non-human primates not only failed to confer protection in humans, but may have paradoxically increased risk of transmission [3,4,5,6]. This vaccine failure provides greater urgency for determining the precise repertoire of host genetic factors that influence three parameters: risk of HIV acquisition, extent of initial HIV-1 replication (as reflected by the steady-state viral load), and rate of disease progression. This is because these three parameters also may have utility as endpoints for evaluation of vaccine efficacy [7,8,9,10]. In this quest to identify the correlates of protection that might have relevance for vaccine development, the immunological features linked to alleles of class I Human Leukocyte Antigen (HLA) genes, which reside in the Major Histocompatibility Complex (MHC) locus, may be especially informative [11]. This is because genetic association studies have provided incontrovertible evidence demon- strating that HLA alleles are critical determinants of HIV-AIDS pathogenesis [11,12,13,14,15,16,17,18]. For example, HLA-B*57 is among the most intensively studied alleles in the HIV-AIDS field as it is among the most protective HLA class I alleles. In multiple cohorts of HIV-positive individuals possession of a B*57 allele is associated with a slower rate of disease progression and strong virologic control [11,12,14,15,17,19,20,21,22]. Thus, it is conceivable that adminis- tration of a therapeutic (HIV disease-retarding) vaccine that elicits immunologic features similar to those associated with disease protective HLA class I alleles might have benefit in terms of reducing population-level viral load, and consequently, as modeling studies suggest, the pace of the epidemic [1,2,23,24,25,26]. The importance of HLA alleles in control of HIV replication is further illustrated by the results of a recent genome wide association study (GWAS) because nearly 50% of the top 50 single nucleotide polymorphisms (SNPs) that associated with virologic control were in or around the MHC locus at 6p21.3 [27]. The selection criteria of subjects for the GWAS were based on a therapy-na¨ıve HIV+ individual of European descent achieving a steady-state viral load or having a stable low viral load (,1,000 RNA copies/ml), without knowledge of the date of seroconversion [27]. 486 subjects met this criteria from the 30,000 HIV-positive subjects screened retrospectively [27]. The two SNPs that had the strongest association with virologic control were both near HLA-B*57: the first was a SNP in an endogenous retroviral element designated as HLA complex P5 (HCP5), and the second SNP was in the 59-region of the HLA-C gene [27]. Also implicated as major determinants of AIDS progression rates were seven SNPs in and around the genes encoding ring finger protein 39 (RNF39) and zinc ribbon domain–containing 1 (ZNRD1), which are in close proximity to the HLA-A locus [27]. Thus, the main inferences of this GWAS were: (i) HLA-C and ZNRD1 are key independent host determinants associated with control of HIV replication and disease progression, respectively, and (ii) HCP5 may contribute positively to the well-described protective effects attributed to HLA-B*57, as this allele was found to be in nearly 100% linkage disequilibrium (LD) with the HLA- B*5701 allele. This GWAS clearly represents an advance for the HIV-AIDS field, and the results served as a fulcrum to probe further the importance of the MHC locus in HIV-AIDS pathogenesis. To understand further the role of HLA alleles in AIDS pathogenesis we reflected on the following three points. First, the SNPs in HCP5 and HLA-C accounted for ,9.6% and ,6.5% of the variability in steady-state viral load, respectively [27]. Despite these high percentages and the prior observation that viral load is highly predictive of clinical outcome [28,29], these polymorphisms were not among those identified when disease progression was used as a phenotypic endpoint [27]. For example, the association between the HLA-C allele and viral load exceeded HLA, HCP5 and ZNRD1 in HIV the threshold for statistical significance imposed by a GWAS, but the association of this allele for disease progression was not statistically significant [27]. Additionally, the SNP in HCP5 was not identified in the GWAS screen for disease progression, despite being in nearly 100% LD with the HLA-B*5701 allele, which in multiple cohorts has been associated with better virologic control, as well as a slower rate of disease progression [15,17,19,21,27,30]. These observations (strong association for the SNPs in HLA-C and HCP5 for viral load, but not for disease progression) suggested that perhaps epidemiological considerations, such as the combined analyses of subjects who achieve steady-state viral load and HIV controllers [31], might have inadvertently led to an enrichment of subjects with genotypes that might confer enhanced restriction of viral replication, and under-representation of individuals who have progressive disease during the early stages of infection. Hence, alleles that might associate with disease acceleration may not be fully represented in the study sample examined, and conceivably, this may obscure genotype-phenotype associations that might be detected within the context of a natural history cohort of HIV-1- infected subjects. Second, there is a high degree of LD between SNPs in the MHC locus and HLA alleles [32,33]. It thus remained conceivable that the effects attributed to the three SNPs identified in the GWAS (HCP5, HLA-C and ZNRD1) were due, in part, to their LD with HLA class I alleles that affect viral load and/or disease course. Third, we and others have shown that the host genetic determinants of HIV-AIDS pathogenesis may be population- specific [34,35,36]. Since the GWAS was restricted to subjects of European descent, we considered whether the reported SNPs also influenced other populations. For these three reasons, we sought to achieve greater clarity regarding the phenotypic effects associated with the SNPs in the three genes identified in the GWAS on steady-state viral load and disease progression rates. We conducted these analyses in a large, well-characterized U.S.-based natural history cohort of HIV-1- positive individuals, whose clinical characteristics have been described before [34,36,37,38,39]. We evaluated the genotype- phenotype associations attributable to the SNPs in HCP5, HLA-C and ZNRD1 before and after accounting for patterns of LD with HLA alleles that were in close proximity to these SNPs in the entire cohort, and separately in subjects of European and African descent. Results LD patterns of SNPs with HLA alleles We genotyped HIV+ and HIV2 subjects in the Wilford Hall Medical Center (WHMC) cohort for the SNP in HCP5 (rs2395029; designated as HCP5-T.G), the 59-region of the HLA-C gene (rs9264942, designated as HLA-C59-T.C), as well as for the seven SNPs in or near the ZNRD1 and RNF39 genes (Figure 1A). We had previously genotyped subjects from the WHMC HIV+ cohort for HLA class I alleles [38]. We evaluated the pair-wise patterns of LD between the SNPs, as well as between the SNPs and HLA class I alleles in HIV+ individuals. The seven SNPs near the ZNRD1 locus that were found to be associated with inter-subject differences in HIV disease course in the GWAS were all found to be in nearly 100% LD with each other (Figure 1B and Figure 1C, bottom). Predictably, consistent with these very high LD patterns, the genotype-phenotype associations reported below did not differ significantly when we analyzed each of the seven SNPs near ZNRD1 separately (data not shown). Hence, the results reported here are for a single SNP near ZNRD1 (rs9261174; third ZNRD1-RNF39 SNP shown in Figure 1B and Figure 1C, bottom), and this polymorphism is designated here as the ZNRD1-T.C SNP. PLoS ONE | www.plosone.org 2 November 2008 | Volume 3 | Issue 11 | e3636