Isolation and characterization of double stranded RNA containing infectious viral genome RNA from cells infected with Semliki Forest virus

Summary

A double stranded virus specific RNA sedimenting at about 19S on sucrose density gradients has been isolated from BHK-21 cells infected with Semliki Forest virus (SFV). The molecule consists of double stranded RNA (ds RNA) since it is labeled with³H-uridine, is souble in 2m LiCl, resistant against treatment with DNase and RNase at 2×SSC, hydrolyzed by alkali treatment, has a sharp thermal melting point at 89° in 1/10 SSC, and an extended appearance under non denaturing conditions in the electronmicroscope. The following findings show that it consists of intact, infectious 42S RNA similar or identical to the genome RNA of SFV complexed to a complementary 42S minus strand RNA: 1. Denaturation converts the ds RNA into molecules cosedimenting with 42S RNA isolated from SFV particles. 2. About 50 per cent of the radioactivity of³H-uridine labeled 42S RNA molecules generated from 19S ds RNA by denaturation hybridizes to 42S viral RNA. 3. The specific infectivity of denatured 19S ds RNA is about half of that of similarly treated viral 42S RNA. Further properties of this molecule are discussed.