Resistance of Major Histocompatibility Complex Class B (MHC-B) to Nef-Mediated Downregulation Relative to that of MHC-A Is Conserved among Primate Lentiviruses and Influences Antiviral T Cell Responses in HIV-1-Infected Individuals

Abstract

Patient-derived HIV-1 subtype B Nef clones downregulate HLA-A more efficiently than HLA-B. However, it remains unknown whether this property is common to Nef proteins across primate lentiviruses and how antiviral immune responses may be affected. We examined 263 Nef clones from diverse primate lentiviruses including different pandemic HIV-1 group M subtypes for their ability to downregulate major histocompatibility complex class A (MHC-A) and MHC-B from the cell surface. Though lentiviral Nef proteins differed markedly in their absolute MHC-A and MHC-B downregulation abilities, all lentiviral Nef lineages downregulated MHC-A, on average, 11 to 32% more efficiently than MHC-B. Nef genotype/phenotype analyses in a cohort of HIV-1 subtype C-infected patients (n = 168), together with site-directed mutagenesis, revealed Nef position 9 as a subtype-specific determinant of differential HLA-A versus HLA-B downregulation activity. Nef clones harboring nonconsensus variants at codon 9 downregulated HLA-B (though not HLA-A) significantly better than those harboring the consensus sequence at this site, resulting in reduced recognition of infected target cells by HIV-1-specific CD8⁺ effector cells in vitro Among persons expressing protective HLA class I alleles, carriage of Nef codon 9 variants was also associated with reduced ex vivo HIV-specific T cell responses. Our results demonstrate that Nef's inferior ability to downregulate MHC-B compared to that of MHC-A is conserved across primate lentiviruses and suggest that this property influences antiviral cellular immune responses.IMPORTANCE Primate lentiviruses encode the Nef protein that plays an essential role in establishing persistent infection in their respective host species. Nef interacts with the cytoplasmic region of MHC-A and MHC-B molecules and downregulates them from the infected cell surface to escape recognition by host cellular immunity. Using a panel of Nef alleles isolated from diverse primate lentiviruses including pandemic HIV-1 group M subtypes, we demonstrate that Nef proteins across all lentiviral lineages downregulate MHC-A approximately 20% more effectively than MHC-B. We further identify a naturally polymorphic site at Nef position 9 that contributes to the MHC-B downregulation function in HIV-1 subtype C and show that carriage of Nef variants with enhanced MHC-B downregulation ability is associated with reduced breadth and magnitude of MHC-B-restricted cellular immune responses in HIV-infected individuals. Our study underscores an evolutionarily conserved interaction between lentiviruses and primate immune systems that may contribute to pathogenesis.

Keywords: HLA; Nef; human immunodeficiency virus; immune evasion; lentiviruses.

FIG 1

Downregulation of MHC-A and MHC-B in primate lentiviruses. (A) Jurkat cells stably expressing A02 and A02_GGΔCKV were transfected with plasmid DNAs encoding GFP only or GFP plus the indicated lentiviral Nef clones and stained with HLA-A2 antibody. Values shown on flow cytometry plots represent Nef downregulation activities calculated as described in Materials and Methods. The greater the downregulation activity is, the lower the residual cell surface expression of A02 or A02_GGΔCKV is. (B) Phylogenetic analysis of lentivirus Nef clones. A maximum likelihood phylogenetic tree of lentivirus Nef clones used, colored according to lentiviral lineage, is shown. (C) Downregulation activities of Nef clones of various lentiviruses determined in Jurkat cells expressing A02 and A02_GGΔCKV. Each point represents the mean of triplicate determinations. Statistical analysis was performed using a Wilcoxon matched-pairs test. (D) Ratios of A02 and A02_GGΔCKV downregulation activity of these lentivirus Nef clones. Bars and whiskers denote the median and interquartile range for each group. Statistical analysis was performed using a Kruskal-Wallis test.

FIG 2

Downregulation of HLA-A and HLA-B by patient-derived Nef clones of group M HIV-1. (A) Maximum likelihood phylogenetic tree of Nef clones of patient-derived Nef clones (n = 20 each) of subtypes A, B, C, and D in group M HIV-1, colored according to subtype. Downregulation (B) and downregulation ratios (C) of A02 and A02_GGΔCKV by these Nef clones were determined. Each plot represents the mean values of triplicate assays. Statistical analysis was performed using a Wilcoxon matched-pairs test (B) or Kruskal-Wallis test (C).

