19 research outputs found

    Opposite effects of subverted cellular DEAD-box helicases on TBSV recombination in yeast and in the CFE assay.

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    <p>(A) TBSV co-opt two groups of cellular helicases that become part of the VRCs: the DDX3-like Ded1/AtRH20 and the eIF4AIII-like AtRH2. These helicases bind to different <i>cis</i>-acting replication elements present at the 3’ and 5’ ends of the viral (-)RNA as shown. Note that RI(-) carries the promoter for (+)-strand synthesis, while the RIII(-) is a replication enhancer element (REN). (B) Over-expression of AtRH2 enhances recRNAs and degRNA accumulation, while over-expression of AtRH20 decreases the occurrence of these RNAs in wt or ded1–199<sup>ts</sup> yeasts. The Northern blot analysis was done as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g001" target="_blank">Fig. 1</a>. (C) Western blot analysis to detect the expression level of His<sub>6</sub>-tagged cellular helicases and the His<sub>6</sub>-tagged viral replication proteins in the WT and ded1–199<sup>ts</sup> yeasts. Asterisk marks the SDS-resistant p33 dimer. (D) The scheme of the CFE-based TBSV replication assay. Note that the level of Ded1p was depleted in TET::DED1 yeast prior to CFE isolation. (E) Denaturing PAGE analysis of the accumulation of 5’-truncated DI-RIIΔ70 degRNA used as the original template in the CFE assay and the <i>in vitro</i> generated recRNA. See further details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g003" target="_blank">Fig. 3</a>.</p

    Suppression of viral RNA recombination by Ded1 helicase is independent of the Xrn1 pathway.

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    <p>(A) Schematic representation of the previously characterized Xrn1-driven TBSV RNA recombination pathway in yeast. Plasmid-driven expression of the DI-(RI in the presence of p33/p92 replication proteins leads to partial 5’ truncations by the cellular Xrn1p 5’-to-3’ exoribonuclease generating DI-RIIΔ70-like degRNAs as shown. DI-RIIΔ70-like degRNAs then participate in RNA recombination as shown. (B) Recombination profile of DI-(RI RNA in <i>xrn1Δ</i>, <i>met22Δ</i> (as a control) and in ded1–199 yeasts. The original expressed DI-(RI degRNA, DI-RIIΔ70-like degRNAs and recRNAs are depicted with arrowheads and arrows, respectively. Note that the recRNA profile is dramatically different in <i>xrn1Δ</i> yeast when compared to ded1–199 yeast. (C) Half-like of DI-RIIΔ70 degRNA in WT, ded1–95 and ded1–199 yeasts. Note that yeast did not express p33/p92 proteins.</p

    Suppression of TBSV recRNA accumulation by Ded1p in <i>in vitro</i> replication assays.

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    <p>(A) The membrane-enriched fraction (MEF) was isolated from wt or ded1–199<sup>ts</sup> yeast expressing the tombusvirus p33 and p92 replication proteins in combination with the DI-AU-FP repRNA, followed by <i>in vitro</i> tombusvirus replication assay. The denaturing PAGE analysis shows the emergence of recRNAs and degRNA as indicated. (B–C) Northern blot analysis of the polarity of the TBSV RNAs synthesized in the MEF assay (see panel A, except the assay was done with nonlabeled ribonucleotides) using <sup>32</sup>P-labeled probes to detect (-)-strands and (+)-strands, respectively. The ratios between various TBSV RNAs were calculated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g002" target="_blank">Fig. 2</a>. (D) Scheme of the yeast cell-free (CFE) assay for TBSV replication. Note that the assembly of the TBSV replicase takes place in the CFE using recombinant viral components as shown. The TBSV p33 and p92 replication proteins and Ded1p and its mutants (D1 is inactive, D11 has enhanced ATPase activity) were purified from <i>E</i>. <i>coli</i>. (E) Reduced replication of the full-length DI-72 repRNA in TET::DED1 yeast CFEs with high or depleted levels of Ded1p (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g001" target="_blank">Fig. 1</a>). (F-G) Denaturing PAGE analysis of the effect of Ded1p and its mutants on the replication of the full-length recombinogenic DI-AU-FP and the 5’-truncated DI-RIIΔ70 degRNA, respectively, in the CFE assay. The experiments were repeated twice.</p

    Reduced TBSV repRNA accumulation in yeast with depleted Pgk1 level.

