scholarly journals Simian Virus 40 Large T Antigen Disrupts Genome Integrity and Activates a DNA Damage Response via Bub1 Binding

2008 ◽  
Vol 83 (1) ◽  
pp. 117-127 ◽  
Author(s):  
Jennifer Hein ◽  
Sergei Boichuk ◽  
Jiaping Wu ◽  
Yuan Cheng ◽  
Raimundo Freire ◽  
...  
Keyword(s):  
Dna Damage ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Genome Integrity ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Damage Response

ABSTRACT Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shown to lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of γ-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.

scholarly journals Multiple DNA Damage Signaling and Repair Pathways Deregulated by Simian Virus 40 Large T Antigen

2010 ◽  
Vol 84 (16) ◽  
pp. 8007-8020 ◽  
Author(s):  
Sergei Boichuk ◽  
Liang Hu ◽  
Jennifer Hein ◽  
Ole V. Gjoerup
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Viral Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Productive Infection ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin

ABSTRACT We demonstrated previously that expression of simian virus 40 (SV40) large T antigen (LT), without a viral origin, is sufficient to induce the hallmarks of a cellular DNA damage response (DDR), such as focal accumulation of γ-H2AX and 53BP1, via Bub1 binding. Here we expand our characterization of LT effects on the DDR. Using comet assays, we demonstrate that LT induces overt DNA damage. The Fanconi anemia pathway, associated with replication stress, becomes activated, since FancD2 accumulates in foci, and monoubiquitinated FancD2 is detected on chromatin. LT also induces a distinct set of foci of the homologous recombination repair protein Rad51 that are colocalized with Nbs1 and PML. The FancD2 and Rad51 foci require neither Bub1 nor retinoblastoma protein binding. Strikingly, wild-type LT is localized on chromatin at, or near, the Rad51/PML foci, but the LT mutant in Bub1 binding is not localized there. SV40 infection was previously shown to trigger ATM activation, which facilitates viral replication. We demonstrate that productive infection also triggers ATR-dependent Chk1 activation and that Rad51 and FancD2 colocalize with LT in viral replication centers. Using small interfering RNA (siRNA)-mediated knockdown, we demonstrate that Rad51 and, to a lesser extent, FancD2 are required for efficient viral replication in vivo, suggesting that homologous recombination is important for high-level extrachromosomal replication. Taken together, the interplay of LT with the DDR is more complex than anticipated, with individual domains of LT being connected to different subcomponents of the DDR and repair machinery.

scholarly journals T Antigens of Simian Virus 40: Molecular Chaperones for Viral Replication and Tumorigenesis

2002 ◽  
Vol 66 (2) ◽  
pp. 179-202 ◽  
Author(s):  
Christopher S. Sullivan ◽  
James M. Pipas
Keyword(s):  
Cell Cycle ◽  
Viral Replication ◽  
Molecular Chaperones ◽  
Tumor Antigens ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
T Antigens ◽  
Sv40 Large T Antigen

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.

scholarly journals Simian Virus Large T Antigen Interacts with the N-Terminal Domain of the 70 kD Subunit of Replication Protein A in the Same Mode as Multiple DNA Damage Response Factors

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0116093 ◽  
Author(s):  
Boting Ning ◽  
Michael D. Feldkamp ◽  
David Cortez ◽  
Walter J. Chazin ◽  
Katherine L. Friedman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Protein A ◽  
Replication Protein A ◽  
Simian Virus ◽  
T Antigen ◽  
Replication Protein ◽  
Large T Antigen ◽  
Response Factors ◽  
Damage Response

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens

1986 ◽  
Vol 6 (4) ◽  
pp. 1204-1217
Author(s):  
P S Jat ◽  
C L Cepko ◽  
R C Mulligan ◽  
P A Sharp
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Vector System ◽  
Large T Antigen ◽  
T Antigens ◽  
Sv40 Large T Antigen ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

scholarly journals Quantitative Analysis of the Binding of Simian Virus 40 Large T Antigen to DNA

2007 ◽  
Vol 81 (17) ◽  
pp. 9162-9174 ◽  
Author(s):  
Amélie Fradet-Turcotte ◽  
Caroline Vincent ◽  
Simon Joubert ◽  
Peter A. Bullock ◽  
Jacques Archambault
Keyword(s):  
Specific Binding ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sequence Specificity ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Origin ◽  
Single Stranded Dna ◽  
Terminal Domain

ABSTRACT SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5′-GAGGC-3′ sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5′-GAGGC-3′ sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an ∼10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.

scholarly journals Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

1986 ◽  
Vol 6 (4) ◽  
pp. 1204-1217 ◽  
Author(s):  
P S Jat ◽  
C L Cepko ◽  
R C Mulligan ◽  
P A Sharp
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Vector System ◽  
Large T Antigen ◽  
T Antigens ◽  
Sv40 Large T Antigen ◽  
Polyomavirus Middle T Antigen ◽  
Middle T Antigen

