Fig. 1. Addition of mRNA expressing a retroviral protease results in efficient Gag processing but inefficient extracellular VLP production.

(A) Immunoblot analysis of Gag p55 processing in stably Env-expressing HEK293T cells co-transfected with SIV gag mRNA (2 μg per reaction) and pro mRNA at the ratios indicated on the top of the image. The blot was visualized using an anti-SIV Gag antibody that recognizes both uncleaved (p55) and cleaved (p27) SIV Gag. MW, molecular weight. (B) Quantification of total Gag p27 concentration in the culture supernatant of transfected cells. The amount of total Gag p27 (left y axis) was quantified after 48 hours of transfection using an ELISA specific for cleaved p27. Cell viability (right y axis) was measured at 48 hours by flow cytometry using a live-dead dye. (C) Quantification of VLP-associated Gag p27 captured from the culture supernatants of cells transfected as in (B) collected at 48 hours post-transfection using the trimer-specific human bNAb PG16 bound to magnetic beads. The amount of Gag p27 was quantified using the same ELISA as in (B). Data in (B and C) are presented as the mean (± standard error of the mean) from two technical replicates each from two representative experiments.