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

Abstract

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

N-methylisatin-beta-4',4'-diethylthiosemicarbazone, an inhibitor of Moloney leukemia virus protein production: characterization and in vitro translation of viral mRNA

“…Thus, the inhibition of poliovirus by N-methylisatin-P-dibutylthiosemicarbazone was due to the inhibition of viral-RNA-dependent RNA polymerase (15), whereas the activity of IBT against vaccinia virus was caused by inhibition of a late step of virus maturation (11).…”

The Thiosemicarbazones

“…Using methionine 35S labeled protein they found that both types of immature particles sedimented at approximately the same rate. However, tritiated thymidine labeling (KATZ et al 1978 a) revealed that the immature particles made in the presence ofIBT contained very little DNA. Furthermore, using polyacrylamide gel electrophoresis techniques, these investigators also established that there were profound differences in the polypeptide composition of both types of immature particles.…”

“…At least three major late proteins (4 a, 4 b, and 10) comprising more than one-third of the protein mass of vaccinia virions are formed by cleavage of higher molecular weight precursors. KATZ et al (1978 a) found that the immature particles formed in the presence ofIBT lacked two of the main core polypeptides: 4a and 4 b. The KATZ group had previously established by pulse-chase experiments that, while precursors P4 a and P4 b were cleaved in the presence of IBT to produce the structural polypeptides 4 a and 4b (KATZ et al 1973b), the amounts made were only about 50% of those found in the total cytoplasmic fraction (obtained after boiling with detergent and mercaptoethanol) of cells in the absence of drug (KATZ and FELIX 1977).…”

“…KATZ et al (1978 a) found that the immature particles formed in the presence ofIBT lacked two of the main core polypeptides: 4a and 4 b. The KATZ group had previously established by pulse-chase experiments that, while precursors P4 a and P4 b were cleaved in the presence of IBT to produce the structural polypeptides 4 a and 4b (KATZ et al 1973b), the amounts made were only about 50% of those found in the total cytoplasmic fraction (obtained after boiling with detergent and mercaptoethanol) of cells in the absence of drug (KATZ and FELIX 1977). Furthermore, using quantitative immunoprecipitation and polyacrylamide gel electrophoresis techniques, they found an overall quantitative slowdown in late viral protein synthesis (KATZ and FELIX 1977); a conclusion also reached by PENNINGTON in 1977 (the failure of ApPLEYARD et al 1962ApPLEYARD et al , 1965 to observe many late antigens in rabbitpox-infected cells may have been due to the low sensitivity of the immunoprecipitation in agar technique that was used or the inability to detect "insoluble" antigens such as P4a and 4a).…”

Selective repression of v-abl-encoded protein by N-methylisatin-beta-4',4'-diethylthiosemicarbazone and N-allylisatin-beta-4',4'-diallylthiosemicarbazone

“…Methylisatin-p-thiosemicarbazone (M-IBT) prevents the replication of smallpox virus (1) by inhibition of a late step of virus maturation, involving the synthesis of its late proteins (6).…”