Evaluation of Topoisomerase Inhibitors as Potential Antiviral Agents

1993 ◽  
Vol 4 (2) ◽  
pp. 85-91 ◽  
Author(s):  
B. Maschera ◽  
E. Ferrazzi ◽  
M. Rassu ◽  
M. Toni ◽  
G. Palù
Keyword(s):  
Nalidixic Acid ◽  
Topoisomerase Ii ◽  
Antiviral Agents ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Etoposide Treatment ◽  
Cross Links ◽  
Infected Cells ◽  
Cellular Dna ◽  
Sv40 Dna

Anti-eukaryotic topoisomerase drugs, Camptothecin and Etoposide, were tested for their ability of selectively interfering with the replication of simian virus 40 (SV40) DNA. Nalidixic acid was also assayed for a comparison, since the compound has been previously reported to affect papoyavirus growth. Our results indicate that anti-eukaryotic topoisomerase drugs significantly inhibit viral DNA replication but at concentrations that are also toxic for uninfected cells. Etoposide treatment produced a relatively higher number of DNA-protein cross-links in virus-infected cells as compared to uninfected control cells. Nalidixic acid displayed some degree of selectivity for inhibiting SV40 DNA synthesis more effectively than synthesis of cellular DNA without appreciable reduction of cell growth. This activity does not appear to depend on DNA damage or interference with topoisomerase II and deserves further evaluation.

scholarly journals Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide).

1993 ◽  
Vol 13 (10) ◽  
pp. 6190-6200 ◽  
Author(s):  
A L Bodley ◽  
H C Huang ◽  
C Yu ◽  
L F Liu
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Hot Spots ◽  
Topoisomerase Ii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
High Molecular Weight Form ◽  
Cellular Dna ◽  
Sv40 Dna ◽  
Molecular Weight Form

Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.

Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide)

1993 ◽  
Vol 13 (10) ◽  
pp. 6190-6200
Author(s):  
A L Bodley ◽  
H C Huang ◽  
C Yu ◽  
L F Liu
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Hot Spots ◽  
Topoisomerase Ii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
High Molecular Weight Form ◽  
Cellular Dna ◽  
Sv40 Dna ◽  
Molecular Weight Form

Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.

Simian virus 40 minichromosomes contain torsionally strained DNA molecules

1985 ◽  
Vol 5 (11) ◽  
pp. 3048-3057
Author(s):  
J Barsoum ◽  
P Berg
Keyword(s):  
Topoisomerase I ◽  
Simian Virus 40 ◽  
Staphylococcal Nuclease ◽  
Simian Virus ◽  
Infected Cells ◽  
Complete Digestion ◽  
Sv40 Genome ◽  
Negative Supercoiling ◽  
Sv40 Dna ◽  
Nuclease Sensitivity

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.

Monoclonal antibody analysis of the proliferating cell nuclear antigen (PCNA). Structural conservation and the detection of a nucleolar form

1990 ◽  
Vol 96 (1) ◽  
pp. 121-129
Author(s):  
N.H. Waseem ◽  
D.P. Lane
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Expression Vectors ◽  
Nuclear Antigen ◽  
Antigenic Structure ◽  
Cellular Dna ◽  
Sv40 Dna ◽  
Proliferating Cell

The proliferating cell nuclear antigen, PCNA, has recently been identified as the polymerase delta accessory protein. PCNA is essential for cellular DNA synthesis and is also required for the in vitro replication of simian virus 40 (SV40) DNA where it acts to coordinate leading and lagging strand synthesis at the replication fork. The cDNA for rat PCNA was cloned into a series of bacterial expression vectors and the resulting protein used to immunize mice. Eleven new monoclonal antibodies to PCNA have been isolated and characterized. Some of the antibodies recognize epitopes conserved from man to fission yeast. Immunocytochemical analysis of primate epithelial cell lines showed that the antibodies recognized antigenically distinct forms of PCNA and that these forms were localized to different compartments of the nucleus. One antibody reacted exclusively with PCNA in the nucleolus. These results suggest that the PCNA protein may fulfil several separate roles in the cell nucleus associated with changes in its antigenic structure.

scholarly journals Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro.

1992 ◽  
Vol 12 (9) ◽  
pp. 4007-4014 ◽  
Author(s):  
Y Ishimi ◽  
R Ishida ◽  
T Andoh
Keyword(s):  
Molecular Weight ◽  
Dna Replication ◽  
Late Stage ◽  
Topoisomerase Ii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosome Replication ◽  
Topoisomerase Ii Inhibitor ◽  
Sv40 Dna

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.

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 Simian virus 40 minichromosomes contain torsionally strained DNA molecules.

1985 ◽  
Vol 5 (11) ◽  
pp. 3048-3057 ◽  
Author(s):  
J Barsoum ◽  
P Berg
Keyword(s):  
Topoisomerase I ◽  
Simian Virus 40 ◽  
Staphylococcal Nuclease ◽  
Simian Virus ◽  
Infected Cells ◽  
Complete Digestion ◽  
Sv40 Genome ◽  
Negative Supercoiling ◽  
Sv40 Dna ◽  
Nuclease Sensitivity

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.

Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro

1992 ◽  
Vol 12 (9) ◽  
pp. 4007-4014
Author(s):  
Y Ishimi ◽  
R Ishida ◽  
T Andoh
Keyword(s):  
Molecular Weight ◽  
Dna Replication ◽  
Late Stage ◽  
Topoisomerase Ii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosome Replication ◽  
Topoisomerase Ii Inhibitor ◽  
Sv40 Dna

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.

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