Host nuclear proteins expressed in simian virus 40-transformed and -infected cells (original) (raw)
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Journal of virology, 1983
A small fraction of the 94,000-molecular-weight multifunctional large T-antigen of simian virus 40 was associated with the nuclear protein matrix derived from simian virus 40-transformed mouse cells. The interaction between this fraction of T-antigen and the matrix was largely or entirely independent of nuclear DNA. Similar amounts of T-antigen were retained by the nuclei of transformed and revertant cell lines. A 100,000-molecular-weight variant of T-antigen, which has been found to correlate specifically with anchorage-independent growth, was present in the nuclear protein matrix of a transformed cell line. A T-antigen-containing revertant selected for the reacquisition of a high serum requirement and an anchorage requirement for growth retained T-antigen in association with its matrix.
DNA-binding properties of the major structural protein of simian virus 40
1986
We investigated whether the VPl protein of simian virus 40 binds to DNA. In vitro DNA-binding experiments clearly indicate that VP1 bound strongly to double-stranded and single-stranded DNA, with a higher affinity for the latter; additional experiments show that VP1 did not bind to a specific sequence of simian virus 40 DNA.
Biological activity of purified simian virus 40 T antigen proteins
Proceedings of the National Academy of Sciences, 1978
Proteins related to simian virus 40 (SV40) T antigen were isolated from cells infected with adenovirus 2/ SV40 hybrids Ad2+D2 and Ad2+ND1 dp2 as well as from a line of human cells (SV80) transformed by SV40. The 96,000-and 107,000-dalton proteins of SV80 and Ad2+D2, after injection into the cytoplasm of cultured cells, rapidly accumulate in the nuclei, where they remain antigenically reactive for at least 20 hr and trigger DNA synthesis in quiescent cells. By contrast, the 23,000-dalton protein coded by Ad2+ND1 dp2 does not stimulate cellular DNA synthesis. However, all three purified proteins are able to provide helper function for the growth of adenovirus 2 in monkey cells. Thus, purified SV40 T antigen and proteins that share sequences with it retain the ability to carry out at least two functions associated with the product of the A gene of SV40.
Possible nuclease activity of the T-antigen of virus SV-40
Bulletin of Experimental Biology and Medicine, 1975
A preparation of the T-antigen of virus SV-40 was isolated from an extract of golden hamster tumors by precipitation with ammonium sulfate followed by fractionation on DEAE-cellulose. Despite 100-fold purification of the preparation it contained traces of cell proteins as impurities. Treatment of calf thymus DNA with the preparation of T-antigen in the presence of magnesium ions considerably reduced the viscosity of the DNA solution during the first hour of incubation. The T-antigen, if inactivated by heating, or the analogous fraction from normal hamster tissues had no such action. During centirfugation in a sucrose gradient the sedimentation constant of hamster DNA was reduced after treatment with T-antigen from 28 S to 16 S, corresponding to a reduction of about 4-5 times in the molecular weight of the DNA. It can be concluded from these results that the partially purified preparation of T-antigen of virus SV-40 possesses endonuclease activity.
Association of simian virus 40 T antigen with simian virus 40 nucleoprotein complexes
Journal of virology, 1979
Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, ho...
Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin
Journal of Virology
Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight and specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker.
Nonselective Expression of Simian Virus 40 Large Tumor Antigen Fragments in Mouse Cells
Proceedings of The National Academy of Sciences, 1982
To understand the role of various functional domains ofsimian virus 40 early tumor antigens, we have cloned and introduced into mouse cells portions ofearly simian virus 40 DNA. Two types of truncated large tumor. antigen (33 and 12.3 kilodaltons), as well as smalltumor antigen, were identified by immunoprecipitation. Both truncated large tumor antigens have been found to be overproduced with respect to the small tumor antigen, although the 12.3-kilodalton truncated large tumor antigen was more stable than the 33-kdlodalton one. Nonviral 53-kdlodalton protein was not found associated with either truncated large tumor antigen in immunoprecipitations.
Simian virus 40 large tumor antigen: a "RNA binding protein"?
Proceedings of the National Academy of Sciences, 1982
Simian virus 40 large tumor antigen was isolated by immunoaffinity chromatography from monkey or mouse cell cultures undergoing lytic or transforming infection. RNase-treated gel-purified large tumor antigen, on hydrolysis with alkali, gave about equimolar amounts of AMP, GMP, CMP, and UMP. Furthermore, RNA fragments of -45 nucleotides could be isolated from large tumor antigen purified by the same procedure. Mapping of the T1 oligonucleotides showed a high complexity, as indicated by the presence of unique sequences of 15-30 nucleotides and of poly(A). This is compatible with the hypothesis that these RNA fragments are derived from cellular pre-mRNAs or mRNAs. Our results suggest that Simian virus 40 large tumor antigen is a RNA-binding protein and might possibly be involved in regulation of synthesis, maturation, or translation of cellular mRNAs.
Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen
Journal of virology, 1982
Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells. The proteins were different from each other, PAb419 reacting with a 35,000-molecular-weight protein, PAb427 reacting with a 75,000-molecular-weight phosphoprotein, PAb405 reacting with a 150,000-molecular-weight phosphoprotein, and PAb204 reacting with a 68,000-molecular-weight protein. It is suggested that although some of these cross-reactions may be fortuitous, they may, as an alternative, reflect similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.