Fig 1.
Interaction between p33 and p92pol replication proteins and yeast cytosolic Pgk1.
(A) Co-purification of Pgk1 with the viral replicase. Top panel: Western blot analysis of co-purified His6-tagged Pgk1 with FLAG-affinity purified FLAG-p33 and FLAG-p92pol from membrane fraction of yeast. Pgk1 was detected with anti-His antibody. The negative control was His6-tagged p33 and His6-p92 purified from yeast extracts using a FLAG-affinity column. Middle panel: Western blot of purified FLAG-p33 and FLAG-p92pol detected with anti-FLAG antibody. Asterisk marks the SDS-resistant p33 homodimer. Bottom panel: Western blot of His6-tagged Pgk1 and His6-p33 (lane 1) proteins in the total yeast extracts using anti-His antibody. (B) Testing the presence of Pgk1 in the membrane-bound viral replicase after blocking cellular translation by cycloheximide. Top panel: Western blot analysis shows the co-purified His6-tagged Pgk1 with the viral replicase isolated from membrane fraction at the shown time points. See further details in panel A. Middle panel: Western blot analysis of the purified FLAG-p33 with anti-FLAG antibody. Bottom panel: Western blot analysis of His6-Pgk1 in the total yeast lysates with anti-His antibody. Each experiment was repeated three times.
Fig 2.
Recruitment of Pgk1 into the viral replication compartment in yeast and plant cells.
(A) Confocal laser microscopy images show the partial co-localization of TBSV GFP-tagged p33 with the BFP-tagged Pgk1 protein in wt yeast cells. DIC (differential interference contrast) images are shown on the right. Bottom images show the expected cytosolic distribution of BFP-Pgk1 in the absence of viral components in a wt yeast cell. Pex13-RFP is expressed as a peroxisomal marker in experiments presented as the third and fifth panels. Scale bars represent 5 μm. (B) Confocal laser microscopy shows partial co-localization of TBSV YFP-tagged p92pol replication protein with the BFP-NbPgk1 protein in N. benthamiana cells. Expression of the above proteins together with the untagged p33 and the DI-72 RNA from the 35S promoter was achieved after co-agroinfiltration of N. benthamiana leaves. The large structures represent the viral replication compartments. Bottom image: in the control experiment, the BFP-NbPgk1 protein shows the expected cytosolic distribution in the absence of viral components in N. benthamiana cells. Scale bars represent 5 μm (second panel), 10 μm (top panel), and 20 μm (bottom two panels), respectively. (C) Top two panels: In planta interaction between of TBSV p33-cYFP replication protein and the nYFP-NbPgk1 protein. Expression of the above proteins from the 35S promoter was done after co-agroinfiltration into N. benthamiana leaves. Note that p33-cYFP and the nYFP-NbPgk1 proteins were detected by BiFC. The interaction between p33 replication protein and NbPgk1 occurs in the replication compartment decorated by RFP-SKL (peroxisomal luminar marker). Third panel: control BiFC experiments included nYFP-MBP protein in combination with p33-cYFP (bottom panel). Fourth panel: In planta interaction between TBSV p92-cYFP replication protein and the nYFP-NbPgk1 protein. Fifth panel: control BiFC experiments included nYFP-MBP protein in combination with p92-cYFP. Scale bars represent 10 μm (top three panels), and 20 μm (bottom two panels), respectively.
Fig 3.
Reduced TBSV repRNA accumulation in yeast with depleted Pgk1 level.
(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 His6-p33 and His6-p92pol from the copper-inducible CUP1 promoter, and DI-72(+) repRNA from the ADH1 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 GALS 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 CuSO4. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA levels (second panel from top). Middle panel: Northern blot analysis of PGK1 mRNA levels in total RNA samples. Bottom two panels: Western blot analysis of the accumulation level of HA-tagged Pgk1, His6-tagged p33, His6-p92pol proteins using anti-HA and anti-His antibodies, respectively. Each experiment was performed three times. (B) Complementation assay with plasmid-borne expression of His6-Pgk1 in GALS::PGK1 yeast strain. His6-Pgk1 was expressed from the TEF1 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 His6-Pgk1 was expressed from TEF1 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 His6-NbPgk1 was expressed from TEF1 promoter in BY4741 (wt) yeast. See further details in panel B.
Fig 4.
The pro-viral role of cytosolic Pgk1 in tombusvirus replication in N. benthamiana.
(A) Up-regulation of Pgk1 expression in TBSV-infected N. benthamiana 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 N. benthamiana 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 PGK1 mRNA level by VIGS inhibits the accumulation of tombusvirus RNA in N. benthamiana. Top panels: Total RNA samples obtained from N. benthamiana 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 N. benthamiana 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 N. benthamiana 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 His6-tagged NbPgk1 using anti-His antibody. Each experiment was performed three times.
Fig 5.
The cytosolic Pgk1 affects ATP accumulation within the tombusvirus replication compartment in N. benthamiana.
(A) A scheme of the FRET-based detection of cellular ATP within the replication compartment. The enhanced ATP biosensor, ATeamYEMK was fused to TBSV p33 replication protein. (B) Knock-down of PGK1 mRNA level by VIGS in N. benthamiana was done as in Fig 4. Twelve days later, co-expression of p33-ATeamYEMK and RFP-SKL (peroxisomal luminar marker) was done in upper N. benthamiana leaves by agroinfiltration. The CFP signal indicates the distribution of p33-ATeamYEMK, which co-localizes with RFP-SKL to the aggregated peroxisomes. YFP signal was generated by mVenus in p33-ATeamYEMK 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 PGK1 knock-down N. benthamiana using the mitochondrial CIRV p36 replication protein tagged with ATeamYEMK and Tim21-RFP mitochondrial marker protein. See further details in panel B.
Fig 6.
The co-opted function of cytosolic Pgk1 is to provide ATP for viral replicase complex assembly.
(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 Fig 3, panel A. (B) Inefficient activities of the tombusvirus replicases assembled in vitro in CFEs prepared from a yeast strain (GALS::PGK1) with reduced level of Pgk1 expression. Purified recombinant p33 and p92pol replication proteins of TBSV and in vitro transcribed TBSV DI-72 (+)repRNA were added to the CFEs prepared from the shown yeast strains. Top panel: denaturing PAGE analysis of in vitro tombusvirus replicase activity in the CFEs. The 32P-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 32P-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 32P-labeled RNA products synthesized by the purified tombusvirus replicase in vitro. The in vitro assays were programmed with RI/III (-)repRNA, and they also contained ATP/CTP/GTP and 32P-UTP. Note that the original viral template RNA in the replicase from yeast is removed during replicase solubilization/purification. (E) In efficient in vitro activation of the RdRp function of the N-terminally truncated p92pol 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 32P-labeled RNA products obtained in an in vitro 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 S. castellii 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 p92pol. It seems that at least a portion of co-opted Pgk1 is released from the active viral replicase complex.