Genetic and functional analysis of R5X4 human immunodeficiency virus type 1 envelope glycoproteins derived from two individuals homozygous for the CCR5delta32 allele

Abstract

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) isolated from two HIV-1-infected CCR5delta32 homozygotes. Envs from both subjects used CCR5 and CXCR4 for entry into transfected cells. Most R5X4 Envs were lymphocyte-tropic and used CXCR4 exclusively for entry into peripheral blood mononuclear cells (PBMC), but a subset was dually lymphocyte- and macrophage-tropic and used either CCR5 or CXCR4 for entry into PBMC and monocyte-derived macrophages. The persistence of CCR5-using HIV-1 in two CCR5delta32 homozygotes suggests the conserved CCR5 binding domain of Env is highly stable and provides new mechanistic insights important for HIV-1 transmission and persistence.

FIG. 1.

Expression and functional activities of Env clones. (A) Western blot analysis of 293T cells cotransfected with 5 μg of pSVIIIenv plasmid and 1 μg of pSVTat using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following electrophoresis in 8.5% (wt/vol) SDS-PAGE gels. Western blot analysis of cell lysates was performed at 72 h posttransfection using rabbit anti-gp120 polyclonal antisera. pSVIIIenv plasmids containing ADA, HXB2, or 89.6 Env were included as controls. The data are representative of two independent experiments. (B) Fusion assays were conducted by coculturing 1 × 10⁶ TZM-bl cells that express CD4, CCR5, and CXCR4 on the cell surface and stably express the luciferase gene under the control of the HIV-1 long terminal repeat (9, 37, 45) with 1 × 10⁵ 293T cells cotransfected with each pSVIIIenv plasmid plus pSVTat for 8 h at 37°C in 500 μl of culture medium, followed by measurement of luciferase activity (Promega, Madison, WI) in cell lysates, as described previously (20, 34). HXB2, ADA, and 89.6 Envs were included as positive controls, and a nonfunctional Env, ΔKS (deltaKS), was included as a negative control to determine the background level of luciferase activity. The data are representative of two independent experiments and are expressed as means from duplicate experiments, and the error bars represent standard deviations. (C) Single-round entry assays were conducted by infection of Cf2th-CD4/CCR5/CXCR4 cells, which were constructed by transduction of the Cf2th-CD4/CCR5 cell line (47) with pBABE-puro vectors expressing CXCR4 (8, 31), followed by selection and expansion in culture medium containing 1 μg of puromycin per ml, with equivalent amounts of Env-pseudotyped luciferase reporter virus that was produced using pCMVΔP1ΔenvpA and pHIV-1Luc plasmids as described previously (46-48). Luciferase activity was measured in cell lysates at 72 h postinfection (Promega). Luciferase reporter viruses pseudotyped with HXB2, ADA, or 89.6 Env were included as positive controls, and luciferase reporter virus pseudotyped with ΔKS Env was included as a negative control to determine the background level of luciferase activity. The data are representative of two independent experiments and are expressed as means from duplicate infections, and the error bars represent standard deviations.

FIG. 2.

Tropism of Envs and coreceptor preference for HIV-1 entry into primary cells. (A) Single-round entry assays were conducted by infection of 0.5 × 10⁶ phytohemagglutinin-activated, interleukin 2-stimulated PBMC that were depleted of CD8⁺ cells with anti-CD8-conjugated magnetic beads (Invitrogen) or infection of confluent monolayers of MDM cultured for 5 days in 48-well tissue culture plates in medium containing 10% (vol/vol) human serum and 12.5 ng/ml macrophage colony-stimulating factor that were purified from PBMC by plastic adherence with equivalent amounts of Env-pseudotyped luciferase reporter virus for 3 h at 37°C. After washing the cells to remove the virus inoculum and replacement of culture medium, luciferase activity was measured in cell lysates at 72 h postinfection (Promega). Luciferase reporter viruses pseudotyped with HXB2, ADA, 89.6, or ΔKS Env were included as controls. The data are representative of two independent experiments using cells obtained from different donors and are expressed as means from duplicate infections, and the error bars represent standard deviations. CD8-depleted PBMC (B) or MDM (C) that were prepared as described above were left untreated or preincubated with 50 nM TAK-779 (1) and/or 1.2 μM AMD3100 (10, 40) for 1 h at 37°C prior to infection with equivalent amounts of Env-pseudotyped luciferase reporter viruses containing the same concentration(s) of inhibitor. Infections proceeded for 3 h at 37°C. After washing the cells to remove the virus inoculum and replacement of culture medium, luciferase activity was measured in cell lysates at 72 h postinfection (Promega). Luciferase reporter viruses pseudotyped with ADA, HXB2, or 89.6 Env were used as controls in CD8-depleted PBMC infections, and luciferase reporter viruses pseudotyped with ADA or 89.6 Env were used as controls in MDM infections. Luciferase measurements were normalized to the values obtained in untreated infections, which were set at 100. The data are representative of two independent experiments using cells obtained from different donors and are expressed as means from duplicate infections, and the error bars represent standard deviations.

FIG.3.

Env amino acid sequences. The gp120 amino acid sequences of Env clones were deduced from nucleotide sequences obtained by Big Dye terminator sequencing (Applied Biosystems, Foster City, CA). The amino acid alignments were compared to Env from HIV-1 89.6 and the clade B consensus sequence. The dots indicate residues identical to the clade B consensus sequence, and the dashes indicate gaps.

FIG.3.

Env amino acid sequences. The gp120 amino acid sequences of Env clones were deduced from nucleotide sequences obtained by Big Dye terminator sequencing (Applied Biosystems, Foster City, CA). The amino acid alignments were compared to Env from HIV-1 89.6 and the clade B consensus sequence. The dots indicate residues identical to the clade B consensus sequence, and the dashes indicate gaps.