Use of micrococcal nuclease in the purification of highly template dependent RNA-dependent RNA polymerase from brome mosaic virus-infected barley.

The template dependence of RNA-dependent RNA polymerase from brome mosaic virus (BMV)-infected barley was greatly increased by micrococcal nuclease digestion of the endogenous RNA. [32P]UMP incorporation by the nuclease-treated enzyme was stimulated 20-fold when BMV RNA was added as template, while incorporation by the untreated enzyme was stimulated only 5-fold by the addition of BMV RNA. Other properties of BMV polymerase were not changed significantly by nuclease digestion. The extract remained highly active and template specific. Analysis of the products of the reaction showed that separated BMV RNA components could be replicated independently to yield full-length replicative-form RNAs. These data provide strong evidence that the extract is capable of initiating RNA synthesis and that it includes the intact viral replicase. This method should be of general use, allowing the study of cell-free replication of any viral nucleic acid without requiring purification or solubilization of the replicase.