Mechanism of Antiviral Action of Acetone on Rabbitpox Virus Replication

Abstract

Acetone added to the maintenance medium in a 1% concentration reduces the yield of infectious rabbitpox virus in L-cell monolayer cultures by 90 to 97%. This concentration of the inhibitor is not toxic for cells. It was established that there is an inhibitor-sensitive stage late in the infectious cycle. Acetone exerted no significant influence on production of early viral messenger ribonucleic acid, formation of polyribosomes, deoxyribonucleic acid (DNA) replication, or protein synthesis. In the presence of acetone, the assembly of so-called "acetone particles" occurred. These particles are similar to normal virions by morphological and sedimentation properties but are slightly different from them in buoyant density. The amount of virusspecific DNA and the optical density of the "acetone particles" are the same as those of normal virions despite a 10to 25-fold difference in the infectivity of the preparations.It has been shown previously (4) that acetone added to the maintenance medium in a final concentration of 1% significantly inhibits replication of poxviruses. The possibility of the selection of mutants resistant to the inhibitor has been demonstrated (4). The present work was devoted to the study of the mechanism of action of this inhibitor. MATERLIALS AND METHODSCells. An L-cell monolayer culture was grown in medium 199 supplemented with 10% bovine serum and antibiotics.Virus. Rabbitpox virus, Utrecht strain, was propagated in chick embryos, concentrated, and purified by Joklik's method (7). Virus titration was performed on chorioallantoic membranes of 12-dayold chick embryos according to Westwood (23), with the use of five embryos per dilution. The infectious titer of the virus used in the experiments was 1.5 X 1010 to 2 X 1010 plaque-forming units (PFU)/ml. Conditions of cell inoculation and maintenance of in-fected monolayers. Generally, the multiplicity of infection was 30 to 50 PFU/cell. In the case of the experiments on polyribosome analysis and determination of protein synthesis, the multiplicity of infection was 200 to 300 PFU/cell. The virus was sonically treated before inoculation. The virus was allowed to adsorb on the cells at 36 C for 20 min. The unadsorbed virus was removed, and the cells were washed with warm Earle's saline. The time postinfection was counted from the moment of the addition of warm medium 199. At certain intervals postinfection, the cells were disrupted by three cycles of freezing and thawing, and the virus yields were determined.Acetone. In all experiments, unless indicated other-wise, acetone (to a final concentration of 1%, v/v) was added together with medium 199 without serum immediately after adsorption.Preparationof thecytoplasmic extract. The cells were washed with cold Earle's saline, scraped off with a rubber policeman, and sedimented by centrifugation at 1,000 X g for 15 min. Then the cells were resuspended in hypotonic buffer. In the experiments on polyribosomes, the following buffer was used: 0.01 M triethanolamine-HCl, pH 7.8, 0.01 M NaCl, and 0.003 M Mg?+ (refe...