Characterization of RNA-dependent RNA polymerases in uninfected and cowpea chlorotic mottle virus-infected cowpea leaves: selective removal of host RNA polymerase from membranes containing CCMV RNA replicase.

Extracts from Cowpea chlorotic mottle virus (CCMV)-infected Cowpea leaves contained membrane-bound (31,000 g pellet) and soluble (31,000 g supernatant) RNA-dependent RNA polymerase activities. The membrane-bound RNA-dependent RNA polymerase (CCMV RNA replicase) increased 12-fold 4 days after inoculation. The viral RNA synthesis in vitro proceeded linearly for 20 min and required the four nucleoside triphosphates and Mg²⁺ ions for activity. Manganese ion was a poor substitute for Mg²⁺. Optimal enzymatic activity in vitro was unaffected by exogenous RNA or KCI. The CCMV RNA replicase product was predominantly heterodisperse single-stranded RNAs, some of which comigrated with CCMV virion RNA. Small amounts of large double-stranded RNAs were also products of the replicase reaction. The soluble RNA-dependent RNA polymerase from CCMV-infected or healthy Cowpea leaves required the four nucleoside triphosphates and Mg²⁺ ions for activity. Its activity in the in vitro assay was stimulated by adding exogenous RNAs but was inhibited by KCI. The product of the soluble RNA-dependent RNA polymerase was predominantly double-stranded RNA of approximately 4 to 6 S. RNA-dependent RNA polymerase activity, similar to that detected in the soluble fraction, was detected in the membrane pellet. This activity, which complicates the analysis of viral replicase assay, was removed without affecting CCMV RNA replicase activity by washing the 31,000 g pellets with buffer containing 0.5 M KCI. The KCI treatment aids in preparation of membrane-bound fractions devoid of host RNA-dependent RNA polymerase activity and high in viral replicase activity.