scholarly journals The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication

1998 ◽  
Vol 72 (12) ◽  
pp. 9771-9781 ◽  
Author(s):  
Klaus Weisshart ◽  
Poonam Taneja ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Binding Site ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Second Step ◽  
Dna Unwinding ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase α-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

scholarly journals Thermally inactivated simian virus 40 tsA58 mutant T antigen cannot initiate viral DNA replication in vitro.

1990 ◽  
Vol 64 (12) ◽  
pp. 6234-6245 ◽  
Author(s):  
I Reynisdóttir ◽  
D R O'Reilly ◽  
L K Miller ◽  
C Prives
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication

scholarly journals Unusual regulation of simian virus 40 early-region transcription in genomes containing two origins of DNA replication.

1984 ◽  
Vol 4 (9) ◽  
pp. 1915-1928 ◽  
Author(s):  
A R Buchman ◽  
P Berg
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type Virus ◽  
Animal Cells ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Early Region ◽  
Inverted Duplication

As part of our efforts to create multifunctional vectors for the transduction of animal cells, a set of simian virus 40 recombinants were constructed which contain an inverted duplication of the region including the origin of viral DNA replication (ori) and the early-region promoter. The unusual aspects of the structure of these recombinant genomes revealed several unexpected features of their function. In particular, transcription from the early-region promoters on these recombinants occurred primarily after the start of DNA replication, and, in that sense, these promoters behaved as if they were late-region promoters. This behavior results from the fact that these genomes contain multiple ori segments, and, therefore, they replicate earlier and faster than wild-type virus DNA, thereby causing a precocious shift in the initiation of early-region transcription from sites downstream of ori to sites located upstream of ori. The abnormal expression from multiple ori genomes is consistent with our present notions regarding the replication-dependent shift in early-region transcriptional start sites (Buchman et al., Mol. Cell. Biol. 4:1900-1914). Since our experiments demonstrate that RNAs initiated upstream of ori contribute to T-antigen formation late in infection, we suggest that the shift in early-region transcription starts modulates large T-antigen production in concert with viral DNA replication.

scholarly journals Interaction of the Transcription Factor TFIID with Simian Virus 40 (SV40) Large T Antigen Interferes with Replication of SV40 DNA In Vitro

1999 ◽  
Vol 73 (2) ◽  
pp. 1099-1107 ◽  
Author(s):  
Utz Herbig ◽  
Klaus Weisshart ◽  
Poonam Taneja ◽  
Ellen Fanning
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cell Extracts ◽  
Sv40 Dna ◽  
Replication Activity

ABSTRACT Simian virus 40 (SV40) large tumor (T) antigen is the major regulatory protein that directs the course of viral infection, primarily by interacting with host cell proteins and modulating their functions. Initiation of viral DNA replication requires specific interactions of T antigen bound to the viral origin of DNA replication with cellular replication proteins. Transcription factors are thought to stimulate initiation of viral DNA replication, but the mechanism of stimulation is poorly understood. Since the transcription factor TATA-binding protein (TBP) binds to sequences within the origin of replication and interacts specifically with T antigen, we examined whether TBP complexes stimulate SV40 DNA replication in vitro. On the contrary, we found that depletion of TBP complexes from human cell extracts increased their ability to support viral DNA replication, and readdition of TBP complexes to the depleted extracts diminished their activity. We have mapped the sites of interaction between the proteins to residues 181 to 205 of T antigen and 184 to 220 of TBP. Titration of fusion proteins containing either of these peptides into undepleted cell extracts stimulated their replication activity, suggesting that they prevented the T antigen-TBP interaction that interfered with replication activity. TBP complexes also interfered with origin DNA unwinding by purified T antigen, and addition of either the T antigen or the TBP fusion peptide relieved the inhibition. These results suggest that TBP complexes associate with a T-antigen surface that is also required for origin DNA unwinding and viral DNA replication. We speculate that competition among cellular proteins for T antigen may play a role in regulating the course of viral infection.

Unusual regulation of simian virus 40 early-region transcription in genomes containing two origins of DNA replication

1984 ◽  
Vol 4 (9) ◽  
pp. 1915-1928
Author(s):  
A R Buchman ◽  
P Berg
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type Virus ◽  
Animal Cells ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Early Region ◽  
Inverted Duplication

As part of our efforts to create multifunctional vectors for the transduction of animal cells, a set of simian virus 40 recombinants were constructed which contain an inverted duplication of the region including the origin of viral DNA replication (ori) and the early-region promoter. The unusual aspects of the structure of these recombinant genomes revealed several unexpected features of their function. In particular, transcription from the early-region promoters on these recombinants occurred primarily after the start of DNA replication, and, in that sense, these promoters behaved as if they were late-region promoters. This behavior results from the fact that these genomes contain multiple ori segments, and, therefore, they replicate earlier and faster than wild-type virus DNA, thereby causing a precocious shift in the initiation of early-region transcription from sites downstream of ori to sites located upstream of ori. The abnormal expression from multiple ori genomes is consistent with our present notions regarding the replication-dependent shift in early-region transcriptional start sites (Buchman et al., Mol. Cell. Biol. 4:1900-1914). Since our experiments demonstrate that RNAs initiated upstream of ori contribute to T-antigen formation late in infection, we suggest that the shift in early-region transcription starts modulates large T-antigen production in concert with viral DNA replication.

Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation

1984 ◽  
Vol 4 (6) ◽  
pp. 1125-1133
Author(s):  
M M Manos ◽  
Y Gluzman
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Atpase Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Oncogenic Transformation ◽  
Viral Dna ◽  
Viral Dna Replication

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

scholarly journals Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

1984 ◽  
Vol 4 (6) ◽  
pp. 1125-1133 ◽  
Author(s):  
M M Manos ◽  
Y Gluzman
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Atpase Activity ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Oncogenic Transformation ◽  
Viral Dna ◽  
Viral Dna Replication

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.

scholarly journals Simian virus 40 small-t antigen stimulates viral DNA replication in permissive monkey cells.

1994 ◽  
Vol 68 (5) ◽  
pp. 3138-3144 ◽  
Author(s):  
C Cicala ◽  
M L Avantaggiati ◽  
A Graessmann ◽  
K Rundell ◽  
A S Levine ◽  
...  
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Small T Antigen
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