Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC), template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3' terminal promoter region in the viral minus-strand (-)RNA, the other class of eIF4AIII-like RNA helicases bind to a different cis-acting element, namely the 5' proximal RIII(-) replication enhancer (REN) element in the TBSV (-)RNA. We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (-)RNA could unwind the dsRNA structure within the RIII(-) REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(-) REN in stimulation of plus-strand (+)RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(-) REN that promotes bringing the 5' and 3' terminal (-)RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+)-strand synthesis, thus resulting in asymmetrical viral replication.

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Published in PLOS Pathogens, v. 10, issue. 4, e1004051.

© 2014 Kovalev, Nagy. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Figure_S1.pdf (101 kB)
Over-expression of selected yeast RNA helicases enhanced TBSV repRNA accumulation in yeast. (A) The wt yeast strain was used for the overexpression experiments. Top panel: Northern blot analysis of TBSV repRNA accumulation in yeast overproducing the His6-tagged Dbp3p (DDX5-like), Dbp5p, Dbp7p, Fal1p (eIF4AIII-like) and Tif1p (eIF4A-like) DEAD-box helicases from plasmids. These yeast helicases have been identified in previous high throughput screens with TBSV and yeast host. The TBSV repRNA levels were normalized based on rRNA loading. Bottom panel: Northern blot analysis shows the level of ribosomal RNA loading. (B) Top panel: Detection of the overproduced His6-tagged Dbp3p, Dbp5p, Dbp7p, Fal1p and Tif1p DEAD-box helicases by Western blotting using anti-His antibody in yeast. Bottom panel: Detection of Flag-tagged p33 and p92pol by Western blotting using anti-Flag antibody. The total protein level in each sample was analyzed by SDS-PAGE and Coommassie-blue staining. Note that all the helicases expressed in yeast are His6-tagged at the N-terminus.

Figure_S2.pdf (129 kB)
AtRH5, and the yeast Dbp3p and Fal1p helicases bind to the RIII(−) replication enhancer element in the TBSV (−)RNA. (A–C) In vitro binding assay with 0.6 µg of purified AtRH5, the yeast Dbp3p and Fal1p helicases. The assay contained the 32P-labeled DI-72 (−)repRNA (~0.1 pmol) plus increasing amount of unlabeled competitor RNAs, including RI(−), RII(−), RIII(−) or RIV(−). The free or helicase-bound ssRNA was separated on nondenaturing 5% acrylamide gels, followed by quantification of the bound RNA by a Phosphorimager. See further details in Fig. 3.

Figure_S3.pdf (94 kB)
Utilization of full DI-72 RNA/RNA duplex by the tombusvirus replicase is facilitated by cellular helicases in vitro. (A) Schematic representation of the 621 bp DI-72 RNA/RNA duplex used in the tombusvirus replication assay. (B) Representative denaturing gel of 32P-labeled RNA products synthesized in vitro using DI-72 RNA/RNA duplex template by the purified tombusvirus replicase obtained from yeast with depleted Fal1p in the presence of 0.4 µg of purified recombinant cellular helicases (except 1.0 µg in case of AtRH20) is shown. Note that lanes 1–6 show samples from the in vitro replication assays with the combination of two cellular helicases [i.e., AtRH20 (1.0 µg) plus the shown helicase), while lanes 7–13 show samples with only a single helicase in the assay. (C) Detection of (+) and (−)-stranded RNA products produced by the purified TBSV replicase on the DI-72 RNA/RNA duplex template in vitro replication assay containing cellular AtRH5 and AtRH20 helicases (lane 2 in panel B). The blot contains the same amount of cold (+) and (−)-stranded DI-72 RNA, while the 32P-labeled repRNA probes were generated as in panel B. The ratio of (+) and (−)-stranded RNA products was estimated.

