Combined effect of CCR5-Delta32 heterozygosity and the CCR5 promoter polymorphism -2459 A/G on CCR5 expression and resistance to human immunodeficiency virus type 1 transmission

Exposed seronegative individuals (ES) with persistent high-risk sexual behavior may be less susceptible to human immunodeficiency virus type 1 (HIV-1) infection because they carry the chemokine receptor (CR) gene alleles CCR5 open reading frame (ORF) Delta32, CCR5 promoter -2459G, or CCR2 ORF 64I (CCR2-64I), all of which have been found to diminish HIV-1 infectivity and/or disease progression. To investigate this, we determined the haplotypes for these three genetic loci in 93 ES and 247 low-risk control individuals. To test if protective haplotypes exert their effect by modulating CR expression, we measured the protein expression of CCR5 and CXCR4 on circulating CD4+ T cells and CD14+ monocytes in 71 ES and 92 controls. To avoid investigator bias, the analysis was performed without knowledge of each subject's risk and genotype. The CCR5 -2459G allele was significantly enriched in ES Caucasian men, who constituted the majority (84%) of the ES cohort, compared to the control Caucasian men (P = 0.02). This increase was mostly attributable to a higher frequency of the -2459 A/G versus the -2459 A/A genotype in individuals heterozygous for the delta32 allele (P = 0.012). No protective influence of the CCR2-64I allele was observed. The haplotypes CCR5 ORF delta32/CCR5 -2459A (in complete linkage disequilibrium) and CCR5 ORF wt/CCR5 -2459G had a cumulative negative effect on the expression of CCR5, since we measured significantly reduced CCR5 densities on both T-helper cells and monocytes only when both haplotypes were present. Densities of CCR5 on lymphocytes and monocytes were correlated (r = 0.59; P < 0.0001), indicating concordance of CCR5 expression patterns across different cell types. We conclude that the CCR5 ORF delta32/wt-CCR5 -2459 A/G genotype combination offers an advantage in resisting sexual HIV-1 transmission and that this effect is mediated by a relative paucity of CCR5 on potential target cells of HIV-1.     

Statistical estimation and pedigree analysis of CCR2-CCR5 haplotypes

As more SNP marker data becomes available, researchers have used haplotypes of markers, rather than individual polymorphisms, for association analysis of candidate genes. In order to perform haplotype analysis in a population-based case-control study, haplotypes must be determined by estimation in the absence of family information or laboratory methods for establishing phase. Here, we test the accuracy of the Expectation-Maximization (EM) algorithm for estimating haplotype state and frequency in the CCR2-CCR5 gene region by comparison with haplotype state and frequency determined by pedigree analysis. To do this, we have characterized haplotypes comprising alleles at seven biallelic loci in the CCR2-CCR5 chemokine receptor gene region, a span of 20 kb on chromosome 3p21. Three-generation CEPH families (n=40), totaling 489 individuals, were genotyped by the 5'nuclease assay (TaqMan). Haplotype states and frequencies were compared in 103 grandparents who were assumed to have mated at random. Both pedigree analysis and the EM algorithm yielded the same small number of haplotypes for which linkage disequilibrium was nearly maximal. The haplotype frequencies generated by the two methods were nearly identical. These results suggest that the EM algorithm estimation of haplotype states, frequency, and linkage disequilibrium analysis will be an effective strategy in the CCR2-CCR5 gene region. For genetic epidemiology studies, CCR2-CCR5 allele and haplotype frequencies were determined in African-American (n=30), Hispanic (n=24) and European-American (n=34) populations.     

CCR5 Haplotypes Influence HCV Serostatus in Caucasian Intravenous Drug Users

Background

Up to 90% HIV-1 positive intravenous drug users (IDUs) are co-infected with HCV. Although best recognized for its function as a major co-receptor for cell entry of HIV, CC chemokine receptor 5 (CCR5) has also been implicated in the pathogenesis of HCV infection. Here, we investigated whether CCR5 haplotypes influence HIV-1 and HCV seropositivity among 373 Caucasian IDUs from Estonia.

Methods

Of these IDUs, 56% and 44% were HIV and HCV seropositive, respectively, and 47% were coinfected. 500 blood donors seronegative for HIV and HCV were also evaluated. CCR5 haplotypes (HHA to HHG*2) were derived after genotyping nine CCR2–CCR5 polymorphisms. The association between CCR5 haplotypes with HIV and/or HCV seropositivity was determined using logistic regression analysis. Co-variates included in the models were length of intravenous drug use, HBV serostatus and copy number of CCL3L1, the gene encoding the most potent HIV-suppressive chemokine and ligand for CCR5.

