Fig. 4
Residues 40–57 in the N-terminal domain of NL4-3 Nef are required to counteract autophagy initiation. A, D Schematic representation of the domains replaced for the generation HIV-1 NL4-3 and SIVmac239 Nef chimeras. B, E HEK293T cells were co-transfected with EGFP-LC3B and the different nef constructs: SIVmac239 nef, NL4-3 nef and the selected nef chimeras. 48 h post-transfection, cells were analyzed by flow cytometry for autophagosome-associated EGFP-LC3B. Data correspond to the mean and SEM of the percentage of EGFP+ cells from three independent experiments. C, F HEK293T cells were transfected with NL4-3 nef, SIVmac239 nef and the selected chimeras. 48 h later, cells were exposed for 4 h to increasing concentrations of rapamycin (0–4 μM). Next, cells were analyzed by western blot for the levels of GFP, LC3, and ACTB. Densitometric analyses were performed to determine the ratio of LC3-II over LC3-I relative to SIVmac239 nef with no rapamycin treatment. All images are representative of three independent experiments. Significantly different values are indicated by asterisks *P ≤ 0.05; **P ≤ 0.01