103 research outputs found

    The Expanding Functions of Cellular Helicases: The Tombusvirus RNA Replication Enhancer Co-opts the Plant eIF4AIII-Like AtRH2 and the DDX5-Like AtRH5 DEAD-Box RNA Helicases to Promote Viral Asymmetric RNA Replication

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    <div><p>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 <i>cis</i>-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.</p></div

    Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus

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    <div><p>Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.</p></div

    AtRH2 and AtRH5 bind to the RIII(−) replication enhancer element in the TBSV (−)RNA.

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    <p>(A) Schematic representation of the four regions carrying <i>cis</i>-acting sequences in the genomic RNA and DI-72 repRNA used in the binding assay. Specific binding by the various cellular DEAD-box helicases are shown. (B) <i>In vitro</i> binding assay with purified AtRH2. The assay contained the <sup>32</sup>P-labeled DI-72 (−)repRNA (∼0.1 pmol) plus increasing amount of unlabeled competitor RNAs, each used in the same amounts, including RI(−) (3 and 6 pmol), RII(−) (2 and 4 pmol), RIII(−) (5 and 10 pmol) or RIV(−) (4 and 8 pmol). The free or AtRH2-bound ssRNA was separated on nondenaturing 5% acrylamide gels. (C) RNA gel shift analysis shows that AtRH5 binds the most efficiently to RIII(−). <sup>32</sup>P-labeled DI-634 (−)repRNA template (∼0.1 pmol) from FHV and unlabeled competitor RNAs (2 and 4 pmol) representing one of the four regions of TBSV DI-72 RNA from both RNA strands (see panel A) were used in the competition assay. The AtRH5 - <sup>32</sup>P-labeled ssRNA complex was visualized on nondenaturing 5% acrylamide gels. Each experiment was repeated at least three times. Note that we used the heterologous FHV DI-634 (−)RNA in the binding assay to allow comparison of (+) versus (−)RNA regions of TBSV RNA. The template competition assay showed efficient binding/competition by the RIII(−) and RI(+) sequences for AtRH5. (D) Comparable viral RNA binding assay reveals different binding specificity for DDX3-like AtRH20 and the DDX5-like AtRH5 DEAD-box helicases. See additional details in panel C. The template competition assay showed efficient binding/competition by the RI(−), RII(−), RIV(−) and RI(+) sequences for AtRH20. (E) Schematic representation of the long-range RNA-RNA interaction between the “base” sequence in the cPR promoter in RI(−) and the complementary “bridge” sequence in RIII(−) REN <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004051#ppat.1004051-Panavas3" target="_blank">[56]</a>. The competitor RNAs used in panel F are shown schematically. (F) The bridge sequence contributes to binding of RIII(−) to AtRH5 <i>in vitro</i>. Top image: RNA-binding analysis of AtRH5 – DI-72 (−)repRNA interaction after UV cross-linking. <sup>32</sup>P-labeled DI-72 (−)repRNA was used in the absence (lane 1) or presence of various cold competitor RNAs (lanes 2–7) as shown in panel E. Bottom image: SDS-PAGE shows the purified AtRH5 after UV cross-linking to demonstrate comparable sample loading.</p

    Synergistic stimulatory effect of over-expression of the eIF4IIIA-like AtRH5 and the DDX3-like AtRH20 DEAD-box helicases on tombusvirus RNA accumulation in <i>N. benthamiana</i>.

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    <p>(A) Northern blot analysis of the accumulation of CNV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves. Expression of AtRH5 and AtRH20 were done separately or together in <i>N. benthamiana</i> leaves by agroinfiltration. The same leaves were co-infiltrated with <i>Agrobacterium</i> carrying a plasmid to launch CNV replication from the 35S promoter. The control samples were obtained from leaves expressing no proteins (lanes 1–4). Total RNA was extracted from leaves 2.5 days after agroinfiltration after launching CNV replication. The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (middle panel). (B) Co-over-expression of AtRH5 and AtRH20 in <i>N. benthamiana</i> accelerates the rapid necrosis caused by systemic CNV infection. The pictures were taken 12 days after agroinfiltration.</p

    Rab5 is partly co-localized with PE-enriched tombusvirus replication compartment in plant cells.

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    <p>(A) Confocal laser microscopy images show the co-localization of GFP-AtRab5B with the TBSV p33-RFP replication protein in subcellular areas enriched with PE in <i>N</i>. <i>benthamiana</i> protoplasts. Scale bars represent 20 μm in each panel. (B) Confocal laser microscopy images confirm that these subcellular areas are derived from aggregated peroxisomes based on co-localization with either Pex13-GFP peroxisomal membrane marker protein or GFP-SKL peroxisomal luminal marker protein. (C) Control images show the lack of PE enrichment in peroxisomes in the absence of viral components. Note the absence of aggregated peroxisomes in these cells. (D) Confocal laser microscopy images show the co-localization of GFP-AtRab5B with the CIRV p36-RFP replication protein in subcellular areas enriched with PE. (E) Confocal laser microscopy images confirm that these subcellular areas are derived from aggregated mitochondria based on co-localization with Tim21-GFP marker protein. (F) Control images show the lack of PE enrichment in mitochondria in the absence of viral components. Note the absence of aggregated mitochondria in these cells.</p

    AtRH2 is a component of the tombusvirus replicase in yeast.

