Fig. 15

Cell lines expressing gD also restrict HIV-1. 293 and TZM-bl cell lines were transduced with lentivirus vectors expressing HSV-1 gD as described in the Materials and Methods section. Transduced cells were selected using puromycin and gD expressing cell lines were stained for gD and sorted. The number of cells expressing gD was greater than 98%. Cells lines in 6 well plates were inoculated with HIV-1 (NL4-3, complete genome) pseudotyped with the VSV G-protein at an M.O.I of 0.5. At 48 h post-inoculation, the medium was collected and the levels of p24 determined with commercially available p24 ELISA kits and infectious virus released determined using TZM-bl assays. a–d Parental 293 and TZM-bl cell lines and those expressing HSV-1 gD (293-gD, TZM-bl-gD) were fixed and immunostained for gD and examined by confocal microscopy. e–h Parental 293 and TZM-bl cell lines and those expressing EGFP (293-EGFP, TZM-bl-EGFP) were examined for the expression of EGFP by confocal microscopy using a 488 nm filter. i The levels of the p24 released from cells inoculated with VSV-G pseudotyped HIV-1. j The levels of infectious virus released from cells inoculated with VSV-G pseudotyped HIV-1. The experiments were performed at least three times and statistical differences with the 293 or TZM-bl/HIV-1 controls were evaluated using a two-tailed Student’s t-test, with p < 0.01 (filled triangle) considered significant. The numbers above the bars represent the percentage of p24 or infectious virus compared to the control