Interaction between murine type-C virus RNA-directed DNA polymerases and rifamycin derivatives

Abstract

Some rifamycin SV derivatives are inhibitors of purified murine type-C virus RNA-directed DNA polymerase, although other polymerases are also affected. We have previously grouped these compounds into three classes (A, B, and C) based on their relative inhibitory effect on the viral polymerase. The concentration for 50% inhibition by these three classes are 5, 20, and 100 μg/ml, respectively. In this report we examine the mechanism of inhibition of the viral enzyme. The inhibitory effect is independent of the choice of template-primer but is sensitive to the presence of non-ionic detergent. Potent inhibitors bind to the viral enzyme as shown by co-sedimentation of the inhibitor with purified enzyme during glycerol gradient centrifugation. At least two molecules of potent (class C) inhibitors bind to each molecule of the viral enzyme. This binding is reversible with a relatively low concentration of Triton X-100 (0.05%) as judged by a complete restoration of the enzyme activity. Similar results were found with bacterial DNA polymerases but much firmer binding was found between Escherichia coli RNA polymerase and rifamycin derivatives since only 20% recovery of enzyme activity was obtained at a concentration of Triton X-100 as high as 5%. With respect to the purified viral DNA polymerase in decreasing order the rifamycin derivative interact with: the free form of the enzyme, the enzyme—template-primer complex, the enzyme—substrate complex, the initiation complex. The inhibitors were non-competitive with the deoxyribonucleoside triphosphate substrates with K i equivalent to 10 −6 M. The inhibition appears to be at the step before initiation. Therefore, conditions which facilitate initation interfere with the effectiveness of these compounds, and the order of addition of each component becomes critical in assessing their relative inhibitory activity.