Figure_S4.pdf (168 kB)
Cell-free TBSV replication assay supports a role for Fal1p and Dbp3p helicases in plus-strand synthesis. (A) Scheme of the CFE-based TBSV replication assay. TBSV DI-72 (+)repRNA was added to the whole cell extract (CFE) prepared from either WT yeast or Dbp3p-depleted and ts-Fal1p-inactivated yeast strain expressing p33 and p92pol replication proteins. The membrane and soluble fractions were separated at the end of the replication assay by centrifugation. (B) Detection of single- and double-stranded RNA products produced in the cell-free TBSV replication assays. “T” total, “M” membrane fraction, “S” soluble fraction. Note that the ssRNA present in the “S” fraction represents the released (+)repRNA products from the membrane-bound VRCs. (C) Scheme of the CFE-based TBSV replication assay with purified recombinant cellular helicases. (D) Detection of single- and double-stranded RNA products produced in the cell-free TBSV replication assays by denaturing PAGE analysis of the 32P-labeled TBSV repRNA products (See panel C). The assay also contained purified recombinant AtRH2 or AtRH5 (0.15 µg) or MBP (the same molar amount as the helicases) as a control. Odd numbered lanes represent replicase products, which were not heat treated (thus both ssRNA and dsRNA products are present), while the even numbered lanes show the heat-treated replicase products (only ssRNA is present). The % of dsRNA and ssRNA in the samples are shown. Note that, in the nondenatured samples, the dsRNA product represents the annealed (−)RNA and the (+)RNA, while the ssRNA products represents the newly made (+)RNA products. Each experiment was repeated three times.

Figure_S5.pdf (83 kB)
AtRH5 is a component of the tombusvirus replicase in yeast. (A) The membrane-bound tombusvirus replicase was purified via solubilization of the FLAG-tagged p33F from yeast extracts using a FLAG-affinity column (lanes 1–2). Yeast not expressing p33F was used as a control (lane 3). Top panel: Western blot analysis of FLAG-tagged p33F with anti-FLAG antibody. Bottom panel: Western blot analysis of His6-tagged AtRH5 with anti-His6 antibody in the affinity-purified replicase preparations. Note that “soluble” represents the total protein extract from yeast demonstrating comparable levels of His6-AtRH5 in each sample (lanes 4–6). Each experiment was repeated three times. (B) Interaction between yeast DEAD-box helicases and the TBSV p33 replication protein based on the membrane yeast two hybrid assay (split-ubiquitin assay). The bait p33 was co-expressed with the prey full-length host proteins in yeast. The yeast Ssa1p (HSP70 chaperone), and the empty prey vector (NubG) were used as positive and negative controls, respectively. The image shows 10-fold serial dilutions of yeast cultures.

Figure_S6.pdf (171 kB)
Interaction between AtRH2 and AtRH5 and the TBSV p33 replication protein. (A) A schematic representation of viral p33 and its derivatives used in the binding assay (each MBP-tagged at the N-terminus). The various domains include: TMD, transmembrane domain; RPR, arginine-proline-rich RNA binding domain; P; phosphorylated serine and threonine; S1 and S2 subdomains involved in p33:p33/p92 interaction. The results of the in vitro binding experiments are summarized (“+” or “−“, based on two repeats). (B) In vitro pull-down assay of His6-tagged AtRH5 (lanes 1–4), His6-peptide (lanes 5–8) or His6-tagged AtRH2 (lanes 11–14) with MBP-p33 derivatives using amylose resin. Top panel: Western blot analysis with anti-His antibody of His6-tagged helicases pulled down with MBP-p33 derivatives. Lane 9 contains purified His6-tagged AtRH5 as a standard. Bottom panel: Coomasie stained SDS-PAGE gel, showing quality and quantity of purified MBP-p33 derivatives.

Figure_S7.pdf (80 kB)
AtRH2 and AtRH5 do not inhibit the binding of p33 replication protein to the TBSV (+)RNA. (A) Scheme of the in vitro TBSV (+)RNA binding assay. (B) In vitro EMSA binding assay with purified MBP-p33C [an N-terminally truncated version of p33, which shows selective binding to the viral (+)RNA] in the presence of purified AtRH5 or AtRH2. The 32P-labeled RNA template was RII(+)-SL (~0.1 pmol), which is the p33RE [part of RII(+)], and binds selectively to p33. The assay contained 0.02 µg of purified recombinant MBP-p33C, plus 0.02, 0.06, 0.2 or 0.6 µg of purified recombinant AtRH5 or AtRH2, as shown. The samples in lanes 6 and 13 contained 0.6 µg of purified recombinant AtRH5 or AtRH2 in the absence of p33C.