation of the induced protein with antisera, prepared against simian virus 40-induced tumors in hamsters, was used to identify the induced protein as tumor antigen. The same protein can be precipitated from extracts of cells infected by wild-type virus but not from uninfected cells. Furthermore, the mutant-induced protein is more rapidly degraded in vivo and is less tightly bound to intranuclear components than the protein induced by wild-type virus. The presence of the same virus-induced protein in infected cells from different species and the altered behavior of that protein in mutant infection strongly suggest that the protein is virus coded. Because the protein is large enough to account for the entire coding capacity in the early region of the simian virus 40 genome, the 100,000-dalton protein may well be the primary product of the only early gene identified by complementation studies, the A gene. If the 100,000-dalton protein that is overproduced in mutant infection is the A protein and the only early protein, then functional wild-type A protein must regulate its own synthesis in both productive and transforming infections.

. Our findings uncover a direct link between ATM and SV40 LTag that may have implications for understanding the replication cycle of oncogenic polyoma viruses.The eukaryotic DNA damage response represents a series of highly integrated and tightly regulated pathways that coordinate DNA repair, cell cycle, and homeostatic responses to abnormal DNA structures arising endogenously or following exposure to extrinsic genotoxic stimuli. Central to the DNA damage response are a pair of structurally and functionally related protein kinases, designated ATM 2 (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) belonging to the phosphoinositide 3-kinase-related kinase (PIKK) gene superfamily. ATM and ATR share a conserved carboxyl-terminal catalytic domain and display highly overlapping substrate specificities in vitro (1-3). Substrates for ATM and ATR include the p53 and BRCA1 tumor suppressors among many other proteins involved in cell cycle checkpoint activation, DNA repair, and transcriptional regulation (1, 3). Mutations in ATM cause the cancer susceptibility-neurodegeneration syndrome, ataxia-telangiectasia (1, 4). ATM-deficient cells are grossly defective in the ionizing radiation (IR)-induced G 1 /S, intra-S phase, and G 2 /M checkpoints and are profoundly sensitive to IR and other agents that induce DNA double-strand breaks (DSBs) (1). Although structurally and functionally related to ATM, the major functions of ATR pertain to its roles in DNA replication (5). ATR prevents premature firing of DNA replicons, couples the completion of S phase to mitosis, and is required for chromosome maintenance and stabilization of stalled DNA replication forks (6 -10). Although null mutations in ATR are lethal, hypomorphic splicing mutations that reduce ATR protein levels are associated with a rare congenital condition known as Seckel's syndrome (11-13).The catalytic activity of ATM is rapidly up-regulated in response to IR and other DSB-inducing agents. Catalytic activation involves the transautophosphorylation of inactive, dimeric ATM on Ser-1981, followed by dissociation into active monomers (14). The trimeric complex of MRE11, RAD50, and NBS1 (MRN) facilitates ATM activation and ATMdependent substrate phosphorylation through recruitment of ATM to DSBs and/or orientation of the ATM catalytic domain (15-18). Recent studies suggest that the recruitment of ATM is mediated by the carboxyl terminus of NBS1 (19,20). Although ATR isolated from DNA-damaged cells does not show enhanced kinase activity, its recruitment to regions of stalled DNA replication is regulated through binding to the ATRinteracting protein (ATRIP) and replication protein A (RPA) (19,21). Among many key substrates for ATR and ATM are the checkpoint effector kinases, CHK1 and CHK2, which are phosphorylated by ATM and ATR in response to DSBs and DNA replication stress, respectively (6,(22)(23)(24). CHK1 and CHK2 promote checkpoint arrest through phosphorylation and inactivation of CDC25 family phosphatases (25-28).Virus infection can also elicit ATM-dependent...

Defining the ability of simian virus 40 (SV40) to transform human cells has become of even greater importance with the increased understanding that this virus may play a role in some human malignancies. This report documents the requirement for viral small-t (ST) antigen in large-T (LT)-driven transformation of primary fibroblasts, a requirement that cannot be met by a well-known oncogene, c-Ha-ras (EJ-ras), which can cooperate with LT in rodent systems. The cellular gene telomerase is not essential for transformation, although transformed clones are not immortal without it. Similarly, an immortal mesothelial cell line has been developed using LT and telomerase. Immortalized mesothelial cells are morphologically normal, but can be transformed by introduction of ST, or ST + ras, but not by ras alone. It is likely that ST will be required along with LT for transformation of most human cell types.

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.Polyomavirus medium-T antigen, as well as the polyomavirus and simian virus 40 (SV40) small-t antigens (small-t), form complexes with protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase (31, 49) consisting of three subunits, the catalytic subunit and two regulatory subunits, A and B (11). To elucidate the role of these complexes in neoplastic transformation by polyomavirus and SV40, it is important to determine the effect of T-antigen binding on the enzymatic properties of PP2A. In the accompanying article (50), we demonstrate that purified SV40 small-t binds to the free A subunit and the AC form of PP2A but not to the free C subunit or the ABC form. We also show that small-t inhibits dephosphorylation of myelin basic protein and myosin light chains by the AC form but has no effect on the activity of free C or the ABC form. With phosphorylated histone Hi as an exogenous substrate, small-t stimulates the activity of the AC form. The B subunit and small-t appear to bind to the same site on AC and have similar effects on its activity.In this article, we investigated the substrate specificity of PP2A towards SV40 large-T antigen and the cellular protein p53 and how small-t alters this activity. Large-T is phosphorylated at eight or more sit...