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    <p>(A) Northern blot analysis shows decreased TBSV repRNA accumulation in a yeast strain (GALS::PGK1) when Pgk1 was depleted. To launch TBSV repRNA replication, we expressed His<sub>6</sub>-p33 and His<sub>6</sub>-p92<sup>pol</sup> from the copper-inducible <i>CUP1</i> promoter, and DI-72(+) repRNA from the <i>ADH1</i> promoter in the parental (BY4741) and in GALS::PGK1 yeast strains. Note that GALS::PGK1 yeast strain expresses HA-tagged Pgk1 from the galactose-inducible <i>GALS</i> promoter from chromosomal location (i.e., HA-Pgk1 replaces the wt Pgk1 in this haploid yeast strain). The yeast cells were cultured for 16 hours at 29°C in either 2% galactose +2% raffinose [(Raf+Gal), inducing condition for HA-Pgk1] or 2% raffinose (lack of induction of HA-Pgk1 expression) SC minimal media supplemented with 50 μM CuSO<sub>4</sub>. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels (second panel from top). Middle panel: Northern blot analysis of <i>PGK1</i> mRNA levels in total RNA samples. Bottom two panels: Western blot analysis of the accumulation level of HA-tagged Pgk1, His<sub>6</sub>-tagged p33, His<sub>6</sub>-p92<sup>pol</sup> proteins using anti-HA and anti-His antibodies, respectively. Each experiment was performed three times. (B) Complementation assay with plasmid-borne expression of His<sub>6</sub>-Pgk1 in GALS::PGK1 yeast strain. His<sub>6</sub>-Pgk1 was expressed from the <i>TEF1</i> constitutive promoter, whereas the expression of the chromosomal HA-tagged Pgk1 was suppressed via 2% glucose in the growth media. Top panels: Northern blot analysis of repRNA level, middle panel: ethidium-bromide stained gel with ribosomal RNA, as a loading control, whereas bottom panels show Western blot analysis using anti-His antibody. Panels on the right represent samples obtained from yeast grown on the nonfermentable glycerol media. (C) The effect of over-expression of yeast Pgk1 on TBSV repRNA accumulation. The plasmid-borne His<sub>6</sub>-Pgk1 was expressed from <i>TEF1</i> promoter in BY4741 (wt) yeast. See further details in panel B. (D) The effect of heterologous expression of NbPgk1 on TBSV repRNA accumulation in yeast. The plasmid-borne His<sub>6</sub>-NbPgk1 was expressed from <i>TEF1</i> promoter in BY4741 (wt) yeast. See further details in panel B.</p

    Opposite effects of subverted cellular DEAD-box helicases on TBSV recombination in plants.

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    <p>(A) Over-expression of AtRH2 or AtRH20 was done in <i>N</i>. <i>benthamiana</i> leaves by agroinfiltration. The same leaves were agro-infiltrated to co-express CNV helper virus and the DI-AU-FP repRNA from the 35S promoter. The control samples were obtained from leaves not expressing AtRH2 or AtRH20 proteins (lanes 5–8). Total RNA was extracted from leaves 4 days after agroinfiltration. The accumulation of repRNA, recRNAs and degRNA in <i>N</i>. <i>benthamiana</i> leaves was measured by Northern blotting (top panel), see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.g001" target="_blank">Fig. 1</a> for details. The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (bottom panel). (B) Over-expression of AtRH20 inhibits recRNA formation from the degRNAs. AtRH20 was over-expressed in <i>N</i>. <i>benthamiana</i> leaves by agroinfiltration. The same leaves were co-agro-infiltrated to co-express CNV p33 and p92 replication proteins and the DI-RIΔ degRNA from the 35S promoter. The control samples were obtained from leaves not expressing AtRH20 protein (lanes 1–3). See further details in panel A.</p

    The cytosolic Pgk1 affects ATP accumulation within the tombusvirus replication compartment in <i>N</i>. <i>benthamiana</i>.

