Evidence for equimolar synthesis of double-strand RNA and minus-strand RNA in rotavirus-infected cells

Abstract

The genome of the rotaviruses consists of eleven segments of double-strand RNA (dsRNA). Each segment is replicated asymmetrically with viral plus-strand RNA, i.e. messenger (m)RNA, serving as the template for the synthesis of minus-strand RNA to produce dsRNA. To examine the relative frequency of replication of each of the eleven genome segments, MA104 cells were infected with low (3rd) and high (12th) passage stocks of simian rotavirus SA11. The total cytoplasmic RNA of the infected cell was radiolabeled either by maintaining the infected cells in the presence [ 3H]uridine prior to harvest or by 3'-endlabeling the purified RNA with [ 32P]pCp and T4 RNA ligase. The RNA was then analyzed for the presence of 3H- and 32P-labeled dsRNA by electrophoresis on 10% polyacrylamide gels. Total cytoplasmic RNA from infected cells was also 3'-end-labeled with [ 32P]pCp and T4 RNA ligase and examined for the presence of minus-strand RNA by electrophoresis on low pH agarose-urea gels. Bands representing dsRNAs and minus-strand RNAs on autoradiographs of the gels were analyzed for intensity by densitometry. The results showed that the eleven segments of viral dsRNA were present in equimolar concentrations in cells either infected with low or high passage stocks of virus. Like intracellular dsRNAs, full-length minus-strand RNAs were also present in equimolar concentration in cells either infected with low or high passage rotavirus. These data indicate that, despite the non-equimolar levels of viral mRNAs in the cell, the eleven genome segments of rotavirus are replicated with equal frequencies in vivo.