Abstract

The 11 rotavirus mRNAs are capped, but not polyadenylated, have a high AU content, and serve as templates for the synthesis of double-stranded (ds)RNA. Earlier studies using a cell-free replication system showed that the 5'- and 3'-untranslated regions (UTRs) of the mRNAs have cis-acting signals that promote minus-strand synthesis. To identify additional factors that affect RNA replication, chimeric RNAs were made that consisted of portions of the gene 8 mRNA of SA11 rotavirus and of the gene for green fluorescent protein (gfp) or for the N protein of respiratory syncytial virus. Analysis of the chimeras in the cell-free replication system under noncompetitive conditions showed that the open reading frame (ORF) of viral mRNAs contains information that specifically promotes minus-strand synthesis. Results were also obtained indicating that a high AU content may increase the replication efficiency of RNAs and that, in general, an inverse correlation exists between replication efficiency and the length of the RNA template. Replication assays performed under competitive conditions showed that nonviral RNAs can interfere significantly with the replication of viral mRNAs, mostly likely by sequestering nonspecific RNA-binding proteins that are of limited concentration in the replication system and that are essential for dsRNA synthesis. In summary, rotavirus dsRNA synthesis is affected by many factors including cis-acting replication signals located in the 5'-UTR, 3'-UTR, and ORF of the mRNA as well as the size and possibly the AU content of the mRNA. (C) 1999 Academic Press.