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    <p>(A) A scheme of the FRET-based detection of cellular ATP within the replication compartment. The enhanced ATP biosensor, ATeam<sup>YEMK</sup> was fused to TBSV p33 replication protein. (B) Knock-down of <i>PGK1</i> mRNA level by VIGS in <i>N</i>. <i>benthamiana</i> was done as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006689#ppat.1006689.g004" target="_blank">Fig 4</a>. Twelve days later, co-expression of p33-ATeam<sup>YEMK</sup> and RFP-SKL (peroxisomal luminar marker) was done in upper <i>N</i>. <i>benthamiana</i> leaves by agroinfiltration. The CFP signal indicates the distribution of p33-ATeam<sup>YEMK</sup>, which co-localizes with RFP-SKL to the aggregated peroxisomes. YFP signal was generated by mVenus in p33-ATeam<sup>YEMK</sup> via FRET. The FRET signal ratio is shown in the right panels. The more intense FRET signals are white and red (between 0.5 to 1.0 ratio), whereas the low FRET signals (0.1 and below) are light blue and dark blue. We also show the average quantitative FRET values (obtained with ImageJ) for 10–20 samples on the graph. (C) Comparable experiments with <i>PGK1</i> knock-down <i>N</i>. <i>benthamiana</i> using the mitochondrial CIRV p36 replication protein tagged with ATeam<sup>YEMK</sup> and Tim21-RFP mitochondrial marker protein. See further details in panel B.</p

    The pro-viral role of cytosolic Pgk1 in tombusvirus replication in <i>N</i>. <i>benthamiana</i>.

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    <p>(A) Up-regulation of Pgk1 expression in TBSV-infected <i>N</i>. <i>benthamiana</i> leaves. The mRNA levels for the cytosolic Pgk1 were estimated by semi-quantitative RT-PCR (25 and 28 cycles, the latter is shown) in total RNA samples obtained from either TBSV or mock-infected <i>N</i>. <i>benthamiana</i> leaves. Tubulin mRNA was used as a control (second panel). The bottom panel shows an ethidium-bromide stained agarose gel of total RNA samples with ribosomal RNA and TBSV genomic (g)RNA. (B) Western blot analysis of cytosolic Pgk1 level in total protein samples obtained from yeast either supporting TBSV repRNA accumulation or TBSV-free using anti-Pgk1 antibody. (C-E) Knock-down of <i>PGK1</i> mRNA level by VIGS inhibits the accumulation of tombusvirus RNA in <i>N</i>. <i>benthamiana</i>. Top panels: Total RNA samples obtained from <i>N</i>. <i>benthamiana</i> leaves silenced as shown were analyzed by Northern blotting, which shows the accumulation of TBSV gRNA and sgRNAs in panel C, the closely-related cucumber necrosis virus (CNV) RNAs in panel D and the mitochondria-replicating CIRV RNAs in panel E. Bottom images: ethidium-bromide stained gels show ribosomal RNA level. We chose the 12th day after VIGS to inoculate the upper, systemically-silenced leaves with TBSV virions, or agroinfiltrate with pGD-CNV or pGD-CIRV. Samples for RNA extractions were taken 1 day (TBSV) and 2.5 days (CNV or CIRV) post inoculation from the inoculated leaves. The control experiments included the TRV2-cGFP vector. Each experiment was performed three times. (F) Over-expression of the cytosolic NbPgk1 was done in <i>N</i>. <i>benthamiana</i> leaves by agroinfiltration. The same leaves, which were first agroinfiltrated with pGD-NbPgk1 (expressing NbPgk1 from the 35S promoter), were also inoculated with TBSV virions 2.5 days later. Then, total RNA samples were obtained the subsequent day (1 dpi). The control samples were obtained from leaves agroinfiltrated with pGD empty vector (not expressing proteins) (lanes 1–6). Northern blotting was used to detect the accumulation of TBSV RNAs in total RNA samples obtained from <i>N</i>. <i>benthamiana</i> leaves. The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (bottom panel). The bottom image shows a representative Western blot-based detection of His<sub>6</sub>-tagged NbPgk1 using anti-His antibody. Each experiment was performed three times.</p

    Models showing the functions of subverted cellular DEAD-box helicases in TBSV replication and viral RNA recombination.

