HIV-1 Evades a Gag Mutation that Abrogates KIR Binding and Disinhibits NK Cells in Infected Individuals with KIR2DL2⁺/HLA-C*03:04⁺ Genotype

Abstract

HIV-1 sequence variations impact binding of inhibitory killer cell immunoglobulin-like receptors (KIRs) to human leucocyte class I (HLA-I) molecules modulating NK cell function. HIV-1 strains encoding amino acids that mediate binding of inhibitory KIRs might therefore have a selective benefit in individuals expressing the respective KIR/HLA genotypes. Here we demonstrate that HIV-1 clade C avoids a p24 Gag mutation that abolishes binding of KIR2DL2 to HLA-C*03:04 and disinhibits NK cells in individual encoding for this genotype.

Natural killer (NK) cells are an important component of the innate immune system, able to rapidly respond to target cells without prior sensitization. The effector function of NK cells to stimuli are determined by the interplay between activating and inhibitory receptors [1], including killer immunoglobulin receptors (KIRs). KIRs bind to human leukocyte antigen class I (HLA-I) molecules to monitor HLA-I expression levels on cells, and the affinity of these KIR/HLA-I interactions is dependent on the sequence of the HLA-I presented peptide [2, 3]. KIR/HLA-I compound genotypes have been associated with the speed of HIV-1 disease progression [4], and selection of viral sequence polymorphisms associated with specific KIR or KIR/HLA-I compound genotypes in HIV-1 infection have been reported [5–7], indicating KIR+ NK cell-mediated immune selection pressure.

We previously showed selection of two minor HIV-1 sequence polymorphisms in a cohort of 392 clade C HIV-1 infected South Africans and demonstrated that these mutations within p24 Gag were associated with combined expression of KIR2DL3 and HLA-C*03:04 genotypes and mediated escape from NK cell responses through enhanced engagement of inhibitory KIR2DL3 [8]. Here we investigated another sequence polymorphism at position 84 of p24 Gag (T_Gag84V) associated with the combined HLA-C*03:04/KIR2DL2 genotype in this cohort. While 42 infected individuals expressing both KIR2DL2 and HLA-C*03:04 all harbored viruses encoding for the major variant threonine (T) at amino acid position 84 of HIV-1 Gag, the minor variant valine (V) at position 84 was not present in the KIR2DL2⁺/HLA-C*03:04⁺ individuals yet was present in 10% (35/350) of individuals not encoding for KIR2DL2 and HLA-C*03:04 (see section A of Supplementary Material; Fig 1A, P=0.02, Fischer’s Exact test). These data suggest a significant advantage for HIV-1 to encode for T_Gag84 in KIR2DL2⁺/HLA-C*03:04⁺ individuals. The T_Gag84V polymorphism is located within the C-terminal region of the HLA-C*03:04-restricted peptide RSLYNTVATL (RSTL10). The major variant (RSLYNTVATL (RSTL10)) and the minor variant (RSLYNTVAVL (RSVL10)) peptides equally stabilized HLA-C*03:04 expression (method described in section B of Supplementary Material) on transfected TAP-deficient 721.221 cells (221-TAPko-HLA-C*03:04) (T [mean 2.2 ± 0.20 SD] to V [mean 2.3± 0.26SD], P=0.8), and resulted in an up to 3-fold higher expression of HLA-C*03:04 compared to non-peptide-pulsed control cells (Fig 1B). These HLA-C*03:04-stabilization data demonstrated that the T_Gag84V polymorphism identified within HIV-1 p24 Gag did not impact the ability of the respective peptides to bind to HLA-C*03:04.

Figure 1.

Reduced KIR2DL2-binding and enhanced KIR2DL2⁺ NK cell activity in response to the minor V_Gag84 HIV-1 variant. A) Distribution of major T_Gag84 and minor V_Gag84 variant Gag sequences in viruses from individuals not encoding for both KIR2DL2 and HLA-C*03:04 (n=350) and KIR2DL2⁺/HLA-C*03:04⁺ individuals (n=42), P=0.02 calculated using Fischer’s Exact test. B) Equal stabilization of HLA-C*03:04 expression on 221-TAPko-HLA-C*03:04 cells with the RSLYNTVATL peptide (RSTL10) and the RSLYNTVAVL peptide (RSVL10) containing the T_Gag84V sequence polymorphism. The HLA-C*03:04-binding peptides GAVDPLLAL (GAL) and GAVDPLLKL (GKL) were used as controls. HLA-C*03:04 expression is indicated by relative fluorescence intensity normalized to no peptide control. Data represents mean of 3 experiments with error bars indicating SD. C) Binding of KIR2DL2-Fc to 221-TAPko-HLA-C*03:04 cells pulsed with the GAVDPLLAL (GAL), GAVDPLLKL (GKL), RSLYNTVATL (RSTL10) or RSLYNTVAVL (RSVL10) peptides. Results from five independent experiments are shown, lines represent median. P values were calculated using Mann Whitney test. D) Relative inhibition of degranulation (quantified by CD107a expression) of primary KIR2DL2⁺ NK cells by 221-TAPko-HLA-C*03:04 cells pulsed with the GAVDPLLAL (GAL), GAVDPLLKL (GKL), RSLYNTVATL (RSTL10) or RSLYNTVAVL (RSVL10) peptides. CD107a expression of NK cells in response to HLA-I deficient 221 cells was set at 100%, and relative inhibition of KIR2DL2⁺ NK cell in response to target cells presenting the different peptides was calculated. Primary NK cells from eight healthy KIR2DL2⁺ participants were tested, lines represent median. P values were calculated using Mann Whitney test.