Results

Compared to IDUs seronegative for both HCV and HIV (HCV−/HIV-), IDUs who were HCV+/HIV- and HCV+/HIV+were 92% and 82%, respectively, less likely to possess the CCR5-HHG*1 haplotype, after controlling for co-variates (P_adjusted = 1.89×10⁻⁴ and 0.003, respectively). This association was mostly due to subjects bearing the CCR5 HHE and HHG*1 haplotype pairs. Approximately 25% and<10% of HCV−/HIV- IDUs and HCV−/HIV- blood donors, respectively, possessed the HHE/HHG*1 genotype.

Conclusions

Our findings suggest that HHG*1-bearing CCR5 genotypes influence HCV seropositivity in a group of Caucasian IDUs.     

Heterozygous defect in HIV-1 coreceptor CCR5 and chemokine production

CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus 1 (HIV-1). An inactive CCR5 allele with a 32-nucleotide deletion (CCR5Delta32) has been described that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. We found the allele CCR5Delta32 to be not rare in 399 Swiss blood donors with a frequency of 0.080. To assess the influence of defective CCR5 on production of its ligands we determined the capacity to produce the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES in comparison with the production of the CXC chemokine IL-8 which does not bind to CCR5. Production of chemokines was determined during endotoxin stimulation of whole-blood samples ex vivo. Both, basal and LPS-induced chemokine production in 32 blood donors heterozygous for CCR5Delta32 were not significantly different when compared with 55 blood donors who were homozygous for the wild type CCR5 allele.     

Prevalence of the CCR5Delta32 mutation in Brazilian populations and cell susceptibility to HIV-1 infection

We investigated the occurrence of the CCR5Delta32 mutation in various regional ethnic groups in Brazil and tested the resistance of mutant peripheral blood mononuclear cells (PBMCs) to infection by HIV-1 in vitro. The heterozygous prevalence was 5.3% in uninfected African descendents and 8.8% in HIV-1-positive individuals (neither population had Delta32/Delta32). German descendents were 11% heterozygous and l% Delta32/Delta32. Amerindians were exclusively CCR5/CCR5. Heterozygous uninfected PBMCs showed partial resistance to R5-HIV-1 strains in vitro, but no resistance to X4 virus. HIV-1-positive CCR5/CCR5 had higher viral loads than did heterozygous cells.     

细胞内表达CCR5Delta32蛋白对HIV-1感染抑制作用的研究

人CCR5Delta32突变个体能有效抵制HIV-1感染,主要是由于该个体淋巴细胞内表达的CCR5Delta32突变蛋白能通过反式显性失活效应(TDN)抑制细胞表面HIV-1辅受体CCR5和CXCR4的产生．通过构建CCR5Delta32慢病毒载体,体外转染人外周血单个核细胞(PBMCs),研究细胞内表达CCR5Delta32蛋白对HIV-1感染的抑制作用．结果表明,表达CCR5Delta32蛋白的人PBMCs对HIV-1 R5、X4及R5X4毒株感染均具有显著的抑制作用．这些工作为后续的AIDS基因治疗研究奠定了基础．     

Prognostic value of a CCR5 defective allele in pediatric HIV-1 infection

BACKGROUND: A deletion of 32 base pairs in the CCR5 gene (delta32 CCR5) has been linked to resistance to HIV-1 infection in exposed adults and to the delay of disease progression in infected adults. MATERIALS AND METHODS: To determine the role of delta32 CCR5 in disease progression of HIV-1 infected children born to seropositive mothers, we studied a polymerase chain reaction in 301 HIV-1 infected, 262 HIV-1 exposed-uninfected and 47 HIV-1 unexposed-uninfected children of Spanish and Italian origin. Infected children were further divided into two groups according to their rate of HIV-1 disease progression: rapid progressors who developed severe clinical and/or immunological conditions within the second year of life, and delayed progressors with any other evolution of disease. Among the latter were the long-term, non-progressors (LTNP) who presented with mild or no symptoms of HIV-1 infection above 8 years of age. Viral phenotype was studied for 45 delayed progressors. RESULTS: No correlation was found between delta32 CCR5 and mother-to-child transmission of HIV-1. However, the frequency of the deletion was substantially higher in LTNP, compared with delayed (p = 0.019) and rapid progressors (p = 0.0003). In children carrying the delta32 CCRS mutation, the presence of MT-2 tropic virus isolate was associated with a severe immune suppression (p = 0.028); whereas, the presence of MT-2 negative viruses correlated with LTNP (p = 0.010). CONCLUSIONS: Given the rapidity and simplicity of the assay, the delta32 CCR5 mutation may be a useful predictive marker to identify children with delayed disease progression who, consequently, may not require immediate antiretroviral treatment.     

