In Vitro Replication of Cowpea Mosaic Virus RNA I. Isolation and Properties of the Membrane-Bound Replicase

Abstract

A fraction which contained the membrane-bound cowpea mosaic virus RNA replicase was isolated from cowpea mosaic virus-infected cowpea leaves. The replicase activity appeared on day 1 after inoculation, then increased to reach a maximal on day 4. The increase in enzyme activity preceded the most-rapid virus multiplication. The membrane-bound replicase activity was almost completely insensitive to actinomycin D and DNase. The corresponding fraction from healthy leaves had no RNA-dependent RNA polymerase activity. The viral RNA synthesis in vitro proceeded linearly for 20 min and required all four ribonucleoside triphosphates and Mg 2+ ions. Mn 2+ was a poor substitute for Mg 2+ . The reaction was optimal at pH 8.2. During the whole period of RNA synthesis the in vitro synthesized RNA was at least 70% resistant against RNase in 2 × SSC (0.15 M NaCl plus 0.015 M sodium citrate), but completely digestable by RNase in 0.1 × SSC. Analysis of the products by sucrose gradient centrifugation followed by treatment of separate fractions with RNase demonstrated that both single-and double-stranded RNA were present. Double-stranded RNA sedimented at about 20 S , with a shoulder at 16 S to 17 S . A minor part of the double-stranded RNA sedimented below 10 S . Single-stranded RNA sedimented with the same rate as the two viral RNAs, 26 S and 34 S .