Fig. 4

ATG9A-Nef interaction is not required for HIV-1 infectivity. a Schematic representation of HIV-1 NL4-3 Nef as described in Fig. 1a. Red arrowheads indicate residues that were mutated to alanine. Mut1 represents mutation of WL-EEEE to AA-AAAA and Mut2 mutation of EEEE-PxxP to AAAA-AxxA. b HeLa cells were transfected with plasmids encoding WT HIV-1 Nef-FTS, Nef-FTS mutants (G2A, Mut1, or Mut2) or Myrlysin-FTS, and cell extracts were incubated with Strep-Tactin beads. The isolated proteins were subjected to SDS-PAGE and immunoblotting with antibodies to FLAG and ATG9A. The positions of molecular mass markers (in kDa) are indicated on the left. The amount of ATG9A in the isolated samples was quantified relative to the amount of ATG9A in the input. Values were normalized to the ATG9A PD/input ratio in the WT condition. Bar graphs represent the mean ± SD from three independent experiments. Statistical significance was analyzed using a one-way ANOVA test (****p < 0.0001). c RT normalized WT and Nef-mutant NL4-3 viruses produced in HEK-293T cells were used to infect Jurkat cells for 8 h, and the medium was replaced. At 48 h post-infection, cell and virus fractions were harvested and analyzed by immunoblotting with anti-HIV immunoglobulin (HIV-Ig), and antibodies to Nef and tubulin. In b and c, the positions of molecular mass markers (in kDa) are indicated on the left. d Virus release was quantified by determining the amount of virion-associated CAp24 relative to the total amount of CAp24 in cells and virions. Bar graphs represent the mean ± SD from four independent experiments. Statistical significance was analyzed using a one-way ANOVA test; ns: not significant (p > 0.05). e RT-normalized HIV-1 particles were used to infect TZM-bl cells. Infection was determined by detection of LUC activity 2 days post-infection. Data were evaluated for statistical significance using a one-way ANOVA; ns: not significant (p > 0.05), *p < 0.01