Fig 2.
JEV NS2A blocked PKR-mediated growth suppression in PKR-expressing yeast system. (A) Individual cDNAs of JEV viral proteins and HIV Tat were subcloned to pYES2 vector under the GAL1 and T7 promoters. The plasmid protein expression was checked by the T7-coupled TNT rabbit reticulocyte lysate system. Proteins were labeled with [35S]methionine, separated by SDS-PAGE, and analyzed by autoradiography. (B) Growth of yeast RY1-1 transformed with the indicated pYES2 constructs on synthetic dextrose (SD) plate containing 2% glucose. (C) RY1-1 transformants were streaked onto a synthetic galactose (SGAL) plate containing 2% galactose for PKR-mediated growth suppression assay. (D) RY1-1 transformed with pYES2 (vector), JNS2A/pYES2, or HIV Tat/pYES2 were inoculated in SD broth at 30°C overnight and then diluted to OD600 of 0.4/ml in SGAL (10% galactose) broth. Yeast growth was monitored by spectrometer (OD600) at 30°C for the indicated time periods.