Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation
Fig 1
Pdc1 fermentation enzyme is an essential host factor for tombusvirus replication in yeast.
(A) Depletion of pyruvate decarboxylase (Pdc1p) in combination with deletion of the homologous PDC5 inhibits TBSV replicon (rep)RNA replication in yeast. Top panels: northern blot analyses of TBSV repRNA using a 3’ end specific probe demonstrates reduced accumulation of repRNA in GAL::PDC1 pdc5Δ yeast strain with depleted Pdc1p (raffinose-containing media) in comparison with the WT yeast strain or GAL::PDC1 pdc5Δ yeast strain with induced Pdc1p (galactose-containing media). Viral proteins His6-p33 and His6-p92 of TBSV were expressed from plasmids from the copper-inducible CUP1 promoter, while DI-72(+) repRNA was expressed from the constitutive TET1 promoter. Second panel: northern blot with an 18S ribosomal RNA specific probe was used as a loading control. Bottom images: western blot analysis of the level of His6-tagged p33 protein with anti-His antibody. Coomassie blue-stained SDS-PAGE was used for protein loading control. The down-regulation of Pdc1 mRNA was confirmed with RT-PCR. Each experiment was repeated three times. (B-D) Expression of Pdc1p from a plasmid increases tombusvirus replication in pdc1Δ yeast strain. His6-p33 and His6-p92 were expressed from the GAL1 promoter, whereas (+)repRNA was expressed from the GAL10 promoter. For panel C, Flag-p36 and Flag-p95 were expressed from the CUP1 promoter, whereas the repRNA from the GAL10 promoter. The untagged or His6-tagged Pdc1 were expressed from the TET promoter in all these experiments. See further details in panel A above.