scholarly journals Simian Virus 40 Large T Antigen Stabilizes the TATA-Binding Protein–TFIIA Complex on the TATA Element

1998 ◽  
Vol 18 (7) ◽  
pp. 3926-3935 ◽  
Author(s):  
Blossom Damania ◽  
Paul Lieberman ◽  
James C. Alwine
Keyword(s):  
Binding Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Tata Binding Protein ◽  
T Antigen ◽  
Early Gene ◽  
Large T Antigen ◽  
Complex Assembly ◽  
Tata Element

ABSTRACT Large T antigen (T antigen), the early gene product of simian virus 40 (SV40), is a potent transcriptional activator of both cellular and viral genes. Recently we have shown that T antigen is tightly associated with TFIID and, in this position, performs a TATA-binding protein (TBP)-associated factor (TAF)-like function. Based on this observation, we asked whether T antigen affected steps in preinitiation complex assembly. Using purified components in in vitro complex assembly assays, we found that T antigen specifically enhances the formation of the TBP-TFIIA complex on the TATA element. T antigen accomplishes this by increasing the rate of formation of the TBP-TFIIA complex on the TATA element and by stabilizing the complexes after they are formed on the promoter. In addition, DNA immunoprecipitation experiments indicate that T antigen is associated with the stabilized TBP-TFIIA complexes bound to the DNA. In this regard, it has previously been shown that T antigen interacts with TBP; in the present study, we show that T antigen also interacts with TFIIA in vitro. In testing the ability of T antigen to stabilize the TBP-TFIIA complex, we found that stabilization is highly sensitive to the specific sequence context of the TATA element. Previous studies showed that T antigen could activate simple promoters containing the TATA elements from thehsp70 and c-fos gene promoters but failed to significantly activate similar promoters containing the TATA elements from the promoters of the SV40 early and adenovirus E2a genes. We find that the ability to stabilize the TBP-TFIIA complex on thehsp70 and c-fos TATA elements, and not on the SV40 early and E2A TATA elements, correlates with the ability or inability to activate promoters containing these TATA elements.

scholarly journals Stimulation of the Human Heat Shock Protein 70 Promoter in Vitro by Simian Virus 40 Large T Antigen

1989 ◽  
Vol 264 (27) ◽  
pp. 16160-16164
Author(s):  
I C Taylor ◽  
W Solomon ◽  
B M Weiner ◽  
E Paucha ◽  
M Bradley ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Human Heat Shock Protein ◽  
Stimulation Of

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

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.

scholarly journals High-Throughput Cell-Based Screen for Chemicals That Inhibit Infection by Simian Virus 40 and Human Polyomaviruses

2009 ◽  
Vol 83 (11) ◽  
pp. 5630-5639 ◽  
Author(s):  
Edward C. Goodwin ◽  
Walter J. Atwood ◽  
Daniel DiMaio
Keyword(s):  
High Throughput ◽  
Ellagic Acid ◽  
Viral Vector ◽  
Jc Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Large T Antigen ◽  
Human Polyomaviruses

ABSTRACT We developed a high-throughput, cell-based screen to identify chemicals that inhibit infection by the primate polyomaviruses. The screen is based on the detection of compounds that inhibit the ability of a replication-defective simian virus 40 (SV40)-based viral vector to cause growth arrest in HeLa cells by repressing the expression of the endogenous human papillomavirus E7 oncogene in these cells. We identified two compounds, ellagic acid and spiperone, that suppressed the ability of the SV40 recombinant virus to inhibit cellular DNA synthesis. These compounds caused a marked reduction of the ability of wild-type SV40 to productively infect permissive monkey cells, even when the compounds were added several hours after infection. The fraction of cells expressing SV40 large T antigen and the levels of T antigen mRNA were reduced in infected human and monkey cells treated with ellagic acid and spiperone, suggesting that these compounds block a step in the virus life cycle prior to SV40 early gene expression. Ellagic acid and spiperone also inhibited large T antigen expression by BK virus and JC virus, two important, pathogenic human polyomaviruses.

scholarly journals Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

1986 ◽  
Vol 6 (11) ◽  
pp. 3815-3825 ◽  
Author(s):  
R S Decker ◽  
M Yamaguchi ◽  
R Possenti ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Early Gene ◽  
Initial Direction ◽  
Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

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

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.

Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity

1994 ◽  
Vol 14 (9) ◽  
pp. 6244-6252
Author(s):  
J A Frost ◽  
A S Alberts ◽  
E Sontag ◽  
K Guan ◽  
M C Mumby ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
T Antigen ◽  
Mitogen Activated Protein Kinase ◽  
Early Gene ◽  
Mitogen Activated Protein ◽  
Small T Antigen

The simian virus 40 small tumor antigen (small t) specifically interacts with protein phosphatase type 2A (PP2A) in vivo and alters its catalytic activity in vitro. Among the substrates for PP2A in vitro are the activated forms of MEK and ERK kinases. Dephosphorylation of the activating phosphorylation sites on MEK and ERKs by PP2A in vitro results in a decrease in their respective kinase activities. Recently, it has been shown that overexpression of small t in CV-1 cells results in an inhibition of PP2A activity toward MEK and ERK2 and a constitutive upregulation of MEK and ERK2 activity. Previously, we have observed that overexpression of either ERK1, MEK1, or a constitutively active truncated form of c-Raf-1 (BXB) is insufficient to activate AP-1 in REF52 fibroblasts. We therefore examined whether overexpression of small t either alone or in conjunction with ERK1, MEK1, or BXB could activate AP-1. We found that coexpression of small t and either ERK1, MEK1, or BXB resulted in an increase in AP-1 activity, whereas expression of either small t or any of the kinases alone did not have any effect. Similarly, coexpression of small t and ERK1 activated serum response element-regulated promoters. Coexpression of kinase-deficient mutants of ERK1 and ERK2 inhibited the activation of AP-1 caused by expression of small t and either MEK1 or BXB. Coexpression of an interfering MEK, which inhibited AP-1 activation by small t and BXB, did not inhibit the activation of AP-1 caused by small t and ERK1. In contrast to REF52 cells, we observed that overexpression of either small or ERK1 alone in CV-1 cells was sufficient to stimulate AP-1 activity and that this stimulation was not enhanced by expression of small t and ERK1 together. These results show that the effects of small t on immediate-early gene expression depend on the cell type examined and suggest that the mitogen-activated protein kinase activation pathway is distinctly regulated in different cell types.

scholarly journals Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system.

1987 ◽  
Vol 7 (12) ◽  
pp. 4255-4265 ◽  
Author(s):  
W Markland ◽  
A E Smith ◽  
B L Roberts
Keyword(s):  
Rat Liver ◽  
Nuclear Translocation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Rat Liver Nuclei ◽  
Liver Nuclei

An in vitro nuclear translocation system is described in which isolated rat liver nuclei were incubated in a defined buffered medium containing radiolabeled or fluorescently labeled exogenous proteins. The nuclei were rapidly recovered, extracted, and analyzed for the presence of associated radiolabeled or fluorescently labeled proteins. The isolated nuclei exhibited the same specificity for protein uptake as seen previously in vivo, accumulating simian virus 40 wild-type large-T antigen and p53 while excluding a cytoplasmic variant of large-T antigen (d10) and bovine serum albumin. The rapid nuclear accumulation of wild-type large-T antigen was shown to be selective and dependent upon the recognition of a wild-type nuclear location signal, ATP and temperature dependent, and unidirectional. Taken together, the data suggest that in our in vitro system the nuclear translocation of wild-type large-T antigen exhibits some of the characteristics of an active transport process.

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