The GEF1 Proton-Chloride Exchanger Affects Tombusvirus Replication via Regulation of Copper Metabolism in Yeast
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Fig 1Deletion of the GEF1 ClC gene inhibits TBSV repRNA accumulation in yeast. (A) Northern blot analysis of TBSV repRNA using a 3′-end-specific probe shows the reduced accumulation of repRNA in gef1Δ yeast. The viral proteins His6-p33 (six-His-tagged p33) and His6-p92 were expressed from plasmids from the copper-inducible CUP1 promoter, while DI-72 (+)repRNA was expressed from the galactose-inducible GAL1 promoter. We started TBSV replication by growing yeast cells in media containing 2% galactose and 50 μM CuSO4 at 23°C for 48 h. Northern blotting with a 18S rRNA-specific probe was used as a loading control. The mean values of the two lanes are shown below the two lanes in the blot on a gray shaded background. (B) Western blot analysis of the levels of His6-p33 and His6-p92 with anti-His antibody in WT and gef1Δ yeasts (see panel A). The bottom panel shows a Coomassie brilliant blue-stained SDS-PAGE gel as a loading control. (C) Same as panel A, except the yeast cells were grown in media containing 2% galactose and 2 μM CuSO4 at 23°C for 40 h. (D) Northern blot analysis of TBSV repRNA in gef1Δ yeast. Viral His6-p92 was expressed from a plasmid using the constitutive ADH1 promoter, while His6-p33 and DI-72 (+)repRNA were expressed from the galactose-inducible GAL1 and GAL10 promoters. We started TBSV replication by growing yeast cells in media containing 2% galactose at 23°C for 40 h.
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Fig 2Anion channel blocker SITS inhibits TBSV RNA accumulation in N. benthamiana protoplasts. (A) Northern blot analysis of TBSV genomic RNA (gRNA) accumulation in SITS (4 mM)-treated or untreated (control) N. benthamiana protoplasts. The rRNA level is shown as the loading control. (B) Same as panel B with the exception that related TCV gRNA was electroporated into N. benthamiana protoplasts.
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Fig 3SITS anion channel blocker also inhibits TBSV RNA accumulation in whole N. benthamiana plants. (A) Symptom development in N. benthamiana plants infected with TBSV. Leaves of N. benthamiana plants were infiltrated with 7 mM SITS or water, and then the same leaves were inoculated with the TBSV virion preparation. Pictures were taken at the indicated time points after TBSV inoculation. Note that the application of SITS at 7 mM concentration caused mild curliness of the edge of the infiltrated leaves after 4 or 5 days (middle panel [6dpi]). (B) Northern blot analysis of TBSV gRNA accumulation in inoculated leaves treated with SITS at the indicated concentration at 3 days postinoculation (dpi). The bottom panel shows an ethidium bromide-stained agarose gel showing rRNA levels. (C) Same as panel B with the exception that the samples were taken from systemically infected leaves at 6 dpi. (D) SITS treatment of N. benthamiana leaves causes mild curliness of the edge of the infiltrated leaves. The picture was taken 4 days after SITS treatment (applied at 7 mM concentration).
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Fig 4Reduced activity of the affinity-purified TBSV replicase derived from gef1Δ yeast. (A) TBSV replicase was FLAG affinity purified via His6-Flag-tagged p33 viral replication protein from WT and gef1Δ yeast strains expressing p92pol, His6-Flag-tagged p33 expressed from the CUP1 promoter and DI-72 repRNA from the GAL1 promoter for 24 h before purification. Then DI-72 (−)RNA was used to program the purified replicase to produce 32P-labeled cRNA product. In vitro replication was conducted for 4 h. The RNA products were analyzed by denaturing PAGE. (B) Same as panel A, except a short template, called RI/III (−)RNA (which contains the cPR promoter for plus-strand synthesis and the RIII replication enhancer region). (C) Western blot analysis to estimate the amount of His6-Flag-tagged p33 and the copurified His6-p92pol in the FLAG-affinity purified TBSV replicase using anti-His antibody.
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Fig 5Supernatant fraction of the CFE derived from gef1Δ yeast inhibits TBSV repRNA replication in vitro. (A) Scheme of the experiments. The supernatant (soluble [SOL] fraction) and the membrane (ME) fraction of CFE were separated by high-speed centrifugation (35,000 × g), and then the various fractions were mixed as indicated. Purified recombinant MBP-p33, MBP-p92pol, and T7 transcripts of TBSV DI-72 (+)repRNA were added to yeast CFE together with isotope-labeled and unlabeled nucleotides to initiate viral replication. (B) The 32P-labeled repRNA products were analyzed by denaturing PAGE. Note that lanes 11 to 14 show repRNA replication in the original CFEs (i.e., the ME and SOL fractions were not separated). (C) Scheme of the tombusvirus replicase assembly assay. Fractionation was done as in panel A. Step 1 includes VRC assembly, while RNA synthesis by the assembled VRC takes place in step 2 in the presence of 32P-labeled and unlabeled ribonucleotides. (D) The 32P-labeled repRNA products were analyzed by denaturing PAGE.
