Assembly-hub function of ER-localized SNARE proteins in biogenesis of tombusvirus replication compartment
(A) Northern blot analysis of TBSV repRNA using a 3’ end specific probe shows the reduced accumulation of repRNA in GALS::UFE1 or GAL1::USE1 yeast strains grown in glucose media. 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 expression of Ufe1p or Use1p was repressed by glucose (lanes 3–8) or induced by galactose (lanes 13–18). Northern blot with 18S ribosomal RNA specific probe was used as a loading control. Bottom images: Western blot analysis of the level of His6-p33 with anti-His antibody and Ufe1p or Use1p with anti-HA antibody. (B) Reduced TBSV RNA production by the tombusvirus replicase assembled in vitro in cell-free extracts (CFEs) prepared from GALS::UFE1 or GAL1::USE1 yeast strains grown under repressive conditions (in glucose media). 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. Denaturing PAGE analysis shows the 32P-labeled TBSV repRNA products, including the (+)repRNA progeny and the dsRNA intermediate, made by the reconstituted TBSV replicase. Each experiment was repeated. (C) Top images: Confocal laser microscopy analysis of subcellular distribution of CFP-tagged p33 in GALS::UFE1 yeast cells grown under repressive conditions (in glucose media). Note that the co-localization of CFP-p33 with Pho86-RFP (an ER membrane marker protein) demonstrating localization of p33 replication protein in subdomains of the ER membrane. Bottom images: Confocal laser microscopy analysis of subcellular distribution of CFP-tagged p33 in GALS::UFE1 yeast cells grown in galactose media (i.e., inducing condition).