PMC

UL-DL cis-acting element functions as an enhancer element for the replicase assembly (EERA). (A) Scheme of the in vitro TBSV replicase assembly assay performed with yeast CFE. Note that the recombinant p33 and p92^pol are purified from E. coli, while the CFE was prepared from BY4741. After a 1-hour reconstitution, the membrane-bound replicase was solubilized with Triton X-100/SB3-10 detergent, followed by purification on an Ni column of the 6×His/MBP-tagged p33, which is an integral part of the replicase complex. The activity of the affinity-purified TBSV replicase was tested on DI-72(-) RNA added to each sample using the same amount of RNA. (B) Mutations within UL-DL, which interfere with base pairing or reform base pairing due to complementary mutagenesis. (C and D) Representative denaturing gels of ³²P-labeled RNA products synthesized by affinity-purified TBSV replicase preparations obtained in TBSV replicase assembly assays in vitro with yeast CFE in the presence of all four or two ribonucleotide triphosphates. The replicase assembly assay contained the wt DI-73 plus-strand repRNA or versions with mutations in the UL-DL region, as shown in panel B. We used DI-73 plus-strand repRNA in the assembly assay because it contains the UL-DL elements, while DI-72 plus-strand repRNA lacks the corresponding RNA sequences. The replication-competent RNA was DI-73 based, while the replication-incompetent DI-73 carried a debilitating mutation. The template RNA was DI-72(-) repRNA, which produces both full-length (due to terminal initiation [ti]) and shorter (due to internal initiation [ii]) complementary products in the in vitro assay with the purified TBSV replicase. The level of full-length RNA synthesis was compared to that of the replicase activity obtained with DI-73 plus-strand repRNA (100%). (E) No RNA template was added to the in vitro assays with the purified TBSV replicase preparations, while the indicated repRNAs were used during the TBSV replicase assembly assays in vitro with yeast CFE prior to affinity purification of the TBSV replicase. Each experiment was repeated.

A mini-RNA template with RII(+)-SL and RSE-gPR can efficiently support the in vitro assembly of the TBSV replicase. (A) Denaturing-PAGE analysis of the in vitro-reconstituted TBSV replicase in the presence of repRNAs. The replicase reconstitution assay contained CFE, affinity-purified recombinant TBSV p33 and p92^pol, ATP/GTP, and equal amounts of TBSV repRNAs. After assembly and affinity purification, the activity of the replicase preparations was tested on DI-72(-) RNA template in vitro. See further details in . (B) Denaturing-PAGE analysis of the in vitro-reconstituted TBSV replicase in the presence of the indicated mini-repRNAs. Note that each construct had RII(+)-SL, and deletions and mutations were introduced only at the 3′ end (circled) of the DI mini-RNA construct. The only exception is mini-RII(C-G), which carried a single C-to-G mutation within the C·C mismatch in RII(+)-SL. The in vitro replicase assembly assay was performed as described for panel A, except that RI/III(-) was used as a template in the replicase activity assay. (C) Denaturing-PAGE analysis of the CFE-based replication assay. The in vitro replication assay contained yeast CFE, affinity-purified recombinant TBSV p33 and p92^pol, and equal amounts of various TBSV repRNAs (as shown in ). The full-length products are depicted with arrowheads on the right. Note that the repRNA serves as both an assembly factor and a template in this assay. (D) Denaturing-PAGE analysis of the in vitro replicase assay. The assay contained both the purified replicase from yeast coexpressing p33 and p92^pol and the plus-strand repRNAs (as shown). The activity of the affinity-purified TBSV replicase was tested on RI/RIII(-) RNA, which was added to all samples in equal amounts. Note that the assembly of the replicase took place in yeast cells in the presence of coexpressed p33/p92 and actively replicating repRNA. The bottom image shows a Western blot demonstrating equivalent amounts of p33/p92 replication proteins in the purified tombusvirus replicase preparations used for the in vitro assay.

Thermal stability of the replicase. A, time course of RNA replication by replicase purified by the standard method and the effects of incubation before the reaction. The replicase (200 nm) was incubated at 37 °C in the absence of RNA and NTPs for the indicated times (0, 0.5, 2, and 4 min) before the replication assay, and then RNA replication was measured at 37 °C after the addition of 50 nm template RNA (s130) and NTPs. The label “no rep” indicates the results of a control experiment without replicase. B, effects of preincubation on residual replication activity. Each form of replicase was incubated at 37 °C in the absence of template RNA and NTPs before the replication assay for the indicated times. The RNA replication reaction was measured as described in A, and the replication rates were determined by linear regression analysis. The replicase purified by the standard method is indicated by the filled circles. Replicase prepared by dialyzing the standard replicase with 10 mm DTT (U-form) is indicated by circles. Replicase prepared by heat inactivation of the standard replicase followed by gel-filtration chromatography (S-form) is indicated by triangles. The data for the S- and U-forms were fitted to a single exponential decay curve as (gray lines). C, effects of preincubation temperature on residual activity. U-form replicase was incubated at different temperatures before the replication reaction was carried out for 2 min in the absence of RNA and NTPs, and then RNA replication rates of the replicase were measured as described in A.

Mix and match in vitro replicase assays. The CNV replicase preparations represent enriched membrane fractions (lanes 1 to 5), affinity-purified proteins (lanes 6 to 10), and solubilized membrane fractions (lanes 11 to 17) obtained from yeast, except for lane 16, which contains products that were generated by CNV replicase obtained from N. benthamiana plants. The CNV replicase assay depicted in lanes 6 to 17 included exogenous RI/III(−) templates. The coexpressed p33, p92, and DI-72(+) RNA in yeast cells are shown on the top as plus signs, while yeast lacking one of these components is marked with minus signs. The boxed lanes represent the mixed preparations as follows. Preparations containing either p33 or p92 (coexpressed with DI-72 RNA) were obtained from separate yeast lines, which were mixed in 1:1 ratio prior to the CNV replicase reaction (replicase products in lanes 3, 6, 14, and 15 are from mixing preparations corresponding to yeast lines shown in lanes 4 and 5; 7 and 8; and 12 and 13, respectively). Note that the CNV replicase preparation in lane 15 was obtained by mixing p92 and p33 preparations in a 1:4 ratio. The CNV replicase products generated on endogenous DI-72 or exogenous RI/III(−) templates are marked. The lane marked Nb represents the replicase product obtained with the plant-derived CNV replicase preparation.