RNA-dependent RNA polymerases in uninfected and tobacco mosaic virus-infected tobacco leaves: Viral-induced stimulation of a host polymerase activity

Abstract

The soluble RNA-dependent RNA polymerase activities in the 105,000- g supernatant fraction of homogenates prepared from uninfected and tobacco mosaic virus (TMV)-infected tobacco leaves have been compared. The enzymes were purified 40- to 50-fold from both mock-inoculated and TMV-infected leaves and were found to have identical behavior upon (NH 4) 2SO 4 fractionation, Sephadex G-100 gel filtration, and DEAE-Bio-Gel and phosphocellulose chromatography. Although no qualitative differences in polymerase activity were detected, the specific activity of the polymerase from infected leaves was greater than that from mock-inoculated leaves at each step in the purification. Moreover, the most highly purified enzymes from both types of leaf tissue were indistinguishable with respect to a number of catalytic parameters, viz., requirements for RNA synthesis, kinetics of RNA synthesis, lack of template specificity, and character of the RNA products such as strandedness, T m, size, and complementarity to the TMV-RNA used as the template. SDS-polyacrylamide gel electrophoresis of the proteins in the most highly purified enzymes showed that both preparations contained 2 major and at least 13 minor corresponding polypeptides; none were unique to TMV infection. The evidence in toto suggests that the enhanced soluble RNA-dependent RNA polymerase activity following TMV infection is due to a stimulation of a host RNA-dependent RNA polymerase rather than to the genesis of a new viral-coded RNA polymerase. The possible relationships between the soluble host polymerase, the membrane-associated TMV replicase, and a 130,000-MW viral-coded polypeptide thought to be involved in TMV-RNA replication remain to be resolved.