AtRH2 and AtRH5 promote plus-strand synthesis by the affinity-purified tombusvirus replicase.

<p>(A) Scheme of the tombusvirus replicase assay. Yeast with depleted eIF4IIIA-like Fal1p co-expressing p33 and p92^pol replication proteins and DI-72 (+)repRNA were used to affinity-purify the RNA-free tombusvirus replicase. The <i>in vitro</i> assays were programmed with DI-72 (−)repRNA, and they also contained purified recombinant AtRH2, AtRH5 and AtRH20 helicases in addition to ATP/CTP/GTP and ³²P-UTP. (B) Representative denaturing gel of ³²P-labeled RNA products synthesized by the purified tombusvirus replicases obtained from yeast either with high Fal1p (−DOX) level or depleted Fal1p (+DOX) is shown. The level of complementary RNA synthesis on DI-72(−) RNA template producing “repRNA” (marked as “FL”, the full-length product, made via <i>de novo</i> initiation from the 3′-terminal promoter) was compared in each sample. Note that this replicase preparation also synthesizes 3′-terminal extension products (“3′TEX”). Each experiment was repeated three times. (C) Representative denaturing gel of ³²P-labeled RNA products synthesized <i>in vitro</i> using DI-72(−) template by the purified tombusvirus replicase obtained from yeast with depleted Fal1p in the presence of increasing amounts of purified recombinant AtRH2 (0.2 and 0.4 µg), AtRH5 (0.2 and 0.4 µg) and AtRH20 (1.0 µg) helicases is shown. Samples in lane 5 and 10 contain 0.4 µg AtRH2 and AtRH5, respectively, plus 1.0 µg of AtRH20. Each experiment was repeated three times. (D) Time-course experiment with the purified tombusvirus replicase obtained from yeast with depleted Fal1p using DI-72(−)ΔRII/RIV template. The affinity-purified recombinant AtRH2 (0.4 µg), AtRH5 (0.4 µg) and AtRH20 (0.8 µg) helicases were added to the assay as shown. See further details in panel C. (E) Representative denaturing gel of ³²P-labeled RNA products synthesized <i>in vitro</i> using DI-72(−)ΔRIII/RIV template by the purified tombusvirus replicase obtained from yeast with depleted Fal1p in the presence of purified recombinant AtRH2 (0.4 µg), AtRH5 (0.4 µg) and AtRH20 (1.0 µg) helicases is shown. (F) Time-course experiment with the purified tombusvirus replicase obtained from yeast with depleted Fal1p using DI-72(−)ΔRIII/RIV template. The affinity-purified recombinant AtRH2 (0.4 µg), AtRH5 (0.4 µg) and AtRH20 (0.8 µg) helicases were added to the assay as shown. See further details in panel E.