scholarly journals Replication of simian virus 40 (SV40) DNA in virus-infected CV1 cells selectively permeabilized for small molecules by Staphylococcus aureus α-toxin: involvement of mitochondria in the fast O2-dependent regulation of SV40 DNA replication

2005 ◽  
Vol 386 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Hans-Jörg RIEDINGER ◽  
Frank EGER ◽  
Klaus TRUMMLER ◽  
Hans PROBST
Keyword(s):  
Staphylococcus Aureus ◽  
Dna Replication ◽  
Small Molecules ◽  
Mitochondrial Respiration ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Infected Cells ◽  
Sv40 Dna

SV40 (simian virus 40)-infected CV1 cells were permeabilized with Staphylococcus aureus α-toxin for small molecules (<2 kDa) in a medium that supports DNA replication. Incorporation of [α-32P]dATP was shown to proceed at an essentially constant rate for at least 1 h. 32P-labelled DNA replication intermediates and products were analysed by alkaline sucrose density centrifugation. The results suggested that SV40 DNA replication in α-toxin-permeabilized CV1 cells occurred essentially as in vivo. After bromodeoxyuridine 5′-triphosphate-labelling and isopycnic banding, significant amounts of DNA density-labelled in both strands were detected from 110 min of permeabilization onwards, indicating repeated rounds of viral DNA replication in the permeabilized cells. Incubation of permeabilized SV40-infected cells under hypoxic culture conditions caused inhibition of SV40 DNA replication. As seen in unpermeabilized cells, SV40 DNA replication was inhibited at the stage of initiation. The inhibition of DNA replication induced by hypoxia was mimicked by AA (antimycin A), an inhibitor of mitochondrial respiration, and also by the replacement of glutamate, a substrate of mitochondrial respiration, by Hepes in the permeabilization medium. Inhibition of DNA replication was not mediated by intracellular ATP depletion. AA also inhibited SV40 DNA replication in unpermeabilized, normoxically incubated cells. Moreover, as in hypoxically incubated cells, the addition of glucose to SV40-infected cells incubated for several hours with AA induced a burst of new initiations followed by a nearly synchronous round of viral DNA replication. Taken together, these results indicate that mitochondria are involved in the oxygen-dependent regulation of SV40 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.

Minimal subenhancer requirements for high-level polyomavirus DNA replication: a cell-specific synergy of PEA3 and PEA1 sites

1990 ◽  
Vol 10 (9) ◽  
pp. 4996-5001 ◽  
Author(s):  
R Rochford ◽  
C T Davis ◽  
K K Yoshimoto ◽  
L P Villarreal
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
Simian Virus 40 ◽  
Mouse Cell ◽  
Simian Virus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Factor Binding ◽  
Specific Regulation ◽  
High Level

The cell-specific regulation of DNA replication has important implications for the molecular strategy of cellular gene control. Mouse polyomavirus (Py) DNA replication is examined as a model of cell-specific replication control. Using an FM3A-derived mouse cell line which expresses early viral proteins (FOP cells), we determined the minimal sequence requirements for viral DNA replication. FOP cells were observed to have much simpler enhancer requirements than 3T6 and many other cells and did not need a B enhancer for high levels of DNA replication. Using these cells, we show that the individual or tandem binding sites for several unrelated trans-acting factors which are generally subfunctional as transcriptional enhancers (simian virus 40 A core, TGTGGAATG; EBP20, TGTGGTTTT; PEA1 [an AP-1 analog], GTGACTAA; PEA2, GACCGCAG; and PEA3, AGGAAG) stimulated low levels of Py DNA replication. The ordered dimeric combination of PEA3 and PEA1 factor-binding sites, however, acted synergistically to stimulate viral DNA replication to high wild-type levels. This is in contrast to prior results in which much larger enhancer sequences were necessary for high-level viral DNA replication. PEA3/PEA1-stimulated DNA replication showed a distance and orientation independence relative to the origin, which disagrees with some but not other prior analyses of enhancer-dependent DNA replication. It therefore appears that trans-acting factor-binding sites (enhansons) can generally activate DNA replication and that the AP-1 family of sites may act synergistically with other associated trans-acting factors to strongly affect Py DNA replication in specific cells.

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.

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