Fig. 3

ATG9A is required for HIV-1 infectivity. a Flowchart representing the steps to measure HIV-1 release and infectivity. Briefly, RT-normalized HIV-1 NL4-3 particles, produced from WT or ATG9A-KO HeLa or Jurkat cells, were used to infect TZM-bl cells. These cells express the LUC (luciferase) reporter gene under control of the HIV-1 long terminal repeat (LTR). Reporter gene expression is proportional to the number of infectious particles present in the initial inoculum. b, c WT and ATG9A-KO HeLa (b) and Jurkat (c) cells were infected with WT or Nef-defective (ΔNef) pNL4-3 for 8 h and the medium was replaced. Two days post-infection, the culture supernatant was filtered, and viruses were collected by ultracentrifugation. Cell and virus lysates were analyzed by SDS-PAGE and immunoblotting with anti-HIV immunoglobulin (HIV-Ig) to detect viral proteins (Pr55^Gag, CAp24), and with antibodies to HIV-1 Nef, ATG9A and tubulin. The positions of molecular mass markers (in kDa) are indicated on the left. d, e Virus release was quantified by determining the amount of virion-associated CAp24 relative to the level of total CAp24 in cells and viruses. Bar graphs represent the mean ± SD from four (d) or five (e) independent experiments. Values were normalized to the release of WT HIV-1 from WT cells. Statistical significance was calculated using a one-way ANOVA test; ns: not significant (p > 0.05). f, g RT-normalized HIV-1 virus stocks were used to infect TZM-bl cells. Infection of reporter cells was measured by detection of LUC activity at 2 days post-infection. Values were normalized to the infectivity of WT HIV-1 from parental cells. Data were evaluated for statistical significance by using a one-way ANOVA test (ns: p > 0.05, *p < 0.05, ****p < 0.0001)