IntroductionHIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction appears to influence the CD4 down-regulation and might modulate lipid composition of membrane proteins during HIV-1 infection. The Nef regions involved in the association with ACOT8 have been experimentally characterized. The lack of structural information for ACOT8 limits the full comprehension of the biological role of the Nef/ACOT8 association relevant to HIV-1 infection.ResultsIn this work we modelled, through in silico predictions, the ACOT8 structure. A high charge complementarity was observed between Nef and ACOT8 surfaces. This allowed the identification of the ACOT8 aminoacids most likely involved in the interaction with Nef. They map in the Arg45-Phe55 and Arg86-Pro93 ACOT8 regions. Their role has been validated by in vitro assays through the development of ACOT8 deletion mutants. Immunofluorescence and co-immunoprecipitation analyses showed that the ACOT8 K91S mutation is sufficient to abrogate the interaction with Nef. In addition, the ACOT8 Arg45-Phe55 region, as well as the Arg86-Pro93 region, are involved in Nef binding.ConclusionsOur data demonstrate that the ACOT8 Lys91 plays a key role in the interaction with Nef. The observation that both ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are determinant for Nef association suggests that the interaction involves a wider region on ACOT8 surface. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8 and will help elucidating the biological meaning of their interaction.
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