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. 2001 Jun;75(12):5457-64.
doi: 10.1128/JVI.75.12.5457-5464.2001.

Cooperation of the V1/V2 and V3 domains of human immunodeficiency virus type 1 gp120 for interaction with the CXCR4 receptor

B Labrosse  1 , C Treboute, A Brelot, M Alizon
Affiliations

Cooperation of the V1/V2 and V3 domains of human immunodeficiency virus type 1 gp120 for interaction with the CXCR4 receptor

B Labrosse et al. J Virol. 2001 Jun.

Abstract

Human immunodeficiency virus type 1 (HIV-1) entry is triggered by the interaction of the gp120 envelope glycoprotein with a cellular chemokine receptor, either CCR5 or CXCR4. We have identified different mutations in human CXCR4 that prevent efficient infection by one HIV-1 strain (NDK) but not another (LAI) and sought to define these strain-dependent effects at the gp120 level. The lack of activity toward the NDK strain of the HHRH chimeric CXCR4 in which the second extracellular loop (ECL2) derived from the rat CXCR4 and of CXCR4 with mutations at an aspartic acid in ECL2 (D193A and D193R) was apparently due to the sequence of the third variable loop (V3) of gp120, more precisely, to its C-terminal part. Indeed, substitution of the LAI V3 loop or only its C-terminal part in the NDK gp 120 context was sufficient to restore usage of the HHRH, D193A, and D193R receptors. The same result was achieved upon mutation of a single lysine residue of the NDK V3 loop to alanine (K319A) but not to arginine (K319R). These results provide a strong case for a direct interaction between the gp120 V3 loop and the ECL2 domain of CXCR4. By contrast, V3 substitutions had no effect on the inability of NDK to infect cells via a mutant CXCR4 in which the amino-terminal extracellular domain (NT) is deleted. In experiments with a set of chimeric NDK-LAI gp120s, the V1/V2 region from LAI gp120 was both necessary and sufficient for usage of the NT-deleted CXCR4. Different variable domains of gp120 can therefore cooperate for a functional interaction with CXCR4.

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Figures

FIG. 1
FIG. 1
Infection of U373MG-CD4 cells expressing human or rat CXCR4 or the derived chimeric receptors HHRH and RRHR by HIV-1 strains LAI and NDK or derivatives with substitutions of the gp120 V3 loop. Cells were infected with approximately 1,000 I.U. per well (12-well plates) and stained with X-Gal after another 48 h. Blue-stained cells, representing HIV-1-infected cells, were scored in duplicate wells. Bars represent means of three independent transfections.
FIG. 2
FIG. 2
Alignment of the amino acid sequence of gp120 V3 loops of NDK and LAI strains and derived mutants. Numbering is according to the NDK gp120 sequence (52).
FIG. 3
FIG. 3
Effect of V3 substitutions and mutations on usage of rat and human CXCR4. (A) Infections of U373MG-CD4 cells stably expressing human or rat CXCR4 were performed and scored as described in the legend to Fig. 1. (B) Syncytium formation assays between these cell lines and HeLa cells transfected with Env expression vectors with the indicated type of gp120. Cocultures were performed in six-well plates, and cells were stained with X-Gal after 24 h. Blue-stained foci representing syncytia were scored in duplicate wells. Bars represent means of three independent transfections.
FIG. 4
FIG. 4
Effect of V3 substitutions and mutations on usage of human CXCR4 and the derived ECL2 mutants D193A and D193R. Infections (A) and syncytium formation assays (B) were performed and scored as described in the legend to Fig. 3.
FIG. 5
FIG. 5
Effect of a deletion in the amino-terminal domain (NT) of CXCR4 on its coreceptor activity for different HIV-1 strains. Infections of U373MG-CD4 cells stably expressing human CXCR4 or the Δ4-36 mutant were performed in 12-well plates with approximately 2,000 I.U. per well. Cells were stained with X-Gal 48 h later, and blue-stained cells were scored in duplicate wells.
FIG. 6
FIG. 6
Effect of exchanges between the envelope proteins of LAI and NDK on usage of the NT-deleted CXCR4. (A) Schematic organization of chimeric env genes, with restriction sites created to allow substitutions. m.a., membrane anchor domain of gp41. (B) Infections of cells stably expressing human CXCR4 or the Δ4-36 mutant with LAI or NDK virus or recombinant LAI viruses bearing the indicated env gene, performed and scored as in Fig. 5.
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