Effect of BST-2 on LCMV Z-mediated VLP production and vRNP activity.
A. Effect of BST-2 expression on Z-mediated VLP production. 293T cells were transfected with pC-LCMV-Z-FLAG (0.2 μg) together with increasing amounts of pTeth-FL (BST-2). At 48 hrs post transfection, VLPs were recovered from TCS by ultracentrifugation and cell lysates prepared. Levels of Z protein present in VLPs and cell lysates, and BST-2 levels in cell lysates, were detected by WB using an Ab to FLAG. Normalized levels of VLP production were determined by assigning the value of 1 to the ratio of VLP/cell Z protein expression levels in cells transfected with pC-LCMV-Z-FLAG in the absence of BST-2 (n = 3; 2 independent experiments). B. Effect of BST-2 expression on LCMV vRNP activity. 293T cells were transfected with plasmids expressing the components of the LCMV MG-CAT, together with control BST-2 or GFP expression plasmids using the indicated plasmid DNA amounts. CAT expression was measured at 48 hrs post transfection. CAT expression levels were normalized by setting to 1 levels expressed by cells transfected with control plasmid. C, D. Effect of BST-2 over-expression on pol-II mediated expression. 293T cells were transfected with psiCHECK2 together with control plasmid (pcDNFL) or increasing amounts of BST-2 expression plasmid. At 48 hrs post transfection, cell lysates were prepared for detection of both Firefly luciferase (FL) (C) and Renilla luciferase (RL) (D) using a dual luciferase detection system. Expression levels of FL and RL were normalized by setting to 1 the expression levels obtained in cells transfected with FL or RL and the control plasmid. E. Effect of BST-2 expression on 293T cell viability. Cells were transfected with increasing amount of BST-2 expression plasmid, and at 48 hrs post transfection, cell viability was determined by Cell-Titer Glo (Material and Methods). Values were normalized by setting to 1 results from cells transfected with control plasmid (pcDNFL). Data correspond to mean + SD (three independent experiments). Asterisks denote statistical significance (*P < 0.05).
