Distinct molecular pathways to X4 tropism for a V3-truncated human immunodeficiency virus type 1 lead to differential coreceptor interactions and sensitivity to a CXCR4 antagonist

Abstract

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed DeltaV3-X4A.c5 and DeltaV3-X4B.c7, which took distinct molecular pathways for this change. The DeltaV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the DeltaV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to DeltaV3-X4A.c5, DeltaV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.

FIG. 1.

Scheme summarizing the derivation of the ΔV3-X4A and ΔV3-X4B viruses. Mutagenesis and in vitro adaptation steps are indicated in italics. Introduction of the ΔV3(9,9) mutation in HIV-1 R3A and adaptation to SupR5R cells have been described previously (47) (see Materials and Methods). The adapted R3A ΔV3(9,9) viral swarm was serially passaged in a 1:10 mix of SupCCR5 and SupT1 cells, respectively, until infection of greater than 10% of cells (by IFA) occurred. Virus was then serially passaged in SupT1 cells. This adaptation scheme was carried out twice, generating two viral swarms, designated ΔV3-X4A and ΔV3-X4B.

FIG. 2.

Function of ΔV3-X4A and ΔV3-X4B env clones. (A) Coreceptor use in a cell-cell fusion assay for ΔV3-X4A and ΔV3-X4B env clones. Percent fusion was calculated by using luciferase activity normalized to R3A fusion with QT6 cells coexpressing CD4 with CCR5 or CXCR4. The means of three experiments plus the standard error of the means are shown. (B) Growth curves for recombinant NL4-3 viruses containing the R3A, TA1, ΔV3-X4A.c5, or ΔV3-X4B.c7 env in CD4⁺ CXCR4⁺ CCR5⁻ SupT1 cells. RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown.

FIG. 3.

Virus growth using CXCR4-negative SupT1 cells to assess coreceptor use. Growth curves for recombinant NL4-3 viruses containing the R3A (A), TA1 (B), ΔV3-X4A.c5 (C), or ΔV3-X4B.c7 (D) env are shown in parental SupT1, Sup-Zfn/X4⁻, Sup-Zfn/X4⁻R5⁺, and Sup-Zfn/X4⁺ cells. RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown.

FIG. 4.

Alignment of Env sequences for R3A, TA1, ΔV3-X4A.c5, and ΔV3-X4B.c7. Locations of gp120 variable domains (V1/V2, V3, V4, and V5) are indicated. Putative N-linked glycosylation sites are indicated by black dots. Clone TA1 from the adapted R3A ΔV3(9,9) swarm contained the original ΔV3(9,9) mutation with an Ala-to-Val mutation in the Gly-Ala-Gly linker, a deletion of residues 185 to 188 in V1/V2, and three other point mutations in Env. Clone ΔV3-X4A.c5 contained a 7-amino-acid insertion in the truncated V3 loop and 12 other point mutations in Env, compared with the TA1 sequence. Clone ΔV3-X4B.c7 maintained the ΔV3(9,9) truncation in V3 but did not contain a deletion in V1/V2 and contained 18 other point mutations in Env compared with TA1 sequence.

FIG. 5.

ΔV3-X4A determinants for CXCR4 use in cell-cell fusion. Fusion for the TA1 env containing the ΔV3-X4A.c5 7-amino-acid V3 loop insertion with CD4⁺ CXCR4⁺(A) or CD4⁺ CCR5⁺ (B) QT6 cells. Percent fusion was calculated by using luciferase activity normalized to ΔV3-X4A.c5 fusion with CD4⁺ CXCR4⁺ targets (A) or to TA1 fusion with CD4⁺ CCR5⁺ targets (B). The means of three experiments plus standard error of the means are shown.

FIG. 6.

ΔV3-X4B determinants for CXCR4 use in cell-cell fusion. Fusion for the TA1 env containing ΔV3-X4B.c7 mutations alone and in combination with CD4⁺ CXCR4⁺(A) or CD4⁺ CCR5⁺ (C) QT6 cells and for the ΔV3-X4B.c7 env with mutations reverted back to the corresponding TA1 residues with CD4⁺ CXCR4⁺(B) or CD4⁺ CCR5⁺ (D) QT6 cells. Percent fusion was calculated by using luciferase activity normalized to ΔV3-X4B.c7 fusion with CD4⁺ CXCR4⁺ targets (A and B) or to TA1 fusion with CD4⁺ CCR5⁺ targets (C and D). The means of three experiments plus standard error of the means are shown.

FIG. 7.

Sensitivity to the CXCR4 antagonist AMD3100. (A) Inhibition of R3A, ΔV3-X4A.c5, and ΔV3-X4B.c7 by the indicated concentrations of AMD3100 in a cell-cell fusion assay. Percent fusion was calculated by using luciferase activity normalized to fusion with CD4⁺ CXCR4⁺ targets in the absence of drug for each env. The means of three experiments plus standard error of the means are shown. (B to D) Growth curves for recombinant NL4-3 viruses containing the R3A (B), ΔV3-X4A.c5 (C), or ΔV3-X4B.c7 (D) env in the presence of the indicated concentrations of AMD3100. RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown.

FIG. 8.

CXCR4 determinants for ΔV3-X4A and ΔV3-X4B fusion. (A) Use of CXCR4/CXCR2 coreceptor chimeras (21) in a cell-cell fusion assay for R3A, ΔV3-X4A.c5, and ΔV3-X4B.c7 env clones. The 4222 chimera contained a CXCR4 NT domain with the CXCR2 ECLs, and the 2444 chimera contained a CXCR2 NT with the CXCR4 ECLs. Percent fusion was calculated by using luciferase activity normalized to fusion with CD4⁺ CXCR4⁺ QT6 cells for each env. (B) Use of CXCR4 and 2444 in the absence or presence of 1,000 nM AMD3100 in a cell-cell fusion assay for R3A, ΔV3-X4A.c5, and ΔV3-X4B.c7 env clones. Percent fusion was calculated by using luciferase activity normalized to fusion on each coreceptor in the absence of drug. The means of three experiments plus standard error of the means are shown.