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    <p>(A) We propose that the DDX3-like Ded1/AtRH20 helicase facilitates the release of the pausing viral RdRp (shown as p92) at the end of the template, leading to switch from (-)-strand synthesis to (+)-strand synthesis. The previously defined additional functions of Ded1p and the eIF4AIII-like AtRH2 helicase is to open the dsRNA at the promoter region (cPR plus promoter proximal replication enhancer, PPE REN, indicated by rectangles) and the RIII(-)replication enhancer, as shown schematically [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.ref049" target="_blank">49</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.ref054" target="_blank">54</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004680#ppat.1004680.ref061" target="_blank">61</a>]. Also shown is the long-range RNA-RNA interaction between the RIII(-) REN and the cPR region, which is proposed to help repeated use of the (-)RNA of the dsRNA as template for (+)RNA synthesis. The hairpin structure indicates the critical RII(+)-SL sequence required for the p33 replication protein-driven recruitment of the viral (+)RNA into replication. The RI(+) and the complementary RI(-) RNA sequences bound by Ded1p are shown with wide bars. (B) Our model suggests that Ded1/AtRH20 suppresses viral RNA recombination by facilitating the release of the pausing p92 at the end of degRNA recombinogenic template (created by cellular nucleases as shown). This activity of Ded1p results in suppression of RNA recombination (template-switching by p92), see the boxed portion of the model. Depletion of Ded1p or when Ded1p is mutated (ded1–199<sup>ts</sup>), p92 could stay on the degRNA template that likely promotes template-switching to a new (+)degRNA as shown as acceptor degRNA or to the 3’ end of the same (+)degRNA. On the other hand, in the absence of Ded1p, the eIF4AIII-like AtRH2 helicase could facilitate (-)RNA synthesis by opening up the dsRNA intermediate of degRNA that might lead to re-loading p92 RdRp to the 3’end of the (+)-strand for a new round of (-)degRNA synthesis. Similar activity by AtRH2 on degRNA might also facilitate template-switching by p92 RdRp for more efficient recombination. Altogether, these models take into account the co-ordinated actions of the two groups of subverted cellular helicases during replication and recombination.</p

    The co-opted function of cytosolic Pgk1 is to provide ATP for viral replicase complex assembly.

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    <p>(A) Northern blot analysis shows decreased TBSV (+) and (-)repRNA accumulation in a yeast strain (GALS::PGK1) when Pgk1 was depleted (raffinose) in comparison when Pgk1 expression is induced (Gal+Raf). See further details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006689#ppat.1006689.g003" target="_blank">Fig 3</a>, panel A. (B) Inefficient activities of the tombusvirus replicases assembled <i>in vitro</i> in CFEs prepared from a yeast strain (GALS::PGK1) with reduced level of Pgk1 expression. Purified recombinant p33 and p92<sup>pol</sup> replication proteins of TBSV and <i>in vitro</i> transcribed TBSV DI-72 (+)repRNA were added to the CFEs prepared from the shown yeast strains. Top panel: denaturing PAGE analysis of <i>in vitro</i> tombusvirus replicase activity in the CFEs. The <sup>32</sup>P-labeled TBSV repRNA products of the reconstituted replicases are shown. Note that the full cycle of replicase activity of the in vitro reconstituted TBSV replicase depends on Pgk1 levels in the CFEs. The CFEs contained the same amounts of total yeast proteins. (C) Reduced production of <sup>32</sup>P-labeled dsRNA intermediate, consisting of complementary (+) and (-)repRNA strands, by the in vitro reconstituted TBSV replicase when the CFEs were obtained from GALS::PGK1 yeast when Pgk1 was depleted (yeast was cultured in glucose media for 4 h) in comparison when Pgk1 expression is induced (yeast was cultured in galactose media for 4 h). Heat treatment (marked by “+”) was used to show the dsRNA nature of the shown RdRp products. Each experiment was performed three times. (D) Reduced activity of the purified tombusvirus replicase from yeast with depleted Pgk1 level. The membrane-bound replicase complex was collected by centrifugation, followed by solubilization and FLAG-affinity purification from yeasts. Representative denaturing gel of <sup>32</sup>P-labeled RNA products synthesized by the purified tombusvirus replicase <i>in vitro</i>. The <i>in vitro</i> assays were programmed with RI/III (-)repRNA, and they also contained ATP/CTP/GTP and <sup>32</sup>P-UTP. Note that the original viral template RNA in the replicase from yeast is removed during replicase solubilization/purification. (E) In efficient <i>in vitro</i> activation of the RdRp function of the N-terminally truncated p92<sup>pol</sup> replication protein. The soluble fraction of CFEs were obtained from GALS::PGK1 yeast when Pgk1 was depleted (yeast was cultured in glucose media for 4 h) in comparison when Pgk1 expression is induced (yeast was cultured in galactose media for 4 h). Denaturing PAGE analysis of the <sup>32</sup>P-labeled RNA products obtained in an <i>in vitro</i> assay with recombinant p92-Δ167N RdRp. The samples contained or lacked affinity-purified yeast Ssa1p Hsp70 protein (7 pmol). The faster migrating RNA represents a prematurely terminated product, whereas the slower migrating RNA is terminated at the 5’ end of the template. Each experiment was performed three times. (F) Co-expression of <i>S</i>. <i>castellii</i> AGO1 and DCR1 in GALS::PGK1 yeast (BY4741 background) when Pgk1 was depleted, reduces TBSV repRNA accumulation to a similar extent as in wt yeast (BY4741). Top panel: Replication of the TBSV repRNA was measured by Northern blotting 32 h after initiation of TBSV replication. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Each sample is obtained from different yeast colonies. Yeast strain not expressing RNAi components is taken as 100% in each experiment. Average value and standard deviation is calculated from all the biological repeats. Each experiment was repeated twice. Ribosomal RNA is shown as a loading control. (G) A model on the role of the co-opted glycolytic Pgk1 in tombusvirus replication. Direct interaction of the cytosolic Pgk1 with the viral p33 and p92 replication proteins leads to recruitment of Pgk1 into the membranous replication compartment, where Pgk1 generates ATP to fuel the function of the co-opted cellular Hsp70 molecular chaperone. Hsp70 (Ssa1p and Ssa2p in yeast) has been shown to facilitate the assembly of the viral replicase complex, insertion of the replication proteins into peroxisomal membranes and activation of the RdRp function of p92<sup>pol</sup>. It seems that at least a portion of co-opted Pgk1 is released from the active viral replicase complex.</p