Sequence variants of chemokine receptor genes and susceptibility to HIV-1 infection

Genetic susceptibility to HIV infection was previously proven to be influenced by some chemokine receptor polymorphisms clustering on chromosome 3p21. Here the influence of 5 genetic variants was studied: Δ32CCR5, G(-2459)ACCR5, G190ACCR2, G744ACX3CR1 and C838TCX3CR1. They were screened in a cohort of 168 HIV-1 positive adults [HIV(+) group] and 151 newborns [control group] from northwestern Poland. PCR-RFLP was performed to screen for the variants (except for A32CCR5 polymorphism, where PCR fragment size was sufficient to identify the alleles) and then electrophoresed on agarose gel to determine fragment size. Distribution of genotypes and alleles was not significantly different between the groups except for theCCR5 polymorphisms, with the A32 allele and the (-2459)ACCR5 allele more frequent among neonates than in the HIV(+) group. No Δ32/Δ32 homozygotes were found in the HIV(+) group, but 16.1% were Δ32/wt heterozygotes. In the control group, 1.3% were Δ32/Δ32 homozygotes and 26.0% were Δ32/wt heterozygotes. Linkage between the chemokine polymorphisms was calculated using the most informative loci for haplotype reconstruction. Haplotypes containing Δ32 CCR5,190GCCR2 and 744ACX3CR1 were found to be significantly more common in the control group. This suggests an association between these haplotypes and resistance to HIV-1 infection.     

CCR5 promoter haplotypes differentially influence CCR5 expression on natural killer and T cell subsets in ethnically divergent HIV-1 uninfected South African populations

CCR5 plays a critical and central role in HIV-1 infection and, to date, a number of genetic mutations and haplotypes within the gene have been found to positively or negatively influence an individual’s susceptibility and rate of disease progression. In this study, we have evaluated the influence of CCR5 haplotypes, HHA, HHC, HHD, and HHE, on CCR5 expression in healthy HIV-1 uninfected individuals from two populations, South African Africans (SAA, n?=?22) and South African Caucasians (SAC, n?=?31). CCR5 haplotypes were determined through sequencing and real time polymerase chain reaction. Flow cytometry was used to quantitate CCR5 surface expression, as both CCR5 density and percentage of CCR5-expressing cells, on B, T, natural killer (NK) cells and monocytes. SAA individuals positive for the HHA haplotype had significantly lower percentages of CCR5-expressing CD8+ T cells in comparison to individuals without HHA (P?=?0.001). HHC+ SAC individuals had significantly higher CCR5 molecules per cell (density) on NK (CD56+) and CD16+ CD56+ NK cell subsets (P?=?0.030 and P?=?0.024, respectively) compared to HHC? SAC individuals. Haplotypes HHD and HHE had no impact on CCR5 expression. Overall, our data highlight that the protective effect of the HHC haplotype in Caucasians might be explained by higher density of CCR5 expression on NK cells that is not evident in HHC+ SAA individuals. Findings raise the question as to the role of CCR5-expressing cells other than CD4+ T cells in protection from HIV-1 acquisition and disease progression.     

Increased CCR5 affinity and reduced CCR5/CD4 dependence of a neurovirulent primary human immunodeficiency virus type 1 isolate

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.     

Linkage of the CCR5 Delta 32 mutation with a functional polymorphism of CD45RA

A 32-bp deletion in CCR5 (CCR5 Delta 32) confers to PBMC resistance to HIV-1 isolates that use CCR5 as a coreceptor. To study this mutation in T cell development, we have screened 571 human thymus tissues for the mutation. We identified 72 thymuses (12.6%) that were heterozygous and 2 (0.35%) that were homozygous for the CCR5 Delta 32 mutation. We found that thymocyte development was normal in both CCR5 Delta 32 heterozygous and homozygous thymuses. In 3% of thymuses we identified a functional polymorphism of CD45RA, in which cortical and medullary thymocytes failed to down-regulate the 200- and 220-kDa CD45RA isoforms during T cell development. Moreover, we found an association of this CD45 functional polymorphism in thymuses with the CCR5 Delta 32 mutation (p = 0.00258). In vitro HIV-1 infection assays with CCR5-using primary isolates demonstrated that thymocytes with the heterozygous CCR5 Delta 32 mutation produced less p24 than did CCR5 wild-type thymocytes. However, the functional CD45RA polymorphism did not alter the susceptibility of thymocytes to HIV-1 infection. Taken together, these data demonstrate association of the CCR5 Delta 32 mutation with a polymorphism in an as yet unknown gene that is responsible for the ability to down-regulate the expression of high m.w. CD45RA isoforms. Although the presence of the CCR5 Delta 32 mutation down-regulates HIV-1 infection of thymocytes, the functional CD45RA polymorphism does not alter the susceptibility of thymocytes to HIV-1 infection in vitro.     

