Soluble RNA polymerase complex from poliovirus-infected HeLa cells

Abstract

A stable, soluble poliovirus RNA-polymerase complex has been isolated from particulate structures in poliovirus-infected HeLa cells with an anionic-nonionic detergent mixture. The complex sediments in a sucrose gradient with a sedimentation constant of approximately 70 S. The isolated enzyme activity is proportional to the rate of viral RNA synthesis in vivo throughout the infectious cycle. Inhibition of protein synthesis with cycloheximide during time of maximal virus replication results in a rapid fall of RNA synthesis and a proportional exponential decrease in isolated polymerase complex activity, the decay occurring with a t 1 2 = 15 min . Properties of the in vitro polymerase reaction are described. The products synthesized by the enzyme complex include RNase-resistant material sedimenting at 20 S, and RNase-sensitive material that cosediments with RNA extracted from purified virions at 35 S. A large fraction of the RNase-resistant material becomes RNase-sensitive after heating for 10 min at 45 °; the remainder requires heating at temperatures greater than 100 °. The polymerase complex has been purified approximately 85-fold. Activity is independent of exogenous polio RNA template.