Involvement of HLA-C binding stability to β2-microglobulin in HIV-1 infection

MHC-I is a heterotrimeric complex composed by HLA/β2-microglobulin/peptide. HLA-C binding to β2-microglobulin is weaker compared to HLA-A and -B. The HLA-C gene encodes different allotypes classified in stable and unstable clusters based on their binding stability to β2-microglobulin. Unstable HLA-C molecules release more β2-microglobulin than stable variants. The ratio of complete heterotrimers and HLA-C free chains at the cell surface is involved in controlling HIV-1 infection. We recently reported that HIV-1 Env associates with HLA-C during viral budding, increasing viral infectivity and spreading. We aimed to address how the binding stability of different HLA-C allotypes to β2-microglobulin influences HIV-1 infectivity. We showed that HIV-1 virions produced in PBMCs with unstable HLA-C variants are more infectious then those produced in PBMCs with stable ones. To deeply investigate the role of each HLA-C variant in HIV-1 infection modulation, 293T HLA-C-/- packaging cells were developed using CRISPR/Cas9. The different HLA-C alleles were then restored, developing cell lines expressing specific HLA-C allotypes. To analyse the binding stability to β2-microglobulin of each HLA-C variant, an acid wash treatment will be performed to assign a binding stability score ranking. Moreover, to investigate how HLA-C variants modulate HIV-1 infectivity, the homozygous HLA-C cells will be employed to produce HIV-1 pseudotyped viruses to evaluate their infectivity through a luciferase infectivity assay. This study will allow us to stratify the HLA-C alleles according to their binding stability to β2-microglobulin. Furthermore, these analyses will be fundamental to clarify the relationship among HLA-C allotypes binding stability and HIV-1 infection progression.