In cell genetic complementation assay/virus production, and target cell transduction

The gRNA packaging and propagation efficiencies of mutants were investigated using a previously established in cell genetic complementation assay (Supplemental Fig. S1; Browning et al. 2001). This assay uses three expression plasmids: MB22 (FIV Gag/Pol expression plasmid) (Browning et al. 2001), MD.G (vesicular stomatitis virus glycoprotein [VSV-G] expression plasmid) (Naldini et al. 1996), and FIV subgenomic transfer vector TR394 (Browning et al. 2001). TR394 contains the cis-acting sequences vital for RNA packaging, reverse transcription, and integration, and also expresses the hygromycin B phosphotransferase gene from the simian virus 40 (SV40) promoter as a marker to assess successful transduction (transfer or propagation of the packaged RNA) into target cells. A fourth plasmid DNA (pSEAP2-control vector, Clontech) expressing the secreted alkaline phosphatase (SEAP) gene was used to determine the transfection efficiency. The four plasmids were cotransfected into the virus-producing human embryonic kidney cells (HEK293T) using a commercially available calcium phosphate transfection kit (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. The pseudotyped virus particles thus generated were purified as previously described (Browning et al. 2001), and the viral RNA was extracted using TRIzol (Thermo Fisher Scientific), as per the manufacturer's instructions. A portion of the virus stock was used to infect HeLaT4 target cells in the presence of 1 µg/mL diethylaminoethyl (DEAE)-dextran, a polycation polymer to increase virus uptake by the cells. Selection of target cells and scoring of Hyg^R colonies were performed as described previously (Browning et al. 2001). The viral titers were normalized to the transfection efficiency of each transfected culture assessed by the relative SEAP values observed. The number of Hyg^R colonies obtained should be directly proportional to the amount of RNA packaged into the virus particles, providing an indirect estimate of RPE.