    The DDX3-like Ded1p DEAD-box helicase is a suppressor of TBSV RNA recombination.

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    <p>(A) The previously defined viral RNA recombination pathway during TBSV replication. The replication-competent TBSV repRNA is cleaved by cellular endoribonucleases, such as the RNase MRP complex, followed by limited 5’ truncations by the cellular Xrn1p exoribonuclease. These processes lead to the generation of a pool of replication-competent degRNAs that serve as recombinogenic templates in template-switching events driven by the viral replicase. The sequences in recRNAs are shown schematically. (B) Depletion of Ded1p level in yeast leads to the rapid emergence of TBSV recRNAs and degRNAs. Note that doxycycline (+dox samples) leads to depletion of Ded1p expressed from the regulatable <i>TET</i> promoter. Replication of the TBSV DI-AU-FP repRNA (see panel A) in wt and TET::DED1 yeasts co-expressing the tombusvirus p33 and p92 replication proteins was measured by Northern blotting 24 h after initiation of TBSV replication. Note the emergence of different species of recRNAs and degRNA (see panel A) in samples with depleted Ded1p. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA (bottom panel). The bottom images show the results with semi-quantitative RT-PCR, which was used to demonstrate knock-down of Ded1 mRNA levels in TET::Ded1 yeast in the presence of doxycycline. Each sample is obtained from independent yeast colonies. The experiments were repeated two-to-three times. Throughout the paper, +/- means standard deviation. (C) Measuring recRNA levels in yeast expressing wt Ded1p, ded1–95<sup>ts</sup> or ded1–199<sup>ts</sup> mutants at 23°C (permissive temperature for yeast growth) or 29°C (semi-permissive temperature). Top panel: The accumulation of TBSV DI-AU-FP repRNA, recRNAs and degRNA was measured by Northern blotting at the 24 h time point. Middle panel: The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Bottom panel: The accumulation levels of His<sub>6</sub>-p92 and His<sub>6</sub>-p33 were tested by Western blotting. Each experiment was repeated. Asterisk marks the SDS-resistant p33 homodimer. (D) Accumulation levels of degRNA and recRNAs in Ded1<sup>ts</sup> yeasts were measured by Northern blotting. The expressed TBSV template RNA was DI-RIIΔ70 degRNA, which represents a frequently isolated degRNA species lacking RI and part of RII (Panel A). See further details in panel B.</p
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