Replication of Sindbis Virus II. Multiple Forms of Double-stranded RNA Isolated from Infected Cells

Abstract

Three species of double-stranded RNA (replicative forms) have been isolated from cells infected with Sindbis virus after treating the extracts with pancreatic ribonuclease. Their molecular weights have been estimated to be 8.8 x 10^6, 6.6 x 10^6 and 2.9 x 10^6 daltons (RFI, RFII and RFIII, respectively). RFII and RFIII were found to exist in a one-to-one molar ratio throughout the infection cycle, although the total amounts of these replicative forms varied. The molar ratio of RF1 to the other two replicative forms was different at various times after infection. However, in the middle of the virus life-cycle, from two to six hours post-infection, this ratio was constant at 0.4 to 1. Sedimentation analysis of pulse-labeled RNA from Sindbis-infected cells indicated that all replicative intermediates had molecular weights greater than or equal to 8.8 x 10^6 daltons. Since all three replicative forms were liberated from the peak of replicative intermediates by treatment with ribonuclease, this suggested that RFII and RFIII were joined together before digestion. The constant one-to-one molar ratio of RFII and RFIII supported this hypothesis. Kinetic experiments demonstrated that while RFI and RFIII were labeled equally rapidly, RFII was labeled some 20 to 50 times more slowly. Hybridization competition experiments showed that the minus strand of RFIII is complementary to 26 s RNA, a species of single-stranded RNA found in infected cells, and that the minus strand of RFII is complementary to the remainder of the 49 s viral genome. We postulate, therefore, that a precursor-product relation exists between RFI and 49 s RNA, between RFIII and 26 s RNA and between RFII and a third species of Sindbis-specific RNA, which we have not yet detected. Our results are consistent with the hypothesis that two classes of replicative intermediates exist in Sindbis-infected cells. One serves as the template for the synthesis of 49 s RNA, and the second manufactures two smaller segments of viral-specific RNA.

Additional details