Next, the RSTL10 and RSVL10 peptides were assessed for their ability to mediate KIR2DL2-binding to HLA-C*03:04, using a KIR2DL2-Fc fusion construct, as previously described [8], (see also section C of Supplementary Material). The HLA-C*03:04-presented GAVDPLLAL (GAL) peptide derived from the human importin-α−1 subunit (residues 204–212) [9], is known to bind KIR2DL2 [9] and was used as positive control, whereas the variant GAVDPLLKL (GKL) peptide which stabilizes HLA-C*03:04 at similar levels but does not bind KIR2DL2 [9] was used as a negative control. Results from 5 independent KIR2DL2-Fc-binding assays consistently demonstrated weaker binding of KIR2DL2-Fc to HLA-C*03:04 presenting the minor variant peptide RSVL10, while the major variant peptide RSTL10 mediated significantly higher binding of KIR2DL2-Fc to HLA-C*03:04 (Fig 1C, P=0.008, Mann Whitney test). Higher KIR2DL2-Fc-binding furthermore had significant functional consequences for KIR2DL2⁺ NK cells, as demonstrated by increased inhibition of CD107a expression as a read-out for NK cell activation [10], (Supplementary Figure 1), of KIR2DL2⁺ primary NK cells following co-incubation of 221-TAPko-HLA-C*03:04 cells pulsed with the major variant RSTL10 peptide compared to the minor variant RSVL10 peptide (method described in section D of Supplementary Material) (Fig 1D, P=0.007, Mann Whitney test, N = 8). In summary, the results from these KIR2DL2/HLA-C*03:04 binding assays and functional assays using KIR2DL2⁺ primary NK cells demonstrate that the minor V_Gag84 HIV-1 variant that was circulating in KIR2DL2/HLA-C*03:04 negative individuals, but not detected in individuals encoding for KIR2DL2/HLA-C*03:04, mediated significantly reduced binding of KIR2DL2 to HLA-C*03:04, resulting in stronger activation of primary KIR2DL2⁺ NK cells.

Previous studies have reported minor HIV-1 sequence polymorphisms selected in individuals expressing specific KIRs or KIR/HLA-I combinations that enhanced binding of inhibitory KIRs and inhibit KIR⁺ NK cells, consistent with KIR⁺ NK cell-mediated selection of viral escape variants [3, 6–8]. Here we report a sequence polymorphism within HIV-1 Gag (T_Gag84V) for which the major variant (T_Gag84) was encoded in all individuals expressing KIR2DL2/HLA-C*03:04, while the minor variant (V_Gag84) was detected in some individuals not encoding for this KIR/HLA compound genotype. The combined KIR2DL2/HLA-C*03:04 genotype is frequent in the studied population of HIV-1 clade C infected individuals from South Africa (42 of 392 individuals, 10.7%), and these observations are consistent with the selection and potential accumulation of the T_Gag84 variant over time, similar to what has been described for HLA-B*51- restricted epitope TAFTIPSI in different populations [11]. The current study was a cross sectional analysis in chronically HIV-1 clade C infected individuals in which the timing of HIV-1 infection was not known. It was therefore not possible to determine whether the T_Gag84 polymorphism was indeed selected in KIR2DL2⁺/HLA-C*03:04⁺ individuals infected with the minor V_Gag84 variant; and the fact that less than 10% of circulating viruses in these 392 individuals encoded for V_Gag84 suggests that most individuals were initially infected with the T_Gag84 polymorphism. Longitudinal studies with accurate timing of primary infection will be required to determine whether the T_Gag84 variant is indeed selected in KIR2DL2⁺/HLA-C*03:04⁺ individuals infected with the minor V_Gag84 variant, leading to the accumulation of the T_Gag84 variant over time. In summary, these data suggest potential accumulation of sequence polymorphisms that reduce recognition by KIR⁺ NK cells over time, eventually representing the major variant in the circulating HIV-1 strains, similar to the enrichment of HIV-1 variants selected in response to CD8⁺T-cell-mediated immune pressure [11].