Interference with the maturation of vaccinia virus by isatin β-thiosemicarbazone

Isatin-f.-thiosemicarbazone (IBT) In the present study, we followed the fate of viral DNA and the synthesis of early and late viral polypeptides in the presence of IBT, when more than 99% of infectious virus are inhibited (E. Katz et al., J. Gen. Virol., in press). MATERIALS AND METHODSCell culture. HeLa S-3 cells were grown in monolayer cultures in Eagle medium (2) supplemented with 10% calf serum.Virus. Stocks of WR strain (wild type, wt) and IBT-resistant mutant (IBTR) of vaccinia virus, were prepared in HeLa cells and titrated on BSC1 mono-44 layers, as described elsewhere (Katz et al., in press).Infection procedure. Monolayers of HeLa S-3 cells in 60-mm plastic dishes (Nunc, Denmark) were washed with saline and infected with virus, at a multiplicity of infection of 5, in a volume of 0.2 to 0.3 ml. After incubation for 45 min at 37 C, the plates were washed, and Eagle medium containing 2% calf serum was added.Polyacrylamide gel electrophoresis. Gel electrophoresis was performed essentially as described by Summers et al. (17). Gels (10 by 0.6 cm) were prepared of 7.5% acrylamide, 0.27% N',N-methylenebisacrylamide in 0.1 M sodium phosphate (pH 7.1), and 0.1% sodium dodecyl sulfate (SDS). Prior to use, excess catalyst was removed from the gels by electrophoresis at 5 mA/gel for 1 h. Cytoplasmic extracts were prepared by Dounce homogenization and then reduced and dissociated with 2% SDS and 1% mercaptoethanol for 1 min at 100 C. Sucrose was added to the solubilized cytoplasm to 10%, and 200 gliters was applied to each gel. Electrophoresis was carried out at 3.5 mA/gel for 17 h. After electrophoresis, the gels were placed in 10% trichloroacetic acid, stained with 0.1% Coomassie blue in 10% trichloroacetic acid, and washed in 7.5% acetic acid. The gels of every experiment were sliced longitudinally, dried, and placed together in contact with X-ray film (4).3H-thymidine (18.9 mCi/mmol) and 35S-methionine (130 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, England. 5-Iododeoxyuridine (IUdR) was purchased from Nutritional Biochemicals Corp., Cleveland, Ohio. Rifampin was a gift from Lapetit, Milan, Italy. IBT (Schwarz-Mann, Orangeburg, N.Y.) was freshly prepared before use by dissolving 5 mg in 1 ml of acetone and then 4 ml of 0.25 N NaOH was added.

“…1). Synthesis of DNA during infection with vaccinia or rabbit pox viruses, in the presence of IBT, has been observed previously (1,3,18).…”

Section: Methodssupporting

confidence: 53%

mentioning

confidence: 99%

Synthesis of Vaccinia Virus Polypeptides in the Presence of Isatin-β-Thiosemicarbazone

Katz

Margalith

Winer

et al. 1973

“…Since IBT affects late stages during vaccinia virus growth, we examined the viral antigens formed during this period. Cells were labeled with [35S]methionine between 4 and 7 h after infection with vaccinia virus and then were harvested. The [35S]methionine-labeled polypeptides of the cell cytoplasm were separated by polyacrylamide gel electrophoresis ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Viral deoxyribonucleic acid (DNA) and early viral proteins are synthesized in the presence of the drug; however, the viral DNA fails to be coated with proteins and remains sensitive to deoxyribonuclease (4,26). Easterbrook (4), who studied vaccinia virus infection in KB cells in the presence of IBT, did not observe inhibition in viral antigen synthesis, even at the late period after infection, using complement fixation and fluorescent-antibody staining techniques. On the other hand, Appleyard et al (1) followed rabbit pox infection in HeLa cells by precipitation in agar and found that several late viral antigens were missing in the presence of IBT.…”

mentioning

confidence: 99%

Synthesis of Vaccinia Viral Antigens in HeLa Cells in the Presence of Isatin Beta-Thiosemicarbazone

Katz

Felix

1977

Vaccinia virus antigens, in HeLa cells treated with the antipox virus drug isatin p-thiosemicarbazone (IBT), were analyzed by immunoprecipitation, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The total radioactivity in the immunoprecipitate was decreased to almost 50% in the presence ofthe drug as compared to its absence. An inhibition also occurred with an IBT-dependent mutant (IBP) when growing in the absence of IBT. However, similar levels of radioactivity were observed in the immunoprecipitates from an IBT-resistant mutant (IB1') grown in either the absence or presence ofthe drug. When the antigens within the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was shown that the inhibition observed in the wild-type-infected cells in the presence of IBT and in the IBP-infected cells in the absence of the drug, was quantitative, affecting the amounts of the different polypeptides more or less equally.Isatin (-thiosemicarbazone (IBT) is an efficient drug active against pox-viruses, which inhibits late stages during virus growth (21). Viral deoxyribonucleic acid (DNA) and early viral proteins are synthesized in the presence of the drug; however, the viral DNA fails to be coated with proteins and remains sensitive to deoxyribonuclease (4,26). Easterbrook (4), who studied vaccinia virus infection in KB cells in the presence of IBT, did not observe inhibition in viral antigen synthesis, even at the late period after infection, using complement fixation and fluorescent-antibody staining techniques. On the other hand, Appleyard et al. (1) followed rabbit pox infection in HeLa cells by precipitation in agar and found that several late viral antigens were missing in the presence of IBT.The aim of the present study was to analyze the antigens formed in the absence and presence of the drug, using the combined method of immunoprecipitation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. An IBT-resistant mutant (IBTR), recently isolated by us@, served as a control in this study. MATERIALS AND METHODS Cells

“…The inhibition of leucine uptake and the potential inhibitory effect on protein synthesis versus the relatively minor effect on the uptake of thymidine are consistent with results reported for methisazone (N-methylisatin thiosemicarbazone). This compound has been shown to inhibit the late protein synthesis of viral particles to a greater extent than DNA synthesis (4). Although methisazone has been demonstrated to be active against T. cruzi in vitro (18), it was not found to significantly inhibit the uptake of either label in this system.…”

Section: Discussionmentioning

confidence: 78%

Activity of 2-acetylpyridine thiosemicarbazones against Trypanosoma rhodesiense in vitro

Casero

Klayman

Childs

et al. 1980

Twenty-seven 2-acetylpyridine thiosemicarbazones and analogs were tested for antitrypanosomal activity against Trypanosoma rhodesiense using a semiautomated in vitro assay system. Activity was determined by relative inhibition of uptake of two radiolabeled macromolecular precursors, [methyl-3H]thymidine and L-[U-14C]leucine, as compared to untreated controls. It was observed that the nitrogen atom of the pyridyl moiety of the 2-acetylpyridine thiosemicarbazones was essential for antitrypanosomal activity. The 2-acetylpyridine thiosemicarbazones generally inhibited the uptake of L-[U-14C]leucine to a greater extent than they inhibited [methyl-3H]thymidine uptake. Twenty-four of the 27 compounds tested exhibited activity comparable to that found for the antitrypanosomal agent ethidium bromide.