1. Partial recovery of TBSV RNA replication in yeast overexpressing selected subverted proviral host proteins.
Partial recovery of TBSV RNA replication in yeast overexpressing selected subverted proviral host proteins. (A) For the top panel, Northern blot analysis to detect DI-72(+) repRNA accumulation in yeast coexpressing Cdc34p and the WW-domain protein was performed. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA. To launch TBSV repRNA replication, we expressed Flag-p33 and His6-p92 from the copper-inducible CUP1 promoter and DI-72(+) repRNA from the galactose-inducible GAL1 promoter. While Cdc34p was expressed from GAL1 promoter from the chromosome, the WW-domain protein (the WW domain of Rsp5p containing three WW repeats) was expressed from a plasmid based on the GAL1 promoter. For the bottom panel, Northern blot analysis of CDC34 mRNA level before launching TBSV repRNA replication was performed. (B) For the top panel, Northern blot analysis was used to detect DI-72(+) repRNA accumulation in yeast coexpressing the shown host factor (or YFP as a control) and the WW-domain protein. The His6-WW-domain protein was expressed from GAL1 promoter from a plasmid, while the shown host susceptibility factor (also His6 tagged) was expressed from the constitutive ADH1 promoter from the same plasmid as the WW-domain protein. See further details in panel A. For the middle and bottom panels, Western blot analysis of total protein extracts with anti-His or anti-FLAG antibodies was performed.
Daniel Barajas et al. J. Virol. 2015;89: