The effect of ribonuclease on the replicative forms of Sindbis virus RNA

Three species of double-stranded RNA, designated RF I, RF II, and RF III in order of decreasing size (25), are produced by ribonuclease treatment of extracts of chicken embryo cells infected for 6 hours with Sindbis virus. Only one class of replicative form RNA is present in extracts not treated with ribonuclease; this class contains some molecules which can be enzymatically cleaved to produce the other two replicative forms. At a low level of enzyme (0.001 microgram/ml) the major species obtained was RF I, the replicative form of the genome. When the enzyme concentration was increased 10-, 100-, and 1000-fold, there was a progressive increase in the proportions of RF's II and III and a concomitant decrease in the proportion of RF I. The generation of RF's II and III by nuclease resulted in the ratio expected for these two species if they are produced by cleavage of RF I-like molecules. In preparations of isolated double-stranded RNA, only RF I and replicative intermediate RNA were present. Mild nuclease treatment of these preparations converted the replicative intermediates primarily to RF I. Higher enzyme levels generated greater proportions of RF II and RF III, but RF I-like molecules were the major source for these increased proportions. Treatment of the isolated naturally occurring replicative form with 0.01 microgram of ribonuclease per ml cleaved some molecules migrating as RF I during gel electrophoresis into molecules which migrated as RF II and RF III.