Functional interplay between ACOT8 and Nef enhances HIV-1 infectivity and remodels the viral envelope

Background: ACOT8 is an acyl-CoA thioesterase involved in lipid metabolism, and it has been implicated in the HIV-1 replication cycle through interactions with the viral protein Nef. Here, we aimed to study the nature of this interaction by investigating the lipid composition of the virus envelope in the ACOT8-deficient condition and how this affects infectivity. Materials and methods: To assess the role of ACOT8 in HIV-1 infectivity, we generated ACOT8 knockout (KO) HEK293T cells using CRISPR/Cas9. Editing was confirmed by Sanger sequencing and Western blot. HIV-1 pseudotyped viral particles (PVPs) were produced in wild-type (WT) and ACOT8 KO cells, as well as in the presence of Nef variants that either have the capacity or do not interact with ACOT8. Infectivity was evaluated in TZM-bl cells using equivalent p24 input. HIV-1 envelope lipids were extracted with methanol/MTBE and analyzed by uHPLC/MS. Results: PVPs generated in ACOT8 KO cells showed a significant reduction in infectivity compared to WT-derived particles. This effect was envelop-specific, as VSV-G PVPs were unaffected by the absence of ACOT8. Re-expression of ACOT8 fully restores HIV-1 PVPs infectivity. Notably, enhanced infectivity was observed in PVPs produced in the presence of a Nef variant that interacts with ACOT8. In addition, Nef–ACOT8 interaction significantly altered HIV-1 lipid composition, increasing phospholipids (PC, PE) and glycosphingolipids (HexCer), consistent with substantial lipid remodeling. Conclusions: These findings identify ACOT8 as a host factor required for optimal HIV-1 infectivity and further indicate that the Nef-ACOT8 interaction enhances the production of highly infectious viral particles. Lipidomic analysis suggests that the Nef–ACOT8 interaction reshapes the HIV-1 envelope by enriching lipid molecules associated with membrane fluidity and lipid raft organization, potentially promoting a more fusion-competent membrane and thereby increasing viral infectivity