Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Jan;72(1):279-85.
doi: 10.1128/JVI.72.1.279-285.1998.

Amino-terminal substitutions in the CCR5 coreceptor impair gp120 binding and human immunodeficiency virus type 1 entry

T Dragic  1 , A Trkola, S W Lin, K A Nagashima, F Kajumo, L Zhao, W C Olson, L Wu, C R Mackay, G P Allaway, T P Sakmar, J P Moore, P J Maddon
Affiliations

Amino-terminal substitutions in the CCR5 coreceptor impair gp120 binding and human immunodeficiency virus type 1 entry

T Dragic et al. J Virol. 1998 Jan.

Abstract

The CC-chemokine receptor CCR5 is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of CD4+ cells and interacts directly with the viral surface glycoprotein gp120. Although receptor chimera studies have provided useful information, the domains of CCR5 that function for HIV-1 entry, including the site of gp120 interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of CCR5 to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish CCR5-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair Env-mediated membrane fusion and the gp120-CCR5 interaction. Of these three residues, only D11 is necessary for CC-chemokine-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites on CCR5 are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal CCR5 domain.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Mutagenesis of the predicted four extracellular domains of CCR5. The amino acid sequences of the Nt region and three ECL (ECL1 to ECL3) of hCCR5 to hCCR3 are indicated. The polarity (+ or −) of charged residues is indicated below the main sequence, as are the identities of residues which differ in mCCR5. hCCR5 residues with negatively charged (white squares) and positively charged (black squares) side chains, and residues whose charges differed in mCCR5 (white circles), were all modified to alanine by PCR or site-directed mutagenesis. Fidelity was confirmed by sequencing both strands of the entire CCR5 coding region. In some cases, double mutants, K171A/E172A, K191A/N192A, and R274A/D276A, were made to preserve the overall net charge of their domain. The Nt double and triple mutants D2A/D11A and D2A/D11A/E18A were based on initial results with single-residue mutants.
FIG. 2
FIG. 2
Expression of CCR5 coreceptors in lipofected U87MG-CD4 cells. CCR5 proteins in detergent lysates of whole cells (A) or plasma membranes (B) were detected and compared by Western blotting with anti-HA-tag antibodies. Mature receptor proteins migrate with an apparent molecular mass of approximately 40 kDa. Lanes: 1, wt CCR5; 2, D2A; 3, D11A; 4, E18A; 5, D2AD11A; 6, D2AD11AE18A. The amount of mutant receptor relative to that of CCR5 was quantitated by comparing the integrated fluorescence intensities of 42-kDa bands in respective lanes. Molecular mass markers (in kilodaltons) are at left.
FIG. 3
FIG. 3
HIV-1 coreceptor function of CCR5 mutants. Substitutions in negatively charged residues (a), positively charged residues (b), and selected murine residues differing from the human sequence (c) were tested for their effects on HIV-1 entry. U87MG-CD4 cells were transiently lipofected with CCR5 mutants and then infected with NLluc-ADA (dark hatched bars), NLluc-JR-FL (light hatched bars), or NLluc-DH123 (white bars) luc-expressing chimeric viruses. Luc activity (luc cps) was measured 72 h postinfection and standardized for lipofection efficiency and receptor expression levels. The coreceptor activity of each mutant designated on the x axis is expressed as a percentage of the wt coreceptor activity (100%). Values are means ± standard deviation (indicated by error bars) of three independent experiments, each performed in quadruplicate. The asterisks indicate that the amino acids are also different in mCCR5. Similar results (not shown) were obtained with SCL1-CD4 cells.
FIG. 4
FIG. 4
Membrane fusion activity of CCR5 Nt mutants. HeLa-CD4 cells were lipofected with the Nt mutants indicated (or the pcDNA3.1 negative control plasmid) and tested 12 h later for their ability to fuse with HeLa cells expressing the JR-FL env gene (black bars). The vTF7pol system was used to enhance coreceptor expression (hatched bars). The extent of cell-cell fusion was determined by RET assay. The % RET values shown are the means ± standard deviations (indicated by error bars) of three independent experiments, each performed in duplicate. *E18A, the amino acid is also different in CCR5.
FIG. 5
FIG. 5
Competition between gp120 and CCR5 MAb 2D7 for CCR5 binding. HeLa cells cotransfected with CD4 and either wt or mutant CCR5, and infected with vTF7pol to enhance receptor expression, were preincubated with or without 10 μg of gp120/ml (JR-FL) before addition of the PE-labeled 2D7 MAb (200 ng/ml) and FACS analysis to determine mfi.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alkhatib G, Combadiere C, Broder C C, Feng Y, Kennedy P E, Murphy P M, Berger E A. A RANTES, MIP-1α, MIP-1β receptor as a fusion cofactor for macrophage tropic HIV-1. Science. 1996;272:1955–1958. - PubMed
    1. Atchison R E, Gosling J, Monteclaro F S, Franci C, Digilio L, Charo I F, Goldsmith M A. Multiple extracellular elements of CCR-5 and HIV-1 entry: dissociation from response to chemokines. Science. 1996;274:1924–1926. - PubMed
    1. Berson J F, Long D, Doranz B J, Rucker J, Jirik F R, Doms R W. A seven-transmembrane domain receptor involved in fusion and entry of T-cell-tropic human immunodeficiency virus type 1 strains. J Virol. 1996;70:6288–6295. - PMC - PubMed
    1. Bieniasz P D, Fridell R A, Aramori I, Ferguson S S G, Caron M C, Cullen B R. HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR5 co-receptor. EMBO. 1997;16:2599–2609. - PMC - PubMed
    1. Binley J, Moore J P. The viral mousetrap. Nature. 1997;387:346–348. - PubMed

Publication types

MeSH terms