G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4(+)-transformed cells or pharmacological inhibition of G(alpha)i proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Delta32/Delta32). Expression of wild-type (wt) CCR5 in ccr5 Delta32/Delta32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4(+) cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4(+) T lymphocytes or macrophages.     

CCR5 N-terminus peptides enhance X4 HIV-1 infection by CXCR4 up-regulation

The HIV-1 envelope glycoprotein gp120 interacts consecutively with CD4 and CCR5 to mediate the entry of R5-HIV-1 strains into target cells. The N-terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in gp120-CCR5 binding and, consequently, in viral entry. Here, we demonstrate that a tyrosine sulfated peptide, reproducing the entire N-terminal extracellular region of CCR5, its unsulfated analogue, and a point-mutated peptide are unable to inhibit R5-HIV-1 mediated infection, competing with the entire CCR5 in the formation of gp120-CD4-CCR5 complex. Surprisingly, these peptides show the capability of enhancing HIV-1 infection caused by X4 strains through the up-regulation of both CD4 and CXCR4 receptors.     

Genetic protection against hepatitis B virus conferred by CCR5Delta32: Evidence that CCR5 contributes to viral persistence

Recovery from acute hepatitis B virus (HBV) infection requires a broad, vigorous T-cell response, which is enhanced in mice when chemokine receptor 5 (CCR5) is missing. To test the hypothesis that production of a nonfunctional CCR5 (CCR5Delta32 [a functionally null allele containing a 32-bp deletion]) increases the likelihood of recovery from hepatitis B in humans, we studied 526 persons from three cohorts in which one person with HBV persistence was matched to two persons who recovered from an HBV infection. Recovery or persistence was determined prior to availability of lamivudine. We determined genotypes for CCR5Delta32 and for polymorphisms in the CCR5 promoter and in coding regions of the neighboring genes, chemokine receptor 2 (CCR2) and chemokine receptor-like 2 (CCRL2). Allele and haplotype frequencies were compared among the 190 persons with viral recovery and the 336 with persistence by use of conditional logistic regression. CCR5Delta32 reduced the risk of developing a persistent HBV infection by nearly half (odds ratio [OR], 0.53; 95% confidence interval [CI], 0.33 to 0.83; P = 0.006). This association was virtually identical in persons with and without a concomitant human immunodeficiency virus infection. Of the nine individuals who were homozygous for the deletion, eight recovered from infection (OR, 0.25; 95% CI, 0.03 to 1.99; P = 0.19). None of the other neighboring polymorphisms examined were associated with HBV outcome. These data demonstrate a protective effect of CCR5Delta32 in recovery from an HBV infection, provide genetic epidemiological evidence for a role of CCR5 in the immune response to HBV, and suggest a potential therapeutic treatment for patients persistently infected with HBV.     

CCR5Delta32 protein expression and stability are critical for resistance to human immunodeficiency virus type 1 in vivo

Human immunodeficiency virus type 1 (HIV-1) infection of individuals carrying the two alleles of the CCR5Delta32 mutation (CCR5(-/-)) has rarely been reported, but how the virus overcomes the CCR5Delta32 protective effect in these cases has not been delineated. We have investigated this in 6 infected (HIV(+)) and 25 HIV(-) CCR5(-/-) individuals. CD4(+) T lymphocytes isolated from HIV(-) CCR5(-/-) peripheral blood mononuclear cells (PBMCs) showed lower levels of CXCR4 expression that correlated with lower X4 Env-mediated fusion. Endogenous CCR5Delta32 protein was detected in all HIV(-) CCR5(-/-) PBMC samples (n = 25) but not in four of six unrelated HIV(+) CCR5(-/-) PBMC samples. Low levels were detected in another two HIV(+) CCR5(-/-) PBMC samples. The expression of adenovirus 5 (Ad5)-encoded CCR5Delta32 protein restored the protective effect in PBMCs from three HIV(+) CCR5(-/-) individuals but failed to restore the protective effect in PBMCs isolated from another three HIV(+) CCR5(-/-) individuals. In the latter samples, pulse-chase analyses demonstrated the disappearance of endogenous Ad5-encoded CCR5Delta32 protein and the accumulation of Ad5-encoded CCR5 during the chase periods. PBMCs isolated from CCR5(-/-) individuals showed resistance to primary X4 but were readily infected by a lab-adapted X4 strain. Low levels of Ad5-encoded CCR5Delta32 protein conferred resistance to primary X4 but not to lab-adapted X4 virus. These data provide strong support for the hypothesis that the CCR5Delta32 protein actively confers resistance to HIV-1 in vivo and suggest that the loss or reduction of CCR5Delta32 protein expression may account for HIV-1 infection of CCR5(-/-) individuals. The results also suggest that other cellular or virally induced factors may be involved in the stability of CCR5Delta32 protein.     

