Cucumber mosaic virus-induced RNA replicase: solubilization and partial purification of the particulate enzyme

Cucumber seedlings infected with cucumber mosaic virus (CMV) contain a virus-induced RNA-dependent RNA polymerase (RNA replicase) in both soluble and particulate fractions of plant extracts; no such activity is found in uninfected plants. The extensive purification and characterization of the soluble RNA replicase has been described (R. Kumarasamy and R. H. Symons, 1979, Virology 96, 622-632) while the characterization of the particulate enzyme is reported here. The particulate RNA replicase was solubilized-by incubation in 150 mM MgS04 at 37 degrees for 5 min; both magnesium and sulfate ions were required for maximum release. Only 5-7% of the protein of the particulate fraction was released under these conditions either in the presence or absence of a variety of salts. The solubilized enzyme was purified about 60-fold by a three-step procedure to a specific activity similar to that of the 10,000-fold purified soluble enzyme. CMV RNA and poly(C)-copying activities copurified through the three steps. Purification was followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the in vitro 3H-labeled proteins. The most prominent virus-induced components were two proteins of Mr 100,000 and 110,000 which correspond in size to two virus-induced proteins in the extensively purified soluble RNA replicase. The Mr 110,000 protein comigrated with the full length in vitro translation product of CMV RNA 2 while the Mr 100,000 was detectably larger than the full length in vitro translation product of CMV RNA 1.