Replication of tobacco mosaic virus. V. Properties of the bound and solubilized replicase

Insoluble, bound tobacco mosaic virus (TMV) replicase, isolated from leaves of TMV-infected plants has been shown to catalyze the synthesis of nucleotide sequences of both TMV-RNA and its complement. Treatment of the complex containing the bound enzyme with Nonidet P-40 and KCl releases a TMV replicase which may then be further purified by sedimentation into a glycerol gradient. The partially purified enzyme is markedly stimulated by added RNA templates, but it exhibits little specificity for any of the 9 RNAs tested. The product of the partially purified enzyme was complementary to the RNA used as template and was bound to the template forming a ribonuclease-resistant, double-stranded molecule. The partially purified enzyme exhibits characteristics expected of an RNA-dependent RNA polymerase; viz., the requirement for four ribonucleoside triphosphates for maximum activity, insensitivity to added actinomycin D, rifampicin, deoxyribonuclease, and orthophosphate ion, sensitivity to added pyrophosphate or ribonuclease, and stimulation by RNA. The partially purified replicase cosediments in glycerol gradients approximately with human gamma globulin, suggesting it has a molecular weight of about 160,000.