Fig. 5

Nef uses residues 48–49 to prevent autophagosome biogenesis. A Alignment comprising residues 40–57 in NL4-3 Nef. Substitutions introduced in each mutant are indicated in red. B HEK293T cells were co-transfected with EGFP-LC3B and either an empty vector, NL4-3 nef or the selected nef mutants. 48 h post-transfection, cells were analyzed by flow cytometry for autophagosome-associated EGFP-LC3B. C HEK293T cells were transfected with NL4-3 nef or the selected mutants. 48 h later, cells were treated with rapamycin (4 μM) for 4 h and analyzed by western blot for HA, LC3, and ACTB. Densitometric analyses determine the ratio of LC3-II:I relative to NL4-3 nef are shown underneath the blots. D HEK293T cells were co-transfected with BECN1 and either an empty vector, NL4-3 nef or 48–49 NL4-3 nef. 48 h later, cells were harvested and BCL2 was immunoprecipitated. The pulldown fraction was examined for BCL2 and BECN1. Lysates were also analyzed by western blot for BECN1, HA, BCL2, LC3, and ACTB. Densitometric analyses indicate the relative BCL2-BECN1 interaction. E HEK293T cells stably expressing EGFP-LC3 were transfected with an empty vector, NL4-3 nef, a Nef mutant harboring alanine substitutions at the residues responsible for blocking autophagy maturation (36–39 Nef) or 48–49 NL4-3 nef. 48 h later, cells were treated with DMSO or rapamycin (4 μM) for 4 h prior to microscopy visualization of EGFP-LC3-coated autophagosomes. Graph: number of autophagosomes per cell from 15 randomly selected cells. Scale bar: 10 μm. Images are representative of three independent experiments. Significantly different values are indicated by asterisks *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001