The Specificity of Dimethylbenzylrifampicin as an Inhibitor of Viral Induced Transformation

Abstract

The effect of 2',6'-dimethyl-N(4')-benzyl- Certain derivatives of rifampicin (1-7) actively inhibit oncornavirus RNA-dependent DNA polymerase (reverse transcriptase) (8,9). Two rifampicin derivatives, dimethylbenzylrifampicin (DMB) and rifazacyclo-16, inhibit viral reverse transcriptase (7) and at comparable concentrations also inhibit murine sarcoma virus (MSV) transformation of mouse cells in tissue culture (10)(11)(12)(13)(14). This correlation between biological and biochemical data suggests that these drugs block transformation by inhibiting the viral induced reverse transcriptase; alternatively the drugs may inhibit other cellular enzymes required for transformation. To further explore the latter possibility, we have compared the effect of DMB on transformation by MSV with its effect on transformation induced by simian virus 40 (SV40), a small DNA-containing virus which does not have reverse transcriptase activity. We found that SV40-induced transformation was completely normal under conditions where MSV transformation of the same cells was greatly inhibited. MATERIALS AND METHODSCell Culturing. Balb/3T3 clone A31 cells (obtained from Dr. G. Todaro) were used as the parental cell stock. The cells were grown in Dulbecco's modification of Eagle's medium (Gibco no. H21) with 10% calf serum (Colorado Serum Co.) (complete medium). To obtain a drug resistant variant, 100 Balb/3T3 cells were plated into a 60-mm dish with complete medium containing 3jug/ml of DMB and allowed to grow for 3 weeks with biweekly fluid changes. The cells were passaged one time to a Falcon flask containing complete medium plus 3 jug/ml of DMB and then passaged 4 times in the same medium containing 20;zg/ml of DMB. The cells that survived this treatment grew well in DMB and were considered resistant variants. SV40 Virus. SV40 (small plaque variant) was prepared by innoculating Vero cells in Dulbecco's medium plus 2% fetalcalf serum with 1 X 10-4 plaque-forming units per cell of SV40 and harvesting the cell supernatant 2 weeks later at the time of maximum cytopathic effect. Viral stocks were titered on TC-7 monkey cells by means of a fluorescent antibody assay. Three days after infection with various dilutions of virus, the cells (grown on coverslips) were fixed and stained for V antigen (monkey anti-V serum was kindly supplied by Dr. H. Ozer. The fluorescein-conjugated antibody to monkey globulin was obtained from Sycco Co.). SV40 Transformation. For the transformation assay, 60-mm tissue culture dishes of confluent resistant cells grown in complete medium plus DMB, 3,4g/ml, were infected by removing the medium, adding 0.5 ml of either SV40 or mockinfected cell supernatants and incubating at 370 for 3 hr with periodic shaking. The virus or mock virus was removed and fresh complete medium added for 3 hr. Cells were treated with trypsin and plated at various dilutions; cells were allowed to become confluent or colonies were obtained as described (15). In brief, the petri dishes were fixed and stained with hematoxylin (Harris-Lillie...

Different modes of inhibition of purified ribonucleic acid directed deoxyribonucleic acid polymerase of avian myeloblastosis virus by rifamycin SV derivatives

“…Inhibition of cell transformation and/or oncornavirus replication by rifamycin derivatives has been reported by several laboratories (Barlati and Vigier, 1972;Ting et al, 1972;Green et al, 1972Green et al, , 1974Bissel et al, 1974;Smith and Hackett, 1974;Shannon et al, 1974; Szabo et al, 1976). Thus far, it appears that when cytotoxic effects are avoided the antiviral activity observed may be the result of the inhibition of the reverse transcriptase and/or interference with other postpenetrational viral functions.…”