South African Mutations of the CCR5 Coreceptor for HIV Modify Interaction With Chemokines and HIV Envelope Protein

Abstract

The CCR5 chemokine receptor is the major coreceptor for HIV-1 and the receptor for CC-chemokines, MIP-1alpha, MIP-1beta, and regulated upon activation normal T-cell-expressed and secreted. Individuals, who are homozygous for the nonfunctional CCR5Delta32 allele, are largely resistant to HIV-1 infection. Four unique mutations that affect the amino acid sequence of CCR5 have been identified in South Africa. We have assessed the effect of these mutations on CCR5 interactions with chemokines and HIV Envelope protein. The LeuPhe mutation did not affect CCR5 expression, chemokine binding, intracellular signaling, or interaction with Envelope. The ArgGln mutant was similar to wild-type CCR5, but ligand-independent intracellular signaling suggests that it is partially constitutively active. The AspVal mutation decreased chemokine-binding affinity, chemokine-stimulated intracellular signaling, and receptor expression. It also decreased HIV Envelope-mediated cell fusion. The ArgStop mutant showed no measurable chemokine binding or signaling and no measurable expression of CCR5 at the cell surface or within the cell. Consistent with lack of cell surface expression, it did not support envelope-mediated cell fusion. These results show that South African CCR5 variants have a range of phenotypes in vitro that may reflect altered chemokine responses and susceptibility to HIV infection in individuals who carry these alleles.

Constitutively Active CCR5 Chemokine Receptors Differ in Mediating HIV Envelope-dependent Fusion

“…The chimeric G protein construct, Gαqi, which allows receptors that usually activate the Gi/o family of G proteins to stimulate inositol phosphate (IP) signaling [30] was prepared by site-directed mutagenesis, cloned into the pcDNA3.1(+) expression vector (Invitrogen, Carlsbad, CA) and stably expressed in HEK 293 cells (HEK-Gqi) as previously described [22]. The HIV-1C env construct pTHr.gp150CT [31] was a gift from Carolyn Williamson (University of Cape Town).…”

“…Basal and MIP-1β-stimulation of IP second messenger production was assessed as previously described [22], [35]. Briefly, HEK-Gqi cells (3×10 6 per 10 cm dish), transfected with wild type or mutant CCR5 receptor constructs, were distributed into 12-well plates (Corning, 2 plates/10 cm dish), incubated overnight and then incubated with 3 [H] myo -inositol (1 µCi/ml, Amersham Life Sciences, Buckinghamshire, England, 16–18 h).…”

“…HEK 293 cells (3×10 6/ 10 cm dish), transiently transfected with wild type or mutant CCR5 receptor constructs were detached (5 mM EDTA, 50 mM HEPES, pH 7.4, 100 mM NaCl), re-suspended (3×10 5 cells/tube) in binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl 2 , 5 mM MgCl 2 , 0.5% BSA) and incubated, in triplicate, with [ 125 I]-MIP-1β (50 000 cpm, approximately 0.05 pmol) and increasing concentrations of unlabelled MIP-1β (0 to 10 −7 M) in a total volume of 0.2 ml (60 min, 27°C), as previously described [22], [37]. Bound tracer was separated by filtration through glass-fiber filters (GF/C, Whatman, Maidstone, England) presoaked in 1% BSA.…”

“…Different mutations of the Thr 2.56(82) and Pro 2.58(84) residues of the conserved TxP motif, stabilized CCR5 mutants in inactive [20] or constitutively active conformations [21]. A naturally-occurring Arg 6.32(225) Gln mutation causes partial constitutive activity in CCR5 [22].…”

C-C chemokine receptor type five (CCR5): An emerging target for the control of HIV infection

“…In 2010, mutant receptor constructs from four novel variants (L107F, R225Q, D2V, and R225X) identified in the Peterson paper were analyzed in vitro for their effects on expression, chemokine binding and response, and HIV co-receptor properties (Folefoc et al, 2010). The D2V variant found in the N-terminal showed decreased CCR5 expression, reduced chemokine binding and response, and lowered HIV infection.…”