Dy. Chen et Jt. Patton, De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in acell-free system involves a novel mechanism of initiation, RNA, 6(10), 2000, pp. 1455-1467
The replicase activity of rotavirus open cores has been used to study the s
ynthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replica
tion system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly
conserved and are necessary for efficient (-) strand synthesis in vitro, C
haracterization of the cell-free replication system revealed that the addit
ion of NaCl inhibited (-) strand synthesis. By preincubating open cores wit
h (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UT
P, the salt-sensitive step was overcome. Thus, (-) strand initiation, but n
ot elongation, was a salt-sensitive process in the cell-free system. Furthe
r analysis of the requirements for initiation showed that preincubating ope
n cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, al
lowed (-) strand synthesis to occur in the presence of NaCl, Mutagenesis su
ggested 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 f
rom the 3' end of the (+) strand RNA. During preincubation with GTP, format
ion of the dinucleotides pGpG and ppGpG was detected; however, no such prod
ucts were made during preincubation with ATP, CTP, or UTP, Replication assa
ys 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 tem
plate-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.