Functional analysis of interaction between primary HIV-1 nef alleles and host proteins

The HIV-1 accessory protein Nef plays a pivotal role in viral pathogenesis due to its multifunctional character. Nef facilitates immune evasion by promoting the down-modulation of cell surface molecules, including major histocompatibility complex class I (MHC-I), CD4 and a member of the serine incorporator family, SERINC5. Furthermore, the human peroxisomal thioesterase 8 (ACOT8) has been shown to be an important cellular partner of Nef. However, these mechanisms has not yet been fully understood. This study investigated the impact of primary nef alleles on the modulation of MHC-I, CD4 and SERINC5 and their capability to interact with ACOT8. 26 nef alleles were isolated from ART-naïve HIV-1-positive individuals, corresponding to subtypes B, F1, G, C, and CRF02_AG within the M group, and then cloned into a pIRES-eGFP vector for functional analysis. Receptor modulation of MHC-I and CD4 at an endogenous level was evaluated in HEK293T and/or Jurkat T cells using flow cytometry. Additionally, Nef's capability to modulate SERINC5 surface expression was examined by transfecting HEK293T cells to express nef alleles together with SERINC5. The interaction between Nef and ACOT8 was assessed through a co-immunoprecipitation approach in an ACOT8-mScarlet overexpression context. Lastly, Nef polymorphisms potentially associated with these activities were identified and correlated with their respective functions. This study showed that the majority of primary nef alleles led to a significant downregulation of MHC-I, CD4, and SERINC5 from the cell surface, that correlated with a high conservation of the known functional domains. Moreover, it demonstrated for the first time an interaction between primary nef alleles and ACOT8, revealing that this interaction was highly variable among viral subtypes and might depend on amino acid residues within the central core domain of Nef. Overall, the obtained data contributed to enhancing the understanding of Nef's biological functions and identified new mechanisms potentially involved in HIV pathogenesis.