Isolation and Characterization of an RNA-Dependent RNA Polymerase from Black Beetle Virus-Infected Drosophila melanogaster Cells

Abstract

Crude lysates of black beetle virus (BBV)-infected cells of Drosophila melanogaster contain an RNA-dependent RNA polymerase activity not detectable in uninfected cells. The activity (designated BBV polymerase) sedimented at 20,000 × g , indicating an association with particulate material. It was solubilized from the pellet by sonication in a magnesium-deficient buffer. Differential centrifugation resulted in a 43-fold purification with 84% recovery of polymerase activity. The effects of divalent and monovalent cations, time, temperature, and pH on the activity of the partially purified polymerase were examined. RNA synthesis was not stimulated by the addition of exogenous BBV RNA, suggesting that an enzyme-template complex existed. Analysis of the RNA products of the RNA polymerase reaction indicated that full-length “negative” strand BBV RNAs were synthesized.