Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites
(A) The split ubiquitin assay was used to test binding between p33 and six Arabidopsis VAP proteins in scs2Δ yeast. The bait p33 was co-expressed with the shown prey proteins. SCS2 and the empty prey vector (NubG) were used as positive and negative controls, respectively. The left panel shows p33: VAP interactions, the right panel demonstrates that comparable amounts of yeasts were used for these experiments. (B) Expression of Arabidopsis VAP proteins can complement the defect in TBSV repRNA accumulation in scs2Δ yeast. Northern blot analysis of DI-72(+) repRNA accumulation in scs2Δ yeast expressing Arabidopsis VAP proteins from the native SCS2 promoter. Note that pYC expresses a short peptide and used as a negative control. (C) Stimulation of tombusvirus RNA accumulation in plants by expression of two Arabidopsis VAP proteins. Expression of the Arabidopsis VAP proteins was done in N. benthamiana leaves, which were co-infiltrated with Agrobacterium carrying a plasmid to launch CNV replication from the 35S promoter. The control samples were obtained from leaves expressing no VAP proteins (35S, lanes 1–2 and 7–8). Total RNA was extracted from leaves 3 days after agroinfiltration. The accumulation of CNV RNAs in N. benthamiana leaves was analyzed by Northern blot. The ribosomal RNA (rRNA), visualized by ethidium-bromide staining, was used as a loading control.