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Fig 6Iron starvation does not influence TBSV repRNA accumulation in gef1Δ or WT yeast strains. (A) Northern blot analysis shows the accumulation of TBSV repRNA in WT yeast without iron chelator (BPS) or with iron chelator (7.5 μM). See further details in the legend to Fig. 1. (B) Positive control for the BPS treatment. The assay is based on the lack of yeast growth under iron depletion in gef1Δ yeast strain. The growth of the yeast cultures shown at 23°C was measured at OD600 at the following time points: 12, 24, 40, and 48 h.
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Fig 7Deletion of CCC2 ATPase, an intracellular copper pump, hinders TBSV repRNA accumulation in yeast. (A) Northern blot analysis was used to detect the accumulation of repRNA in WT and ccc2Δ yeast strains. See further details in the legend to Fig. 1. (B) Western blot analysis of the expression level of His6-p92pol and His6-p33 in WT and ccc2Δ yeast strains.
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Fig 8Inhibition of in vitro replication of TBSV RNA in CFE by CuSO4. (A) Scheme of the experiments shown in panels B and C. Purified recombinant MBP-p33, MBP-p92pol, and T7 transcripts of TBSV DI-72 (+)repRNA were added to yeast CFE together with 32P-labeled and unlabeled ribonucleotides to initiate viral replication. The CFEs were prepared from untransformed WT or gef1Δ yeast strains. (B and C) The 32P-labeled repRNA products were analyzed by denaturing PAGE. The amounts of CuSO4 (B) or FeSO4 (C) added at the beginning of the assay are indicated above the lanes. Each experiment was performed 2 or 3 times.
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Fig 9Inhibition of in vitro VRC assembly by CuSO4. (A) Scheme of the in vitro CFE-based tombusvirus replicase assembly assay. Note that CuSO4 was added either during step 1 or step 2 as indicated. See further details in the legend to Fig. 5C. (B) The 32P-labeled repRNA products from the replication assay described in the legend to Fig. 5C were analyzed by denaturing PAGE.
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Fig 10Glutathione stimulates the in vitro replication of TBSV RNA in CFEs. (A) Scheme of the experiments. Glutathione (GSH) (37 μM) and 5 min later, purified recombinant MBP-p33, MBP-p92pol, and T7 transcripts of TBSV DI-72 (+)repRNA were added to yeast CFEs together with 32P-labeled and unlabeled ribonucleotides to initiate viral replication. The CFEs were prepared from untransformed WT or gef1Δ yeast strains. (B) The 32P-labeled repRNA products were analyzed by denaturing PAGE. The experiments were performed 2 times.
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Fig 11Inhibition of in vitro activities of the purified tombusvirus replicase and the TCV RdRp by CuSO4. (A) Denaturing PAGE shows the 32P-labeled cRNA product from the tombusvirus replicase assay performed in the presence of 10 mM MgSO4 plus an increasing amount of CuSO4 as shown. The recombinant tombusvirus replicase expressed in yeast was solubilized and affinity purified, rendering the preparation template dependent. (B) Denaturing PAGE shows the 32P-labeled cRNA product from the TCV p88C RdRp assay performed in the presence of 10 mM MgSO4 plus an increasing amount of CuSO4 as shown. The MBP-tagged p88C was purified from E. coli. The template was the 3′ end (SL1/SL2/SL3) of the TBSV DI-72 (+)repRNA, which contains the promoter region and the replication silencer element. The RdRp activity in the absence of CuSO4 was set at 100% (lanes 1 and 2). Each experiment was performed 2 or 3 times.
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Fig 12Model on the role of Gef1p in TBSV replication in yeast. (A) In WT yeast cells, the Gef1p proton-chloride exchanger is inactive, while H+ and Cu2+ ions are transported into the lumen of post-Golgi vesicles, prevesicles, and late endosomes. At a certain membrane potential, the Gef1p proton-chloride exchanger becomes activated and transports Cl− ions to these compartments, which in turn allows further Cu2+ and H+ uptake into the lumen of these organelles. During this state, the Cu2+ concentration in the cytosol is kept at a low level that allows robust tombusvirus replicase (VRC) assembly and RNA replication. (B) Yeast with gef1Δ background or with blocked Gef1p function is not able to transfer Cl− ions into the organelles, thus blocking the additional uptake of H+ and Cu2+ ions into the lumen of these organelles. Ultimately, this leads to increased Cu2+ accumulation in the cytosol, which in turn impairs the assembly of the TBSV VRC and inhibits viral RNA synthesis. Note that gef1Δ or ccc2Δ yeast strains will likely experience higher exposure to cytosolic copper ions, even if these Cu2+ions are not free (bound to glutathione and small metal-binding proteins).
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