scholarly journals Assembly of T-Antigen Double Hexamers on the Simian Virus 40 Core Origin Requires Only a Subset of the Available Binding Sites

1998 ◽  
Vol 18 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
Woo S. Joo ◽  
Henry Y. Kim ◽  
John D. Purviance ◽  
K. R. Sreekumar ◽  
Peter A. Bullock
Keyword(s):  
Structural Changes ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
The Core ◽  
Structural Distortions ◽  
In The Wild ◽  
Sv40 Dna ◽  
Initial Assembly

ABSTRACT Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on “active pairs” of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

scholarly journals Relationship among Location of T-Antigen-Induced DNA Distortion, Auxiliary Sequences, and DNA Replication Efficiency

2003 ◽  
Vol 77 (19) ◽  
pp. 10651-10657 ◽  
Author(s):  
Susan Okuley ◽  
Mindy Call ◽  
Tara Mitchell ◽  
Bugen Hu ◽  
Mary E. Woodworth
Keyword(s):  
Dna Replication ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Single Copy ◽  
Simian Virus ◽  
T Antigen ◽  
Replication Efficiency ◽  
Cellular Sequence ◽  
In The Wild ◽  
Sv40 Dna

ABSTRACT T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork.

Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

1984 ◽  
Vol 4 (8) ◽  
pp. 1476-1482
Author(s):  
H Ariga
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Cell Extract ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

E1A induces phosphorylation of the retinoblastoma protein independently of direct physical association between the E1A and retinoblastoma products

1991 ◽  
Vol 11 (8) ◽  
pp. 4253-4265
Author(s):  
H G Wang ◽  
G Draetta ◽  
E Moran
Keyword(s):  
Cell Cycle ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
Physical Interaction ◽  
Resting Cells ◽  
Wild Type ◽  
Quiescent Cells ◽  
Kinase Expression

We have studied the initial effects of adenovirus E1A expression on the retinoblastoma (RB) gene product in normal quiescent cells. Although binding of the E1A products to pRB could, in theory, make pRB phosphorylation unnecessary for cell cycle progression, we have found that the 12S wild-type E1A product is capable of inducing phosphorylation of pRB in normal quiescent cells. The induction of pRB phosphorylation correlates with E1A-mediated induction of p34cdc2 expression and kinase activity, consistent with the possibility that p34cdc2 is a pRB kinase. Expression of simian virus 40 T antigen induces similar effects. Induction of pRB phosphorylation is independent of the pRB binding activity of the E1A products; E1A domain 2 mutants do not bind detectable levels of pRB but remain competent to induce pRB phosphorylation and to activate cdc2 protein kinase expression and activity. Although the kinetics of induction are slower, domain 2 mutants induce wild-type levels of pRB phosphorylation and host cell DNA synthesis and yet fail to induce cell proliferation. These results imply that direct physical interaction between the RB and E1A products does not play a required role in the early stages of E1A-mediated cell cycle induction and that pRB phosphorylation is not, of itself, sufficient to allow quiescent cells to divide. These results suggest that the E1A products do not need to bind pRB in order to stimulate resting cells to enter the cell cycle. Indeed, a more important role of the RB binding activity of the E1A products may be to prevent dividing cells from returning to G0.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro

1987 ◽  
Vol 7 (10) ◽  
pp. 3694-3704
Author(s):  
C Prives ◽  
Y Murakami ◽  
F G Kern ◽  
W Folk ◽  
C Basilico ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Early Gene ◽  
Wild Type ◽  
The Core ◽  
Cell Extracts ◽  
Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

scholarly journals Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

1985 ◽  
Vol 5 (8) ◽  
pp. 2019-2028 ◽  
Author(s):  
T Michaeli ◽  
C Prives
Keyword(s):  
Xenopus Laevis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocytes ◽  
Large T Antigen ◽  
Viral Dna ◽  
Infected Monkey ◽  
Sv40 Dna

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.

scholarly journals Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

1985 ◽  
Vol 5 (5) ◽  
pp. 1043-1050 ◽  
Author(s):  
R E Lanford ◽  
C Wong ◽  
J S Butel
Keyword(s):  
Cell Lines ◽  
Mouse Embryo ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Mouse Embryo Fibroblasts ◽  
Established Cell Lines ◽  
Established Cell ◽  
Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

Regulation of simian virus 40 gene expression in Xenopus laevis oocytes

1985 ◽  
Vol 5 (8) ◽  
pp. 2019-2028
Author(s):  
T Michaeli ◽  
C Prives
Keyword(s):  
Xenopus Laevis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Oocyte Nucleus ◽  
Xenopus Laevis Oocytes ◽  
Large T Antigen ◽  
Viral Dna ◽  
Infected Monkey ◽  
Sv40 Dna

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.

Circular and linear simian virus 40 DNAs differ in recombination

1985 ◽  
Vol 5 (4) ◽  
pp. 869-880
Author(s):  
D Dorsett ◽  
I Deichaite ◽  
E Winocour
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Linear Forms ◽  
Rolling Circle ◽  
Linear Dna ◽  
Sv40 Dna

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.

Separation of simian virus 40 large-T-antigen-transforming and origin-binding functions from the ability to block differentiation

1988 ◽  
Vol 8 (3) ◽  
pp. 1380-1384 ◽  
Author(s):  
V Cherington ◽  
M Brown ◽  
E Paucha ◽  
J St Louis ◽  
B M Spiegelman ◽  
...  
Keyword(s):  
Dehydrogenase Activity ◽  
Adipocyte Differentiation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Lipid Binding ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
Glycerophosphate Dehydrogenase ◽  
Triglyceride Accumulation

Wild-type simian virus 40 large T antigen is very effective at blocking adipocyte differentiation in 3T3-F442A cells as assayed by triglyceride accumulation, induction of glycerophosphate dehydrogenase activity, and expression of mRNAs for glycerophosphate dehydrogenase, the adipocyte serine protease adipsin, and the putative lipid-binding protein adipocyte P2. Point mutants defective for either origin-specific DNA binding or transformation blocked differentiation as completely as wild type.

scholarly journals Large T-Antigen Double Hexamers Imaged at the Simian Virus 40 Origin of Replication

2000 ◽  
Vol 20 (1) ◽  
pp. 34-41 ◽  
Author(s):  
Mikel Valle ◽  
Claudia Gruss ◽  
Lothar Halmer ◽  
José M. Carazo ◽  
Luis Enrique Donate
Keyword(s):  
Simian Virus 40 ◽  
Initial Step ◽  
Simian Virus ◽  
T Antigen ◽  
Structural Domain ◽  
Large Tumor ◽  
Sv40 Origin ◽  
Sv40 Dna ◽  
First Time ◽  
Inner Channel

ABSTRACTThe initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg2+and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag–DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.

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