Monoclonal antibodies specific for simian virus 40 tumor antigens.

SUMMARY Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen—a most amazing molecule.

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Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein

Molecular and Cellular Biology ◽

10.1128/mcb.4.2.232-239.1984 ◽

1984 ◽

Vol 4 (2) ◽

pp. 232-239

Author(s):

F Van Roy ◽

L Fransen ◽

W Fiers

Keyword(s):

Monoclonal Antibodies ◽

Immune Complex ◽

Immune Complexes ◽

Kinase Activity ◽

P53 Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.

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Simian virus 40-transformed adherent cells from human long-term marrow cultures: cloned cell lines produce cells with stromal and hematopoietic characteristics

Blood ◽

10.1182/blood.v70.2.464.464 ◽

1987 ◽

Vol 70 (2) ◽

pp. 464-474 ◽

Cited By ~ 47

Author(s):

JW Singer ◽

P Charbord ◽

A Keating ◽

J Nemunaitis ◽

G Raugi ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Cell Lines ◽

Stromal Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Adherent Cells ◽

Semisolid Medium ◽

Microscopic Appearance ◽

Marrow Cultures

Abstract Adherent cells from long-term marrow cultures from 23 individuals were transformed with wild-type simian virus 40 (SV40). After transformation, cloned cell lines were developed that even after rigorous subcloning invariably produced both stromal cells and round cells. The stromal cells expressed cytoskeletal filaments similar to those of long-term marrow culture adherent cells and produced interstitial and basal lamina collagen types. The round cells had the electron microscopic appearance of primitive hematopoietic cells and when examined with cytochemical stains and monoclonal antibodies to hematopoietic differentiation antigens had reaction patterns suggestive of cells from several lineages. Most round cells expressed the pan- hematopoietic T-200 determinant, and lesser percentages expressed the early T cell antigens CD-1 and CD-3, HLA-DR determinants, the monocytic antigen recognized by Leu M3, and the myeloid antigens detected by monoclonal antibodies 1G10 and 12.8. In addition, when plated in semisolid medium in the presence of a source of colony-stimulating activity, up to 11% of the cells formed colonies consisting of blastlike cells that also expressed hematopoietic cell surface determinants. The data suggest that adherent cells in long-term marrow cultures contain a cell that after transformation by SV40 obligately produces cells with hematopoietic as well as stromalike features.

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Protein kinase activities in immune complexes of simian virus 40 large T-antigen and transformation-associated cellular p53 protein.

Molecular and Cellular Biology ◽

10.1128/mcb.4.2.232 ◽

1984 ◽

Vol 4 (2) ◽

pp. 232-239 ◽

Cited By ~ 4

Author(s):

F Van Roy ◽

L Fransen ◽

W Fiers

Keyword(s):

Monoclonal Antibodies ◽

Immune Complex ◽

Immune Complexes ◽

Kinase Activity ◽

P53 Protein ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen

Immune complex kinase assays in the simian virus 40 system were performed by incubation of immunoprecipitates containing tumor antigens with [gamma-32P]ATP, followed by analysis of any phosphoacceptor proteins. These assays yielded mainly the viral large T-antigen and, in particular, the associated cellular p53 as endogenous substrates. The nature of these substrates was confirmed by proteolysis techniques. Under specific conditions, casein could be used as an exogenous substrate as well. The kinase reactions showed preference for ATP and MgCl2 instead of GTP or MnCl2. Both phosphoserine and phosphothreonine, but in no case phosphotyrosine, were detected after an immune complex kinase reaction. Apparently, several in vivo phosphorylation sites were recognized in vitro in both large T-antigen and p53, but the presence of some artifactual sites could not be completely excluded. Although contaminating kinases were detectable in the immune complexes, at least the p53 molecules were phosphorylated in vitro in a more specific way. This followed from several characteristics of the immune complex kinase reactions and especially from the strong inhibition of p53 phosphorylation by two anti-large-T monoclonal antibodies. It was shown that large T-antigen showed associated kinase activity, although none of our results could unambiguously demonstrate an intrinsic kinase activity of this protein. Finally, anti-p53 monoclonal antibodies only slightly affected in vitro phosphorylation reactions, whereas a p53 molecule from a simian virus 40-free, chemically transformed human cell line was not phosphorylated in vitro under any condition tested. Thus, it is highly unlikely that the p53 molecule per se carries intrinsic or even associated kinase activities.

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