Assembly-hub function of ER-localized SNARE proteins in biogenesis of tombusvirus replication compartment
(A) Northern blot analysis of TBSV repRNA shows the reduced accumulation of repRNA in wt yeast strain expressing RFP-Ufe1ΔTM. Viral proteins His6-p33 and His6-p92 were expressed from plasmids from the copper-inducible CUP1 promoter, while DI-72(+) repRNA was expressed from the ADH1 promoter. TBSV replication was induced by growing yeast cells in media supplemented with 50 μM CuSO4 at 23°C for 36 hours, whereas the co-expression of RFP-Ufe1ΔTM was induced by galactose (lanes 5–8). Northern blot with 18S ribosomal RNA specific probe was used as a loading control. Bottom image: Western blot analysis of the expression levels of His6-p33, His6-p92 and RFP-Ufe1ΔTM with anti-His antibody. (B) Northern blot analysis of TBSV repRNA shows the lack of inhibition of repRNA accumulation in wt yeast strain when RFP-Ufe1ΔTM was expressed 6 hours after launching TBSV repRNA replication. See further details in panel A. Each experiment was repeated. (C) Northern blot analysis of TBSV repRNA shows the reduced accumulation of repRNA in pex19Δ yeast strain expressing RFP-Ufe1ΔTM. See further details in panel A. Each experiment was repeated. (D) Confocal laser microscopy analysis of subcellular distribution of GFP-tagged p33 and RFP-Ufe1ΔTM in wt yeast cells. Note that the different pattern of localization of GFP-tagged p33 and RFP-Ufe1ΔTM, which is mostly cytosolic due to the deletion of the transmembrane region. (E) The split ubiquitin assay was used to test binding between the TBSV p33 replication protein and Ufe1ΔTM in yeast. The bait p33 was co-expressed with N-terminally-tagged Ufe1ΔTM protein. SSA1 (HSP70 chaperone), and the empty prey vector (NubG) were used as positive and negative controls, respectively.