Fig. 6

Nef uses residues 48–49 to prevent Gag redistribution to autophagosomes, restoring Gag and virion levels. A HEK293T cells stably expressing EGFP-LC3 were co-transfected with NL4-3 Δnef and either HA-GST (irrelevant protein), NL4-3 nef, or 48–49 NL4-3 nef. 48 h later, cells were stimulated with rapamycin (4 μM) for 4 h and autophagosome formation was examined by fluorescence microscopy. B The Pearson’s correlation coefficient for the Gag-LC3 co-localization was analyzed from three biological replicates. Scale bar: 10 μm. Images are representative of three independent experiments. C HEK293T were co-transfected with NL4-3 Δnef and either an empty vector, NL4-3 nef, or 48–49 NL4-3 nef. 24 h later, cells were treated with DMSO or rapamycin (4 μM) for 12 h. The percentage of maximal virus release was determined by p24 ELISA of the culture supernatants. D Lysates were also analyzed by western blot for Gag, HA and ACTB. Significantly different values are indicated by asterisks *P ≤ 0.05