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

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.

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

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.66.2.179-202.2002 ◽

2002 ◽

Vol 66 (2) ◽

pp. 179-202 ◽

Cited By ~ 160

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.

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p53 Targets Simian Virus 40 Large T Antigen for Acetylation by CBP

Journal of Virology ◽

10.1128/jvi.78.15.8245-8253.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8245-8253 ◽

Cited By ~ 41

Author(s):

Danielle L. Poulin ◽

Andrew L. Kung ◽

James A. DeCaprio

Keyword(s):

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Dependent Manner ◽

Large T Antigen ◽

T Antigens ◽

Sv40 Large T Antigen ◽

Acetylation Site ◽

P300 Binding

ABSTRACT Simian virus 40 (SV40) large T antigen (T Ag) interacts with the tumor suppressor p53 and the transcriptional coactivators CBP and p300. Binding of these cellular proteins in a ternary complex has been implicated in T Ag-mediated transformation. It has been suggested that the ability of CBP/p300 to modulate p53 function underlies p53's regulation of cell proliferation and tumorigenesis. In this study, we provide further evidence that CBP activity may be mediated through its synergistic action with p53. We demonstrate that SV40 T Ag is acetylated in vivo in a p53-dependent manner and T Ag acetylation is largely mediated by CBP. The acetylation of T Ag is dependent on its interaction with p53 and on p53's interaction with CBP. We have mapped the site of acetylation on T Ag to the C-terminal lysine residue 697. This acetylation site is conserved between the T antigens of the human polyomaviruses JC and BK, which are also known to interact with p53. We show that both JC and BK T antigens are also acetylated at corresponding sites in vivo. While other proteins are known to be acetylated by CBP/p300, none are known to depend on p53 for acetylation. T Ag acetylation may provide a regulatory mechanism for T Ag binding to a cellular factor or play a role in another aspect of T Ag function.

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Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens.

Journal of Virology ◽

10.1128/jvi.34.3.752-763.1980 ◽

1980 ◽

Vol 34 (3) ◽

pp. 752-763 ◽

Cited By ~ 177

Author(s):

E G Gurney ◽

R O Harrison ◽

J Fenno

Keyword(s):

Monoclonal Antibodies ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen ◽

T Antigens

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

Journal of Virology ◽

10.1128/jvi.00384-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9162-9174 ◽

Cited By ~ 22

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.

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Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3093 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3093-3096 ◽

Cited By ~ 61

Author(s):

R L Radna ◽

Y Caton ◽

K K Jha ◽

P Kaplan ◽

G Li ◽

...

Keyword(s):

Cell Lines ◽

Human Fibroblasts ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Cellular Aging ◽

Cellular Growth ◽

Viral Gene ◽

Large T Antigen ◽

Temperature Dependent

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.

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

Journal of Virology ◽

10.1128/jvi.00334-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8007-8020 ◽

Cited By ~ 59

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.

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Requirement for the simian virus 40 small tumor antigen in tumorigenesis in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3382-3390.1988 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3382-3390

Author(s):

Y W Choi ◽

I C Lee ◽

S R Ross

Keyword(s):

Mouse Mammary Tumor Virus ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen ◽

T Antigens ◽

Mammary Tumor Virus ◽

Mouse Mammary Tumor ◽

Tumor Virus ◽

Lung And Kidney

To examine the role of simian virus 40 (SV40) large T and small t antigens in tumorigenesis in animals, we generated transgenic mice which expressed either both the SV40 large T and small t antigens or the SV40 large T antigen alone under the control of the mouse mammary tumor virus long terminal repeat. The mouse mammary tumor virus long terminal repeat directs the expression of transgenes in ductal epithelial cells of several organs, including the mammary gland, lung, and kidney, and in lymphoid cells. The mice which expressed both the T and t tumor antigens developed lung and kidney adenocarcinomas, while those which expressed large T alone did not. Both types of mice developed malignant lymphomas with similar frequencies and latency periods. Our results show that the SV40 small t antigen cooperates with the large T antigen in inducing tumors in slowly dividing epithelial cells in the lung and kidney.

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