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    <p>(A) The membrane-bound tombusvirus replicase was purified via solubilization of the FLAG-tagged p33 and FLAG-p92 from yeast extracts using a FLAG-affinity column (lanes 1–2). Yeast not expressing p33/p92 was used as a control (lane 3). Top panel: Western blot analysis of FLAG-tagged p33 with anti-FLAG antibody. Bottom panel: Western blot analysis of His<sub>6</sub>-tagged AtRH2 with anti-His<sub>6</sub> antibody in the affinity-purified replicase preparations. Note that “soluble” represents the total protein extract from yeast demonstrating comparable levels of His<sub>6</sub>-AtRH2 in each sample (lanes 4–6). Each experiment was repeated three times. (B) Interaction between <i>Arabidopsis</i> 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.</p

    Stimulation of tombusvirus RNA accumulation by over-expression of the eIF4IIIA-like AtRH2 and the DDX5-like AtRH5 DEAD-box helicases in yeast and <i>N. benthamiana</i>.

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    <p>(A) Expression of AtRH2 and AtRH5 in yeast (BY4741) enhances TBSV repRNA accumulation. Top panel: Replication of the TBSV repRNA was measured by Northern blotting 14 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. Middle and bottom panels: The accumulation levels of His<sub>6</sub>-AtRH2 and His<sub>6</sub>-AtRH5 and FLAG-p33 were tested by Western blotting and total protein loading is shown by SDS-PAGE. Each experiment was repeated twice. (B) Expression of AtRH2 and AtRH5 were done separately or together in <i>N. benthamiana</i> leaves by agroinfiltration. The same leaves were co-infiltrated with <i>Agrobacterium</i> carrying a plasmid to launch <i>Cucumber necrosis virus</i> (CNV, a close relative of TBSV) replication from the 35S promoter. The control samples were obtained from leaves expressing no proteins (lanes 5–8). Total RNA was extracted from leaves 2.5 days after agroinfiltration that launched CNV replication. The accumulation of CNV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves was measured by Northern blotting (Top panel). The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (bottom panel).</p

    Recruitment of <i>Arabidopsis</i> Rab5 into the tombusvirus replication compartment in <i>N</i>. <i>benthamiana</i>.

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    <p>(A) Confocal laser microscopy shows partial co-localization of TBSV RFP-tagged p33 replication protein or CIRV RFP-tagged p36 with the GFP-AtRab5B protein in <i>N</i>. <i>benthamiana</i> cells. Expression of the above proteins from the 35S promoter was achieved after agro-infiltration into <i>N</i>. <i>benthamiana</i> leaves. Scale bars represent 20 μm. (B) Partial re-localization of RFP-AtRab5B protein to the peroxisomes (marked by GFP-SKL) in <i>N</i>. <i>benthamiana</i> cells infected with either TBSV or CNV. The bottom image shows the absence of re-localization of RFP-AtRab5B protein to the peroxisome in the mock-infected plant leaves. Scale bars represent 20 μm. (C) Partial re-localization of RFP-AtRab5B protein to the mitochondria (marked by mito-EGFP) in <i>N</i>. <i>benthamiana</i> cells infected with CIRV. The bottom image shows the absence of re-localization of RFP-AtRab5B protein to the mitochondria in the mock-infected plant leaves. Scale bars represent 20 μm. (D) TBSV infection induces membrane proliferation, which is occasionally visualized as aggregated circle-like structures. These membranous structures are enriched in PE in plant cells. The confocal laser microscopy image shows the enrichment of PE and its co-localization with the TBSV p33/p92 replication proteins, which were detected with p33-specific primary antibody and secondary antibody conjugated with Alexa Fluor488. Localization of PE is detected by using biotinylated duramycin peptide and streptavidin conjugated with Alexa Fluor 405. DIC images are shown on the right. Scale bars represent 20 μm. (E) Top image: <i>In planta</i> interaction between TBSV p33-cYFP replication protein and the nYFP-AtRab5B protein. Expression of the above proteins from the 35S promoter was done after agro-infiltration into <i>N</i>. <i>benthamiana</i> leaves. Note that p33-cYFP and the nYFP-AtRab5B protein were detected by bimolecular fluorescence complementation (BiFC). Control BiFC experiments included nYFP-MBP protein. Bottom images: The interaction between p33 replication protein and AtRab5B occurs in the replication compartment decorated by RFP-p33. As expected, the enlarged replication compartment (highlighted via RFP-SKL) also contained the viral dsRNA replication intermediate only in TBSV-infected cells (second panel form the bottom) but not in the mock-inoculated cells (bottom panel). Scale bars represent 20 μm. (F) The corresponding experiments with the CIRV p36 protein and AtRab5B (see panel E for details). Scale bars represent 20 μm.</p

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

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    <p>(A) Scheme of the tombusvirus replicase assay. Yeast with depleted eIF4IIIA-like Fal1p co-expressing p33 and p92<sup>pol</sup> 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 <sup>32</sup>P-UTP. (B) Representative denaturing gel of <sup>32</sup>P-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 <sup>32</sup>P-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 <sup>32</sup>P-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.</p
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