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.