OBJECTIVES: Certain human leucocyte antigen (HLA)-B alleles (protective alleles) associate with durable immune control of HIV-1, but with substantial heterogeneity in the level of control. It remains elusive whether viral factors including Nef-mediated immune evasion function diminish protective allele effect on viral control. DESIGN: The naturally occurring non-Ser variant at position 9 of HIV-1 subtype C Nef has recently exhibited an association with enhanced HLA-B downregulation function and decreased susceptibility to recognition by CD8 T cells. We therefore hypothesized this Nef genotype leads to diminished immune control mediated by protective HLA alleles. METHODS: Nef sequences were isolated from HIV-1 subtype C-infected patients harboring protective alleles and several Nef functions including downregulation of HLA-A, HLA-B, CD4, and SERINC5 were examined. Association between Nef non-Ser9 and plasma viral load was examined in two independent South African and Botswanan treatment-naïve cohorts. RESULTS: Nef clones isolated from protective allele individuals encoding Nef non-Ser9 variant exhibited greater ability to downregulate HLA-B when compared with the Ser9 variant, while other Nef functions including HLA-A, CD4, and SERINC5 downregulation activity were unaltered. By analyzing a cohort of South African participants chronically infected with subtype C HIV-1, Nef non-Ser9 associated with higher plasma viral load in patients harboring protective alleles. Corroboratively, the Nef non-Ser9 correlated with higher plasma viral load in an independent cohort in Botswana. CONCLUSION: Taken together, our study identifies the Nef genotype, non-Ser9 that subverts host immune control in HIV-1 subtype C infection.