The synthesis of infectious RNA with a replicase purified according to its size and density.

Abstract

We report the further purification of Q[beta] replicase by banding in Csd followed by zonal centrifugation In linear gradients of sucrose. The resulting enzyme is effectively free of virus particles, permitting direct assay of the RNA product for infectivlty. The concomitant removal of polynucleotlde contaminants does not alter the ability of the replicase to respond to added Q[beta]-RNA by synthesizing infectious copies. The possibility that this is due to "activation" of pre-existent RNA to higher infective efficiency is therefore virtually eliminated. The replicase activity behaves as a single component with an apparent molecular weight of 110,000 and a density of 1.26 gm/cm3. The data do not encourage involving pre-existent RNA (double- or single-stranded), free or enzyme-associated, in the replicase reaction. Neither do they provide evidence for the possibility that replicase consists of 2 or more separable protein mediating distinct reactions in a 2-step process.