FIG 3

Downregulation of HLA-A and HLA-B by patient-derived subtype C Nef clones. (A) Downregulation of A02 and A02_GGΔCKV by patient-derived Nef clones of subtype C (n = 168). Data represent A02 and A02_GGΔCKV downregulation activities (B) and their corresponding A02/A02_GGΔCKV downregulation ratios (C) when Nef sequences were stratified based on expression of Tyr-202 (consensus) (n = 55) versus that of other residues at this position (n = 113). Each plot represents the mean values of triplicate assays. Bars and whiskers represent median values and interquartile ranges. Statistical analysis was performed using a Wilcoxon matched-pairs test (A) or Mann-Whitney U test (B and C).

FIG 4

Subtype-specific effects of Nef-9 variation on HLA-A and HLA-B downregulation. Subtype C and subtype B Nef clones (n = 10 each) harboring nonconsensus amino acids at position 9 (Others) were cloned into HIV-1 pNL43, and their corresponding Ser-9 consensus reversion mutants were constructed (Ser). Absolute A02 and A02_GGΔCKV downregulation (A) and the corresponding ratios of A02 and A02_GGΔCKV downregulation (B) by these Nef clones are shown. (C) Nef band intensity by Western blotting (normalized to the control strain Nef_SF2 as 100% following subtraction of background band intensity observed in the NL43_ΔNef negative control) is shown. Solid lines connect the pairwise plots for Nef clones SK-346 and SK-367 that were used for subsequent analyses (Fig. 5 to 7). Each plot represents the mean values of triplicate assays. Statistical analysis was performed by a Wilcoxon matched-pairs test. (D) A representative Western blot is shown. Lysates of HEK-293 T cells transfected with the NL43 strain harboring the indicated subtype C Nef alleles (the control strain Nef_SF2 and the NL43_ΔNef negative control are included) were separately stained by anti-Nef and anti-β-actin antibodies.

FIG 5

Effect of Nef-9 variations on downregulation of various HLA-A and HLA-B allotypes in 721.221 transfectants. (A) Cytoplasmic tail sequences of HLA-A and HLA-B molecules used. (B) A representative set of flow cytometry plots showing uninfected 721.221 transfectants stably expressing the indicated HLA allotypes, as well as those infected with HIV-1 encoding SK-367 Nef (Arg-9), its revertant (Ser-9), or ΔNef, stained with pan-specific HLA-I MAb w6/32. (C) Quantification of HLA downregulation ability by patient-derived subtype C Nef clones SK-346 and SK-367 and their respective Ser-9 reversions. Data shown are means ± SDs of a minimum of three independent experiments. Statistical analysis was performed using a Wilcoxon matched-pairs test.

FIG 6

Effect of Nef-9 variations on HLA downregulation in human primary T lymphocytes. (A) A representative set of flow cytometry plots of uninfected human primary T lymphocytes obtained from HIV-negative donor 1 and from donors infected with HIV-1 encoding SK-367 Nef (Arg-9), its revertant (Ser-9), or ΔNef, stained with the serotype-specific MAbs for HLA-A02 and HLA-Bw6. Values on flow cytometry plots denote Nef downregulation activities. (B) Quantification of the HLA downregulation ability of SK-367 (Arg) and its corresponding Ser-9 revertant using human primary T lymphocytes from two HIV-negative donors. Data shown are means ± SDs of a minimum of three independent experiments. Statistical analysis was performed using a Wilcoxon matched-pairs test.

FIG 7

Effect of Nef sensitivity to HLA-A and HLA-B on antigen-specific T cell recognition. (A) Luciferase reporter effector T cells expressing an HLA-A*02-restricted HIV-1 Gag FK10-specific TCR were cocultured with either uninfected target T cells stably expressing A02 or A02_GGΔCKV or target T cells infected with HIV-1 encoding SK-367 Nef (Arg-9), its revertant (Ser-9), or ΔNef at the indicated E/T ratios. T cell recognition of target cells by effector cells is quantified as luminescence signal detected at 6 h. Data represent the means ± SDs of a minimum of three independent assays. Statistical analysis was performed by a Wilcoxon matched-pairs test. Over all experiments, background luminescence signal (obtained by incubation of the effector cells alone) was 151.8 ± 17.3, and HIV-1 infection frequency of target cells, determined by intracellular expression of p24^Gag protein, was 49.7% ± 11.1%. HLA-A- and HLA-B-restricted epitope response breadth (B) and magnitude (C) were measured using a gamma interferon ELISPOT assay in the subset of patients (n = 30) expressing protective alleles HLA-B*57, HLA-B*58:01, and HLA-B*81:01. Box-and-whisker plots show the median (horizontal line), interquartile range (edges of box), and range (whiskers). Data include outliers. Statistical analysis was performed using a Mann-Whitney U test. SFC, spot-forming cells.