HLA Class I Downregulation by HIV-1 Variants from Subtype C Transmission Pairs

Abstract

HIV-1 downregulates human leukocyte antigen A (HLA-A) and HLA-B from the surface of infected cells primarily to evade CD8 T cell recognition. HLA-C was thought to remain on the cell surface and bind inhibitory killer immunoglobulin-like receptors, preventing natural killer (NK) cell-mediated suppression. However, a recent study found HIV-1 primary viruses have the capacity to downregulate HLA-C. The goal of this study was to assess the heterogeneity of HLA-A, HLA-B, and HLA-C downregulation among full-length primary viruses from six chronically infected and six newly infected individuals from transmission pairs and to determine whether transmitted/founder variants exhibit common HLA class I downregulation characteristics. We measured HLA-A, HLA-B, HLA-C, and total HLA class I downregulation by flow cytometry of primary CD4 T cells infected with 40 infectious molecular clones. Primary viruses mediated a range of HLA class I downregulation capacities (1.3- to 6.1-fold) which could differ significantly between transmission pairs. Downregulation of HLA-C surface expression on infected cells correlated with susceptibility to in vitro NK cell suppression of virus release. Despite this, transmitted/founder variants did not share a downregulation signature and instead were more similar to the quasispecies of matched donor partners. These data indicate that a range of viral abilities to downregulate HLA-A, HLA-B, and HLA-C exist within and between individuals that can have functional consequences on immune recognition.IMPORTANCE Subtype C HIV-1 is the predominant subtype involved in heterosexual transmission in sub-Saharan Africa. Authentic subtype C viruses that contain natural sequence variations throughout the genome often are not used in experimental systems due to technical constraints and sample availability. In this study, authentic full-length subtype C viruses, including transmitted/founder viruses, were examined for the ability to disrupt surface expression of HLA class I molecules, which are central to both adaptive and innate immune responses to viral infections. We found that the HLA class I downregulation capacity of primary viruses varied, and HLA-C downregulation capacity impacted viral suppression by natural killer cells. Transmitted viruses were not distinct in the capacity for HLA class I downregulation or natural killer cell evasion. These results enrich our understanding of the phenotypic variation existing among natural HIV-1 viruses and how that might impact the ability of the immune system to recognize infected cells in acute and chronic infection.

Keywords: HIV-1; HLA; NK cells; Nef; Vpu; human immunodeficiency virus; human leukocyte antigen class I; natural killer cells; quasispecies; transmitted/founder virus.

FIG 1

Diversity of Nef and Vpu in characterized viruses. Viral variants from donors of transmission pairs are labeled as pair ID followed by variant number, while recipient transmitted/founder viruses are labeled TF. Each pair is set off by color. The laboratory strains are in gray, while the subtype C LANL consensus is labeled C. BioNJ tree of phylogenetic relationships of Nef (A) and Vpu (B) amino acid (aa) sequences of transmitted/founder and nontransmitted donor variants (NT), along with one subtype C (MJ4) and one subtype B (NL4-3) laboratory strain. The LANL subtype C consensus is included for reference. Number of amino acid differences from the transmitted/founder for each NT donor variant from each transmission pair for Nef (C) and Vpu (D).

FIG 2

HLA downregulation of infected cells in vitro. Infectious molecular clones from six transmission pairs along with two laboratory strains (MJ4, subtype C; NL4-3, subtype B) were assayed for the ability to downregulate HLA-I by flow cytometry with antibodies recognizing HLA-A2, HLA-B7, HLA-C, and pan-HLA-I. The gating strategy is shown as an example from 331 TF (A), and histograms showing CD4⁻ Gag⁺ (red), CD4⁺ Gag⁻ (black), and HLA negative controls (gray) are shown for 4 variants that show a range of activities for HLA-A2 (top row), HLA-B7 (row 2), HLA-C (row 3), and HLA-I (bottom row) downregulation (B). (C) Mean values from pooled triplicates averaged from 2 to 5 experiments of the fold downregulation of HLA by MFI on infected T cells, as described for panels A and B, compared to uninfected CD4⁺ T cells. Standard errors for each HLA class I factor are provided in Materials and Methods. Each viral clone is denoted with a pair ID followed by variant number when amplified from the donor partner or TF for variants derived from the recipient. HLA-A2, A2; HLA-B7, B7; HLA-C, C; pan-HLA-I, HLA-I; TF, transmitted/founder; NT, nontransmitted donor variant.

