Polymorphism in RANTES chemokine promoter affects HIV-1 disease progression

Abstract

RANTES (regulated on activation normal T cell expressed and secreted) is one of the natural ligands for the chemokine receptor CCR5 and potently suppresses in vitro replication of the R5 strains of HIV-1, which use CCR5 as a coreceptor. Previous studies showed that peripheral blood mononuclear cells or CD4(+) lymphocytes obtained from different individuals had wide variations in their ability to secrete RANTES. These findings prompted us to analyze the upstream noncoding region of the RANTES gene, which contains cis-acting elements involved in RANTES promoter activity, in 272 HIV-1-infected and 193 non-HIV-1-infected individuals in Japan. Our results showed that there were two polymorphic positions, one of which was associated with reduced CD4(+) lymphocyte depletion rates during untreated periods in HIV-1-infected individuals. This mutation, RANTES-28G, occurred at an allele frequency of approximately 17% in the non-HIV-1-infected Japanese population and exerted no influence on the incidence of HIV-1 infection. Functional analyses of RANTES promoter activity indicated that the RANTES-28G mutation increases transcription of the RANTES gene. Taken together, these data suggest that the RANTES-28G mutation increases RANTES expression in HIV-1-infected individuals and thus delays the progression of the HIV-1 disease.

Figure 1

Nucleotide sequence of RANTES promoter region. Numbers indicate nucleotide positions relative to the major transcription start site marked by a triangle. The numbers −403 and −28 indicate the two polymorphic positions. Possible consensus sites for NFκB, NFIL6, CCAAT, and TATA are underlined. Primer positions used for PCR amplification and sequence determination are underlined with arrows, indicating the direction of the primers. SacI and KpnI restriction-enzyme sites used to generate the luciferase plasmid are boxed. Asterisks below the sequence indicate the nucleotides that were not present in a previous report (32). Each of our sequences possesses these additional nucleotides. At present, it is unclear whether or not these differences represent sequence polymorphisms between white and Japanese individuals.

Figure 2

Effect of sequence polymorphism in RANTES promoter region on CD4⁺ lymphocyte depletion rate. The bars represent the 25th and 75th percentiles for the CD4⁺ depletion rate; a horizontal line in the bar represents the 50th (median) percentile and small horizontal lines above and below the bar represent the 5th and the 95th percentiles. Homozygotes and heterozygotes of each haplotype are represented by a black bar. Those lacking each respective haplotype are represented by a white bar. The mean CD4⁺ depletion rate of each group is shown by an arrowhead. Statistical significance for each difference is indicated.

Figure 3

RANTES secretion levels in serum (A) and culture supernatants of PHA-stimulated CD4⁺ enriched lymphocytes (B) obtained from 37 uninfected individuals. The symbols +/+ and +/− denote homozygote and heterozygote for haplotype III, respectively, and −/− denotes those lacking haplotype III. Horizontal lines indicate the mean levels. Statistical significance for difference is indicated.

Figure 4

The effect of sequence polymorphism in the RANTES promoter region on promoter activity in SW480 and U937 cells. The promoter regions inserted into the pGL3-Basic vector are shown by solid lines with the first and last nucleotides indicated. Nucleotides at the two polymorphic positions (−403 and −28) are indicated. Boxes indicate firefly luciferase ORFs. The relative luciferase activity of each construct is represented by solid bars. Data shown are representative of four independent experiments with similar results. Error bars account for fluctuations between measurements of relative luciferase activity in two independent clones of each construct.