Figure 1.

Summary of in vitro transcribed repRNA constructs encoding HIV-1 Env genes and confirmation of protein expression by western blotting

(a) Schematic of the TC-83 replicon structure showing the subgenome encoding the gene of interest (GOI). HIV-1 genes were cloned in the TC-83 subgenome, and repRNA constructs were synthesized by in vitro transcribed reactions. (b) In vitro transcribed RNA constructs were analyzed by agarose gel electrophoresis to verify molecular weight and molecule integrity. (c–e) Western blot analyses of cell lysates and supernatants collected from BHK21 cells transfected with synthesized constructs. Gels were labeled with anti-gp120 human monoclonal antibody (mAb) 2G12, anti-gp41 human mAb 4E10 or anti-HIV-1 rabbit polyclonal against Gag polyprotein. pos., recombinant BG505 SOSIP.664 used as a positive control; neg., repRNA-encoding RFP as a negative control; A + B, co-transfection with constructs A and B. All samples were run on the same RNA (b) or protein (c–e) gel. Where applicable, black dividing lines are shown to exclude an irrelevant sample run in between constructs C and D.