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

The regulation of cellular transcription by Simian virus 40 large T-antigen

1985 ◽  
Vol 226 (1242) ◽  
pp. 15-23 ◽  
Keyword(s):  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Productive Infection ◽  
Transformed Cells ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Cellular Genes ◽  
Cellular Transcription

The transforming protein of Simian virus 40 (SV40), large T-antigen, regulates transcription both positively and negatively during the productive infection cycle. We have isolated a number of cellular genes which are expressed at elevated levels in SV40-transformed cells and have used these to study the mechanism or mechanisms by which the viral transforming protein regulates cellular gene expression. Small RNAs homologous to the mouse B2 repetitive sequence family are found at higher levels in transformed cells than in normal cells and we have shown that pure large T-antigen stimulates transcription of such repeats by RNA polymerase III. A class I major histocompatibility complex (MHC) gene is also activated as a result of SV40 transformation and we have used DNA-mediated gene transfer to study how this gene is regulated by large T-antigen.

scholarly journals Excess wild-type p53 blocks initiation and maintenance of simian virus 40 transformation.

1991 ◽  
Vol 11 (7) ◽  
pp. 3472-3483 ◽  
Author(s):  
K Fukasawa ◽  
G Sakoulas ◽  
R E Pollack ◽  
S Chen
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Sv40 Transformation ◽  
Mouse Cells ◽  
Wild Type P53

Wild-type (wt) murine p53 has been tested for its ability to block and reverse the transforming effects of simian virus 40 (SV40) large T antigen. Established and precrisis mouse cells overexpressing exogenously introduced wt p53 became resistant to SV40 transformation. The introduction of excess wt p53 into SV40-transformed precrisis cells reverted their transformed phenotype. However, the phenotype of SV40-transformed established cells was not reverted by excess wt p53. We conclude that an antioncogenic action of wt p53 is exerted during SV40 transformation and that in precrisis cells, the antitransforming action of wt p53 can be exerted both at initiation and during the maintenance of transformation.

Excess wild-type p53 blocks initiation and maintenance of simian virus 40 transformation

1991 ◽  
Vol 11 (7) ◽  
pp. 3472-3483
Author(s):  
K Fukasawa ◽  
G Sakoulas ◽  
R E Pollack ◽  
S Chen
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Sv40 Transformation ◽  
Mouse Cells ◽  
Wild Type P53

Wild-type (wt) murine p53 has been tested for its ability to block and reverse the transforming effects of simian virus 40 (SV40) large T antigen. Established and precrisis mouse cells overexpressing exogenously introduced wt p53 became resistant to SV40 transformation. The introduction of excess wt p53 into SV40-transformed precrisis cells reverted their transformed phenotype. However, the phenotype of SV40-transformed established cells was not reverted by excess wt p53. We conclude that an antioncogenic action of wt p53 is exerted during SV40 transformation and that in precrisis cells, the antitransforming action of wt p53 can be exerted both at initiation and during the maintenance of transformation.

Non-specific elongation of cell cycle phases by cycloheximide in rat 3Y1 cells, and specific reduction of G1 phase elongation by simian virus 40 large T antigen

1988 ◽  
Vol 91 (2) ◽  
pp. 295-302
Author(s):  
A. Okuda ◽  
G. Kimura
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
S Phase ◽  
G1 Phase ◽  
Protein Accumulation ◽  
Large T Antigen ◽  
Coding Region ◽  
Sv40 Large T Antigen ◽  
In Cells

Partial inhibition of protein synthesis by cycloheximide caused prolongation of G1, S and G2 phases in rat 3Y1 fibroblasts. In cells expressing simian virus 40 (SV40) large T antigen, by infection with SV40 in the previous generation, the prolongation of G1 phase in the presence of cycloheximide was suppressed. However, the prolongation of S and G2 phases in the presence of cycloheximide was not suppressed in cells expressing large T antigen, by infection with SV40 in the current generation. Similarly, when density-arrested cells (cells in G0 phase) were infected with SV40 (either wild-type strain or a mutant deleted in the unique coding region for small t antigen) and reseeded sparsely in the presence of cycloheximide, the cycloheximide-induced delay of entry into S phase was suppressed. In this case, the reduction in [35S]methionine incorporation, that in protein accumulation and that in cell volume increase, were not surmounted by SV40 infection. In T-antigen-negative cells, all the regions in G1 phase seemed to be sensitive to cycloheximide, i.e. they suffered elongation. These results suggest that, in comparison with cells that enter S phase by the action of growth factors, cells expressing large T antigen can enter S phase more efficiently through a quite different process.

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