Fig. 1. Establishing transfection conditions for comparing gRNA- and cellular RNA–mediated Gag multimerization by fluorescence imaging.

COS7 cells were cotransfected with pNL4-3ΔPolΔEnv-Gag-mEos3.1 or pCR3.1-Gag-mEos3.1 in a 1:10 ratio with the respective untagged construct. A total of 2 μg of pNL4-3–based constructs and 0.4 μg of pCR3.1-based constructs were transfected to confer similar Gag expression levels in the cell and supernatant between the two transfections (see fig. S2). (A and B) Assessment of Gag expression levels. Western blot was performed with HIV–immunoglobulin (Ig) to detect Gag (55 kDa) and Gag-mEos3.1 (81 kDa) in the (A) cell and (B) supernatant at ~18 hours after transfection of the pNL4-3–based or pCR3.1-based constructs. Gag expression (normalized to the level of Gag yield by pNL4-3–based constructs) and the ratio of Gag and Gag-mEos3.1 in the cell and supernatant were calculated as described in Materials and Methods. Data represent means ± SEM of four experiments. Note that under our transfection conditions, Gag expression levels in the cell and supernatant were similar between the two expression systems, with the ratio of Gag and Gag-mEos3.1 in the cell and supernatant corresponding well to the 1:10 cotransfection ratio in both cases. (C) Assessment of Gag release efficiency (normalized to the level of Gag release yield by pNL4-3–based constructs). Data represent means ± SEM of four experiments.