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. 2000 Feb;74(4):1787-93.
doi: 10.1128/jvi.74.4.1787-1793.2000.

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha

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Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha

Y Maeda et al. J Virol. 2000 Feb.

Abstract

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.

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Figures

FIG. 1
FIG. 1
Sensitivity of a selected mutant to C-C chemokines. MAGI/CCR5 cells were treated with various concentrations of MIP-1α (a), MIP-1β (b), and RANTES (c), followed by an inoculation of wild-type JR-FL (□) and virus selected for 3 months in the presence of increasing concentrations of MIP-1α (■). Blue cells were counted 2 days after infection. The y axis represents the percentages of positive blue cells counted. The x axis represents the concentrations of each C-C chemokine. All experiments were performed in duplicate. The data are expressed as means ± standard deviations.
FIG. 2
FIG. 2
V2 and V3 amino acid sequences from JR-FL-infected cells passaged and selected in MIP-1α for 3 months. Amplified products from infected MOLT-4#8/CCR5 cells passaged for 2 weeks and 3 months and selected by MIP-1α were cloned, and 8 to 22 clones from each sample were sequenced. The wild-type JR-FL amino acid sequences of V2 and V3 are shown in the top line. Numbers on the right are the numbers of clones with the sequence over the total number of clones tested. In each set of clones, the deduced amino acid sequences of the V2 (a) and V3 (b) regions were aligned by the single-amino-acid code. Dashes denote sequence identity.
FIG. 3
FIG. 3
Sensitivities of luciferase reporter HIV strains pseudotyped with mutant envelope to MIP-1α. MAGI/CCR5 cells were infected with luciferase reporter HIVs pseudotyped with the JR-FL envelope wild type (□), an S303G mutant (⧫), a V166M mutant (●), an S303G/V166M mutant (▴), and the NL4-3 envelope (■). Luciferase (Luc) activity was measured 3 days after infection. The y axis represents the relative luciferase activity of each virus. All experiments were performed in duplicate. The data are expressed as means ± standard deviations.
FIG. 4
FIG. 4
Chemokine receptor usage of luciferase reporter HIVs pseudotyped with mutant envelope. CXCR4 (filled bar)- or CCR5 (open bar)-expressing NP-2/CD4 cells were infected with luciferase reporter HIVs pseudotyped with the JR-FL envelope with the indicated mutations or the NL4-3 envelope. Luciferase activity was measured 3 days after infection. The data are geometric means ± standard deviations of duplicate determinations.

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