De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation

Abstract

The replicase activity of rotavirus open cores has been used to study the synthesis of (−) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5′-UGUGACC-3′, are highly conserved and are necessary for efficient (−) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (−) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (−) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (−) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (−) strand synthesis initiated at the 3′-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3′ end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (−) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (−) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5′-phosphorylated dinucleotide, that is, pGpG or ppGpG.