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.     

A haplotype in the CCR5 gene promoter was associated with the susceptibility to HIV-1 infection in a northern Chinese population

It has been reported that single nucleotide polymorphisms (SNPs) in the promoter of the CCR5 gene are associated with the risk for HIV-1 infection and AIDS progression. Using resequencing, we performed a systematic survey of 78 HIV-1 seropositive individuals and 70 population-matched healthy control individuals from northern China to investigate SNPs of the CCR5 gene promoter and evaluated their effects on HIV-1 infection and the progression of AIDS. Linkage disequilibrium (LD) plots and haplotypes were generated using Haploview software. The association analyses were statistically compared using the Chi-square test with SPSS13.0 software for Windows. Seven SNPs (58755A>G, 58791C>T, 58934G>T, 59029A>G, 59353C>T, 59402A>G and 59653C>T) in the region of the CCR5 gene promoter were evaluated in this study. Among the seven SNPs, the minor allele frequencies of 58755G and 58791T were less than 2%. The differences in frequencies of the other five SNPs were not significant between case and control cohorts (P > 0.05). In the case cohort, the association between these SNPs and clinical features (CD4⁺ T-lymphocyte counts and clinical categories) was not significant (P > 0.05); however, there was a significant association between the haplotype GGTAC and susceptibility to HIV-1 infection (P < 0.05), which is not consistent with other reports studied in different populations. The results suggest that the haplotype GGTAC may have a role in the process of HIV-1 infection in the northern Chinese population.     

Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.

BACKGROUND: CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus-1 (HIV-1). Recently, an inactive CCR5 allele (designated here as CCR5-2) was identified that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. The reports conflict on the effect of heterozygous CCR5-2 on HIV-1 susceptibility, and race and risk levels have not yet been fully analyzed. Here we report our independent identification of CCR5-2 and test its effects on HIV-1 pathogenesis in individuals with contrasting clinical outcomes, defined race, and quantified risk. MATERIALS AND METHODS: Mutant CCR5 alleles were sought by directed heteroduplex analysis of genomic DNA from random blood donors. Genotypic frequencies were then determined in (1) random blood donors from North America, Asia, and Africa; (2) HIV-1+ individuals; and (3) highly exposed-seronegative homosexuals with quantified risk. RESULTS: CCR5-2 was the only mutant allele found. It was common in Caucasians, less common in other North American racial groups, and not detected in West Africans or Tamil Indians. Homozygous CCR5-2 frequencies differed reciprocally in highly exposed-seronegative (4.5%, n = 111) and HIV-1-seropositive (0%, n = 614) Caucasians relative to Caucasian random blood donors (0.8%, n = 387). This difference was highly significant (p < 0.0001). By contrast, heterozygous CCR5-2 frequencies did not differ significantly in the same three groups (21.6, 22.6, and 21.7%, respectively). A 55% increase in the frequency of heterozygous CCR5-2 was observed in both of two cohorts of Caucasian homosexual male, long-term nonprogressors compared with other HIV-1+ Caucasian homosexuals (p = 0.006) and compared with Caucasian random blood donors. Moreover, Kaplan-Meier estimates indicated that CCR5-2 heterozygous seroconvertors had a 52.6% lower risk of developing AIDS than homozygous wild-type seroconvertors. CONCLUSIONS: The data suggest that homozygous CCR5-2 is an HIV-1 resistance factor in Caucasians with complete penetrance, and that heterozygous CCR5-2 slows the rate of disease progression in infected Caucasian homosexuals. Since the majority (approximately 96%) of highly exposed-seronegative individuals tested are not homozygous for CCR5-2, other resistance factors must exist. Since CCR5-2 homozygotes have no obvious clinical problems, CCR5 may be a good target for the development of novel antiretroviral therapy.