Production of Specific mRNA Transcripts, Usage of an Alternate Promoter, and Octamer-Binding Transcription Factors Influence the Surface Expression Levels of the HIV Coreceptor CCR5 on Primary T Cells

Abstract

Surface levels of CCR5 on memory CD4+ T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection.

Circulating human CD4 and CD8 T cells do not have large intracellular pools of CCR5

“…First, induction of CCR5 surface expression on activated CD4 ϩ T cells is delayed for 5-8 days after activation, implying that intracellular pools are not readily mobilized to the cell surface. 19,20 Second, recovery of surface CCR5 is very slow after ligand-induced receptor internalization, 9 implying that surface ligation also saturates the intracellular pool that is accessible to the surface.We conclude that intracellular pools of CCR5 in human T cells do not exist and that instead, in agreement with several previous studies, 1,2,6,7 CCR5 expression is restricted to a limited population of circulating T cells and predominantly located at the cell surface. Our findings indicate that the results obtained by Achour et al 11 were almost certainly because of the generation of irrelevant antibody binding sites during the fixing and permeabilization procedure, a phenomenon that is a recognized source of falsepositive results in antibody-based experiments.…”

An Evolutionarily Conserved TNF-α–Responsive Enhancer in the Far Upstream Region of Human CCL2 Locus Influences Its Gene Expression

“…G, Bar chart depicting the relative percentage increase in accessibility of the ECRs and nonconserved regions upon TNF-a treatment compared with unstimulated U87MG astroglioma cells. CCR5 CNS2 (27) was used as a reference to calculate the differences in the relative percentage increase in accessibility. The error bars represent SD from the mean.…”

“…The samples were then incubated overnight in the presence of proteinase K at 37˚C, and genomic DNA was extracted from these samples by phenol-chloroform extraction followed by ethanol precipitation. The digestion (or accessibility) of the genomic DNA was quantified using quantitative PCR (SYBR Greener; Invitrogen) as previously described (27). Optimal primer pairs were selected using the FAST-PCR program (http://www.primerdigital.com/fastpcr.html), and their specificity was confirmed by using Megablast against the human genome; the nucleotide sequences of the primers used to amplify the regions of interest are shown in Supplemental Table I.…”

“…The genomic DNA was quantified using an ND-100 spectrophotometer (NanoDrop Technologies), and 20 ng of the DNA was used as template for each condition. Real-time PCRs were performed in triplicate in an HT7900 system (Applied Biosystems) as described previously (27). The conditions for PCR were as follows: stage 1, 50˚C for 2 min (1 cycle); stage 2, 95˚C for 10 min (1 cycle); stage 3, 95˚C for 15 s, and 60˚C for 1 min (40 cycles).…”

Confirmation of differential binding of Interferon Regulatory Factor-1 (IRF-1) to the functional and HIV disease-influencing −2578 A/G polymorphism in CCL2

“…In panels b and c, in vitro transcription and translations (IVTT) of the IRF-1 cDNA and antibody supershift experiments were performed as described previously. 5 Briefly, radiolabeled oligomers that spanned the À2578A/G polymorphism or contained the consensus IRF-1-binding motif were incubated with in vitro translated IRF-1 in the presence of binding buffer, poly(dI:dC), and 2 ml of the indicated Abs for 45 min before resolution on native polyacrylamide gels. The antibodies used were rabbit isotype control (sc-2027), anti-p65 (sc-109 X) and anti-IRF-1 (sc-497 X), and they were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).…”