Fig. 6

Env-dependence of ATG9A role in HIV-1 infectivity. a WT HIV-1 particles, produced from WT and ATG9A-KO HeLa cells, and cell lysates were analyzed by SDS-PAGE and immunoblotting with HIV-Ig to detect CAp24, and with antibodies to gp41 and ATG9A. The positions of molecular mass markers (in kDa) are indicated on the left. The incorporation of gp41 was quantified by determining the amount of virion-associated gp41 relative to CAp24. RT-normalized HIV-1 virus stocks were used to infect TZM-bl cells. Infection of reporter cells was measured by detection of LUC activity 2 days post-infection. Values were normalized to parental HeLa cells. Statistical significance from three independent experiments was determined using an unpaired Student’s t-test; ns: not significant (p > 0.05), ****p < 0.0001). b VSV-G pseudotyped HIV-1 particles were produced from WT and ATG9A-KO HeLa cells, and cell lysates were analyzed by SDS-PAGE and immunoblotting with HIV-Ig to detect CAp24, and with antibodies to VSV-G and ATG9A. The positions of molecular mass markers (in kDa) are indicated on the left. The incorporation of VSV-G was quantified by determining the amount of virion-associated VSV-G relative to CAp24. RT-normalized HIV-1 particles were used to infect TZM-bl cells. Virus infectivity was determined as in panel B. c Model for the role of ATG9A in HIV-1 infectivity. ATG9A promotes HIV-1 infectivity independently of Nef. ATG9A could promote the removal of an inhibitor or the incorporation of an activator of the infectivity of the viral particles. This effect of ATG9A on HIV-1 infectivity is dependent on HIV-1 Env, as pseudotyping with VSV-G overcomes the infectivity defect