CCR5-32

We have found a distinction in specific binding affinity for separate CCR5P allele sequence motifs to nuclear binding (potential transcription) factors, which suggests a possible mechanism for CCR5P1/P1 epidemiologic consequences. We used electrophoretic mobility-shift analysis (8) (EMSA) to assess DNA-protein interactions with the common CCR5P1 (frequency, f = 0.56) and CCR5P4 (f = 0.35) alleles. Synthetic allele-specific oligonucleotides (~20 bp), representing polymorphic CCR5P sites 208 (G/T), 627 (C/T), 676 (A/G), and 303 (A/G) (2) in the context of their adjacent nucleotides, were incubated with nuclear extracts from phytohemmaglutinin (PHA)-blasted phorbolmyristate acetate (PMA) and ionomyocin-treated human T cells (8). Variants at three sites (303, 627, and 676) showed no difference in binding for alternative allele oligonucleotides; however, the T-bearing oligonucleotide at site 208 (carried in CCR5P3 and P4) (1) displayed 5- to 12-fold greater binding to a specific nuclear binding protein (or proteins) than did the G-bearing oligonucleotide (Fig. 1, complex A). Specificity for the binding was demonstrated by the fact that competitive binding of the cold CCR5P oligonucleotide (208T and 208G), but not of a nonspecific SP-1 oligonucleotide, eliminated complex A formation. Additionally, in cross-competition experiments with 208G versus 208T, as little as 10-fold excess of cold 208T eliminated complex A formation on 208G. However, 100-fold excess of cold 208G only partially competed for complex A formation on 208T.

We examined the sequence surrounding CCR5P site 208 for sequence homology to binding sites for previously described transcription factors (9). The sequence revealed significant homology to sites capable of binding to cRel (a member of the Rel/NF-B family). We incubated specific antibodies to cRel (10), p50 (11), and p65 (11) with the 208T oligonucleotide. We then performed EMSAs, and each antibody resolved unique complexes in addition to complex A (Fig. 1, complex C). The 208G allele also produced a weak supershift complex with the three antisera, which indicates that this sequence is also capable of binding cRel, p50, and p65 (12). The intensity of the supershift complex (complex C) relative to complex A is similar whether 208G or 208T is tested, and neither allele appreciably diminishes the major allele specific complex A, as measured by supershift experiments that use the Rel/NF-B family antisera. Therefore, it seems the predominant interaction of CCR5P4 (that is, complex A) involves other yet to be identified binding factors in addition to the three implicated transcription factors.

The identification of differential binding of nuclear factors to oligonucleotides with CCR5P site 208T (retained by CCR5P3 and P4) as compared with CCR5P site 208G (retained in alleles CCR5P1 and P2) raises the possibility that inclusion of the site 208G in CCR5P1 (and linkage disequilibrium of the site 303A with CCR5P1) would account for the recessive hyper-susceptibility of CCR5P1/P1 homozygotes to rapid progression to AIDS end points (1, 2). If this were so, then CCR5P1/P1, CCR5P1/P2, and CCR5P2/P2 should each be associated with rapid AIDS progression, insofar as both CCR5P1 and P2 alleles contain the 208G nucleotide residue (1). We explicitly tested this prediction by comparing cohort survival curves of CCR5P1/P1 genotypes with CCR5P1/P2 plus CCR5P2/P2, or with the sum of CCR5P1/P1, CCR5P1/P2, and CCR5P2/P2 genotypes (Table 1). In every case, the CCR5P1/P1 genotypes alone were associated with rapid progression, while the CCR5P2-bearing genotypes did not progress more rapidly than other CCR5P genotypes. For this reason, we conclude that the 208G/T polymorphism differential binding to nuclear factors cannot fully explain the reported epidemiological data (1, 2).

Table 1. Influence of CCR5P-208G variant on survival to AIDS end points (outcomes) when included in genotypes bearing CCR5P1 or CCR5P2. Indicated CCR5P1 and CCR5P2 genotypes are on chromosome haplotypes that are wild type (+) for adjacent CCR2-64I and CCR5-32 sites. Results are adjusted for the CCR5-32 and CCR2-64I protective effects as a combined variable in the Cox models. AIDS outcomes P1/P1 P1/P2 or P2/P2 P1/P1, P1/P2, or P2/P2 Cohorts n/events RH P-value RH P-value RH P-value CD4 < 200 Caucasians 634/357 1.34 0.06 0.75 0.17 1.07 0.64 MACS 365/192 1.31 0.20 0.84 0.53 1.10 0.59 MHCS 183/119 1.01 0.98 0.57 0.20 0.84 0.48 AIDS 1993 Caucasians 638/421 1.51 0.005 0.94 0.74 1.25 0.07 MACS 367/242 1.48 0.04 0.94 0.80 1.23 0.20 MHCS 185/126 1.43 0.20 1.00 1.00 1.28 0.32 AIDS 1987 Caucasians 641/324 1.42 0.03 1.02 0.93 1.26 0.11 MACS 370/191 1.39 0.12 0.97 0.90 1.21 0.29 MHCS 185/89  1.38 0.33 1.44 0.41 1.40 0.24 Death Caucasians 641/248 1.31 0.15 1.12 0.64 1.23 0.19 MACS 370/151 1.22 0.40 1.18 0.55 1.21 0.35 MHCS 185/72  1.51 0.24 1.38 0.50 1.47 0.21

Nevertheless, the presence of a mixture of nuclear binding factors which discriminate among CCR5 promoter alleles remains a viable possibility to account for differential availability of CCR5 receptors in various cell populations. The nuclear factors may vary in abundance among different cell types and respond to diverse stimuli that mediate CCR5 transcription. Defining the implications of these events is an important goal of ongoing experiments.

Jay H. Bream
Howard A. Young
Laboratory of Experimental Immunology,
National Cancer Institute-Frederick Cancer
Research and Development Center (NCI-FCRDC),
Frederick, MD 21702, USA
Nancy Rice
Molecular Basis of Carcinogenesis
Laboratory,
Advanced Bioscience Laboratories,
Inc.-Basic Research Program,
NCI-FCRDC
Maureen P. Martin
Michael W. Smith
Mary Carrington
Intramural Research Support Program,
Science Applications International Corporation, NCI-FCRDC
Stephen J. O'Brien
Laboratory of Genomic Diversity,
NCI-FCRDC
E-mail: obrien{at}ncifcrf.gov

REFERENCES AND NOTES

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12. Research sponsored (in part) by the National Cancer Institute, under contract with Advanced Bioscience Laboratories, Inc.