Polymorphisms of CUL5 are associated with CD4+ T cell loss in HIV-1 infected individuals

Abstract

Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (Apobec3) antiretroviral factors cause hypermutation of proviral DNA leading to degradation or replication-incompetent HIV-1. However, HIV-1 viral infectivity factor (Vif) suppresses Apobec3 activity through the Cullin 5-Elongin B-Elongin C E3 ubiquitin ligase complex. We examined the effect of genetic polymorphisms in the CUL5 gene (encoding Cullin 5 protein) on AIDS disease progression in five HIV-1 longitudinal cohorts. A total of 12 single nucleotide polymorphisms (SNPs) spanning 93 kb in the CUL5 locus were genotyped and their haplotypes inferred. A phylogenetic network analysis revealed that CUL5 haplotypes were grouped into two clusters of evolutionarily related haplotypes. Cox survival analysis and mixed effects models were used to assess time to AIDS outcomes and CD4(+) T cell trajectories, respectively. Relative to cluster I haplotypes, the collective cluster II haplotypes were associated with more rapid CD4(+) T cell loss (relative hazards [RH] = 1.47 and p = 0.009), in a dose-dependent fashion. This effect was mainly attributable to a single cluster II haplotype (Hap10) (RH = 2.49 and p = 0.00001), possibly due to differential nuclear protein-binding efficiencies of a Hap10-specifying SNP as indicated by a gel shift assay. Consistent effects were observed for CD4(+) T cell counts and HIV-1 viral load trajectories over time. The findings of both functional and genetic epidemiologic consequences of CUL5 polymorphism on CD4(+) T cell and HIV-1 levels point to a role for Cullin 5 in HIV-1 pathogenesis and suggest interference with the Vif-Cullin 5 pathway as a possible anti-HIV-1 therapeutic strategy.

Figure 1. Gene Map, SNPs, and Haplotypes in the Human CUL5 Gene

Coding exons are marked by black blocks, and 5′ and 3′ UTR by white blocks. Nucleotide changes and frequencies of SNPs and haplotypes (Hap) in AA, EA, and HC are presented. ctSNPs are shown in color.

Figure 2. LD of CUL5 SNPs in AA and EA

LD of CUL5 SNPs is shown in AA (A) and EA (B). Pairwise D′ plots were generated using Haploview with its standard color scheme. Dark-red squares indicate high D′ values, light-blue squares indicate high D′ values with low LOD scores, and light-red and white squares indicate low D′ values. D′ values were indicated for those not equal to 1.0. A single LD block was defined for both AA and EA under the default confidence interval criteria. A reduced-medium network for the genealogical relationship of CUL5 haplotypes is shown in AA (C) and EA (D). The network was inferred in terms of mutational distance, on the basis of 12 CUL5 SNPs and one chimpanzee (Chimp) sequence. Median vector (mv1), the consensus sequences inferred by parsimony criteria, represents possible unsampled sequences or extinct ancestral sequences. Haplotypes (H1–H11) are represented by circles, whose area reflects the number of alleles observed in each population. The solid branches between haplotypes represent mutational events or SNPs (S1–S12). The circles in green show haplotypes with detrimental effect and those in blue show protective effect on AIDS progression in the Cox model analysis; the protective effect of H3 in light blue was of less certainty (see Results). The haplotypes were separated into two clusters, cluster I and II, carrying ctSNP5 A or G, respectively. Cluster I and II in AA are shaded in blue and green, respectively. SNP2 is omitted in (B) and (D) as it was absent in EA.

Figure 3. Kaplan-Meier Survival Analysis of CUL5 Variants on Progression from Seroconversion to CD4⁺ T cells < 200 /mm³ in AA

(A) Shows ctSNP5 (the cluster); (B) shows htSNP6 (Hap10); and (C) shows compound genotypes of ctSNP5 and htSNP6.

Figure 4. Arithmetic Mean of CD4⁺ T Cell Counts over Time in the ALIVE Cohort by ctSNP5 (the Cluster)

The observation period was from seroconversion to the censoring date of July 31, 1997. CD4⁺ T cell counts were measured at 6-month intervals and were square-root transformed with standard error represented by the vertical bar.

Figure 5. EMSA Analysis of CUL5 SNP6 A/G

Nuclear extracts from human T lymphocytes induced by IL-4 were bound to the oligonucleotide containing the SNP6 A allele (lane A) or G allele (lane B), without cold competitors. Lane C contained nuclear extracts, SNP6 A allele probe, and a 100-fold excess of its cold probe as competitor. Lane D contained nuclear extracts, SNP6 G allele probe, and a 100-fold excess of its cold probe as competitor. An arrow indicates the band showing differential binding of nuclear factor(s) to the oligonucleotides.