T-cell expression levels of CC chemokine receptor 5 (CCR5) are a critical determinant of HIV/AIDS susceptibility, and manifest wide variations (i) between T-cell subsets and among individuals and (ii) in T-cell activation-induced increases in expression levels. We demonstrate that a unifying mechanism for this variation is differences in constitutive and T-cell activation-induced DNA methylation status of CCR5 cis-regulatory regions (cis-regions). Commencing at an evolutionarily conserved CpG (CpG −41), CCR5 cis-regions manifest lower vs. higher methylation in T cells with higher vs. lower CCR5 levels (memory vs. naïve T cells) and in memory T cells with higher vs. lower CCR5 levels. HIV-related and in vitro induced T-cell activation is associated with demethylation of these cis-regions. CCR5 haplotypes associated with increased vs. decreased gene/surface expression levels and HIV/AIDS susceptibility magnify vs. dampen T-cell activation-associated demethylation. Methylation status of CCR5 intron 2 explains a larger proportion of the variation in CCR5 levels than genotype or T-cell activation. The ancestral, protective CCR5-HHA haplotype bears a polymorphism at CpG −41 that is (i) specific to southern Africa, (ii ) abrogates binding of the transcription factor CREB1 to this cis-region, and (iii) exhibits a trend for overrepresentation in persons with reduced susceptibility to HIV and disease progression. Genotypes lacking the CCR5-Δ32 mutation but with hypermethylated cis-regions have CCR5 levels similar to genotypes heterozygous for CCR5-Δ32. In HIV-infected individuals, CCR5 cis-regions remain demethylated, despite restoration of CD4+ counts (≥800 cells per mm 3 ) with antiretroviral therapy. Thus, methylation content of CCR5 cis-regions is a central epigenetic determinant of T-cell CCR5 levels, and possibly HIV-related outcomes.HIV | CCR5 | methylation | T-cell activation | polymorphism C C chemokine receptor 5 (CCR5) is the major coreceptor for T-cell entry of HIV-1 (1). CCR5 levels on T cells influence HIV acquisition, disease progression rates, viral load, and immune recovery during antiretroviral therapy (ART), among other traits (1-4) (discussed in ref. 5). In these instances, lower CCR5 levels correlate with beneficial outcomes. Polymorphisms in the ORF and cis-regulatory regions (cis-regions) of CCR5 that correlate with higher vs. lower surface and/or gene expression levels are associated with increased vs. decreased HIV/AIDS risk and immune recovery (4-12). Classic examples are homozygosity Significance Levels of CC chemokine receptor 5 (CCR5) on T cells are a critical factor influencing HIV/AIDS susceptibility. DNA methylation is an epigenetic feature associated with lower gene expression. Here we show that the DNA methylation status of CCR5 cisregulatory regions (cis-regions) correlates inversely with CCR5 levels on T cells. T-cell activation induces demethylation of CCR5 cis-regions, upregulating CCR5 expression. Higher vs. lower sensitivity of CCR5 cis-regions to undergoing T-cell activationinduced...

Polymorphisms within the open reading frame as well as the promoter and regulatory regions can influence the amount of CCR5 expressed on the cell surface and hence an individual's susceptibility to HIV-1. In this study we characterize CCR5 genes within the South African African (SAA) and Caucasian (SAC) populations by sequencing a 9.2 kb continuous region encompassing the CCR5 open reading frame (ORF), its two promoters and the 3′ untranslated region. Full length CCR5 sequences were obtained for 70 individuals (35 SAA and 35 SAC) and sequences were analyzed for the presence of single-nucleotide polymorphisms (SNPs), indels and intragenic haplotypes. A novel SNP (+258G/C) within the ORF leading to a non-synonomous amino acid (Trp → Cys) change was detected in one Caucasian individual. Results demonstrate a high degree of genetic variation: 68 SNP positions, four indels, as well as the Δ32 deletion mutant, were detected. Seven complex putative haplotypes spanning the length of the sequenced region have been identified. These haplotypes appear to be extensions of haplotypes previously described within CCR5. Two haplotypes, SAA-HHE and SAC-HHE were found in high frequency in the SAA and SAC population groups studied (20.0% and 18.6%, respectively) and share four SNP positions suggesting an evolutionary link between the two haplotypes. Only one of the identified haplotypes, SAA/C–HHC, is common to both study populations but the haplotype frequency differs markedly between the two groups (8.6% in SAA and 52.9% in SAC). The two population groups show differences in both haplotype arrangement as well as SNP profile.

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Summary The chemokine receptor CCR5 is pivotal in determining an individual’s susceptibility to HIV‐1 infection and rate of disease progression. To establish whether population‐based differences exist in cell surface expression of CCR5 we evaluated the extent of CCR5 expression across all peripheral blood cell types in individuals from two populations, South African Africans (SAA) and South African Caucasians (SAC). Significant differences in CCR5 expression, both in number of CCR5 molecules per cell (density) and the percentage of CCR5‐expressing cells, were observed between the two study groups, within all cell subsets. Most notably, the percentage of all CCR5+ cell subsets was significantly lower in SAC compared with SAA individuals (P < 0·01) among natural killer (NK) ‐cell subsets (CD56+, CD16+ CD56+ and CD56dim) whereas CCR5 density was significantly higher in SAC compared with SAA individuals in CCR5+ CD8+ T‐cell subsets and CCR5+ NK‐cell subsets (CD56+, CD16+ CD56+ and CD56dim) (all P < 0·05). These relationships were maintained after exclusion of CCR5Δ32 heterozygous individuals (n = 7) from the SAC dataset. The SAA individuals exhibited significantly higher cell activation levels, as measured by HLA‐DR expression, than SAC individuals in CD4+ T‐cell subsets (P = 0·002) and CD56+ NK‐cell subsets (P < 0·001). This study serves to demonstrate that ethnically divergent populations show marked differences in both cell activation and CCR5 expression, which are likely to impact on both susceptibility to HIV‐1 infection and the rate of HIV‐1 disease progression.