FIG 3

Relationship of HLA class I molecules on infected cells. (A) Overall HLA class I downregulation for the tested primary variants showing the means and standard deviations. Bars with asterisks above mark significant differences. TF are blue and NT are red. (B) Spearman correlation analysis between HLA-A2 and HLA-B7 downregulation (left), HLA-A2 and HLA-C downregulation (middle), and HLA-A2 and total HLA-I (right). (C, left) Identification of HLA-A2⁺ HLA-B7⁺ and HLA-A2⁺ HLAB7⁻ cells by flow cytometry by screening of PBMC from 4 different individuals as examples. (Right) Spearman correlation of HLA-A2 downregulation in two different PBMC donors averaged from multiple experiments using TF variants.

FIG 4

Quasispecies-level HLA class I downregulation and clustering by phenotype. (A) Box-and-whisker plots showing median and minimum to maximum per transmission pair (x axis) for downregulation of HLA-A2 (top left), HLA-B7 (top right), HLA-C (bottom left), and total HLA-I (bottom right). Transmitted viruses are in blue, nontransmitted variants are in red, NL4-3 is in gray, and MJ4 is in black. Lines and asterisks represent statistical significance at a P value of <0.05, and all y axes depict fold change of CD4⁻ Gag⁺ MFI divided by CD4⁺ Gag⁻ MFI. (B) Hierarchical clustering analysis of variants by HLA downregulation level independent of their genetic relatedness. Each variant is labeled and colored per transmission pair as described for Fig. 2C.

FIG 5

Amino acids of viruses with high and low levels of HLA-A and HLA-B downregulation. Shown is a Weblogos display of the Nef amino acid sequence composition of primary viruses at the lowest and highest terciles for HLA-A and HLA-B downregulation. Sequence Harmony analysis identified the shown positions with a significant Z score, determined as described in Materials and Methods. A full listing of amino acids is presented in Table 1. Green amino acids denote the subtype C consensus amino acid at that position, and gray numbers are Nef positions common to virus groupings by HLA-A and HLA-B. Position numbers are based on the alignment in Data Set S1 in the supplemental material.

FIG 6

NK cell suppression of virus growth in vitro. (A) TF (blue) and NT (red) residual replication in the presence of NK cells following infection at a multiplicity of infection of 1.0 is shown for four pairs. Each variant is listed on the x axis, and the y axis is calculated as the percentage of replication in the presence of NK cells compared to the no-NK controls equivalent to an effector-to-target ratio of 1:10, as further described in Materials and Methods. The error bars represent the standard errors of the means. (B) Mean quasispecies NK susceptibility, including that for laboratory strains NL4-3 (gray) and MJ4 (black). Lines and asterisks represent a P value of <0.05 using ANOVA followed by Tukey's multiple-comparison test. Wilcoxon test was done to compare TF and NT median for the pairs.

FIG 7

Relationship between NK cell suppression and HLA-C downregulation. A Spearman correlation analysis of residual replication in the presence of NK cells against HLA-C (A), HLA-A2 (B), HLA-B7 (C), and total HLA-I (D) downregulation on the x axis. TF (blue) and NT (red) are colored.

FIG 8

Replicative capacity and NK cell suppression. (A) Residual replication in the presence of NK for Gag-MJ4 chimeric viruses (GC) ordered by replicative capacity scores. The x axis contains names of each chimera according to their replicative capacity score, such that variant ID GC 0.34 stands for gag chimera with a replicative capacity score of 0.34. (B and C) Spearman correlation analysis of replicative capacity (RC) versus NK suppression in vitro of Gag-MJ4 chimeras (B) and primary viruses (C). (D) Spearman correlation of residual replication in the presence of NK cells versus replication without NK cells. % of ctrl, percentage of replication in the absence of NK cells.