(A) Cf2-Luc cells were transfected with pcDNA3-CD4 alone or cotransfected with pcDNA3-CD4 and pcDNA3 expressing CCR2b, CCR3, CCR5, CCR8, CXCR4, Gpr1, Gpr15, Strl33 or Apj and infected with equivalent amounts of each control HIV-1 virus (left panel) or patient-derived virus (right panel). Control cells were transfected with pcDNA3 plasmid only. Mock-infected cells were treated with culture medium. Cell lysates were prepared at 48 h post-infection and assayed for luciferase activity. (B) Cf2-Luc cells transfected with pcDNA3-CD4 alone or cotransfected with pcDNA3-CD4 and plasmids expressing CCR5, CX3CR1, RDC1, CCR7, CXCR1, CXCR2, CXCR3, C5aRC9, D2DRsC9, DARC, D6 or FMLP-R were infected with equivalent amounts of each HIV-1 virus. HIV-1 entry was measured as above. Data are represented as means from duplicate infections. Error bars represent standard deviations. Similar results were obtained in two independent experiments.

Amino acid sequences were obtained from gp160 env genes cloned from genomic DNA of PBMC infected with the primary virus as described in Materials and Methods. Amino acid alignments of Envs from the patient virus are compared to the clade B consensus sequence. Dots indicate residues identical to the clade B consensus and dashes indicate gaps.

(A) V3 and C4 amino acid sequences of the Clade B consensus and patient Env clones were aligned with Clustal X. Dots represent amino acids identical to the Clade B consensus sequence. Residues important for CCR5 binding (Rizzuto et al., 1998) in the C4 region are indicated. Amino acid variants analyzed in mutagenesis studies are highlighted in gray. (B) HIV-1 luciferase reporter viruses pseudotyped with each Env were generated and used to infect Cf2th cells cotransfected with pcDNA3-CD4 and pcDNA3 expressing CCR5, CXCR4, Gpr1, Gpr15, Strl33, or D6. Control virus was produced by pseudotyping with ΔKS Env. Cell lysates were prepared at 60 h post-infection and assayed for luciferase activity. Data were normalized to parental Env-30 activity and are represented as means from two to three independent experiments, each performed in duplicate. Error bars represent standard error of the mean. *, p<0.05; **, p<0.01, student's t test. inset, 293T cells were cotransfected with 15 μg Env-expressing plasmids and 2 μg pLTR-Tat. At 72 h post-transfection, cell lysates were analyzed by Western blotting using goat anti-gp120. The positions of gp160 and gp120 are indicated on the right.

HIV-1 luciferase reporter viruses pseudotyped with each Env were generated and used to infect Cf2th cells cotransfected with pcDNA3-CD4 and pcDNA3 expressing CCR5(Y14N), CCR5(Δ18), or CCR5(G163R). Control virus was produced by pseudotyping with (ΔKS Env. Cell lysates were prepared at 60 h post-infection and assayed for luciferase activity. Results for each mutant CCR5 were normalized to levels of luciferase activity in cells expressing CCR5(wt) for each Env and are represented as means from two independent experiments, each performed in duplicate. Error bars represent standard deviations.

(A) PBMC, CEMx174 cells and MDM were infected with equivalent amounts of each HIV-1 virus, as described in Materials and Methods, and cultured for 28 days. Virus production in culture supernatants was measured by RT assays. (B) PBMC were treated with concentrations of CCR5 (TAK-779) or CXCR4 inhibitors (AMD3100), or both inhibitors increasing 10-fold from 0.01 to 100 μM, and infected with the patient virus in the presence of each concentration of inhibitor. Virus production in culture supernatants at day 14 post-infection was measured by quantitation of soluble HIV-1 p24 Ag. Production of p24 Ag was calculated as a percentage of the amount produced in the absence of inhibitors, and then expressed as the percentage of inhibition relative to cultures containing no inhibitor. (C) MDM were treated with TAK-779 (100 nM), AMD3100 (1.2 μM) or both for 1 h prior to infection. Untreated cells contained no inhibitor. Cells were infected with equivalent amounts of HIV-1 NDK or the patient isolate and cultured for 21 days in the presence of each inhibitor. HIV-1 production in culture supernatants was measured by RT assays. (D) Virus stocks were treated with concentrations of MAbs or CD4-IgG2 increasing 10-fold from 0.01 to 100 μM for 30 min, and used to infect PBMC. Virus production in culture supernatants and calculation of percent neutralization was determined as per (B). Values shown are means from duplicate infections. Error bars represent standard deviations (A, C). Results are representative of two independent experiments using cells obtained from different donors.

(A) 293T cells were cotransfected with 15 μg pCR3.1 Env-expressing plasmid (Envs 6, 12, 16 and 30) or pSVIII Env-expressing plasmid (ADA, HXB2 and 89.6 Envs) and 2 μg pLTR-Tat. At 72 h post-transfection, cell lysates were analyzed by Western blotting using rabbit anti-gp120. The positions of gp160 and gp120 are indicated on the right. (B) 293T effector cells transfected with Env and Tat as above were mixed with Cf2-Luc cells transfected with pcDNA3-CD4 only or cotransfected with pcDNA3-CD4 and pcDNA3 expressing CCR2b, CCR3, CCR5, CCR8, CXCR4, Gpr1, Gpr15, Strl33, Apj or D6 and incubated at 37°C for 12 h. Control 293T cells were transfected with ΔKS Env. Mock-transfected Cf2-Luc cells were transfected with pcDNA3 only. Cell lysates were then prepared and assayed for luciferase activity. (C) HIV-1 luciferase reporter viruses pseudotyped with each Env were generated and used to infect Cf2th cells transfected with pcDNA3-CD4 only or cotransfected with pcDNA3-CD4 and pcDNA3 expressing CCR2b, CCR3, CCR5, CCR8, CXCR4, Gpr1, Gpr15, Strl33, Apj or D6. Control virus was produced by pseudotyping with a non-functional Env (ΔKS Env). Cell lysates were prepared at 60 h post-infection and assayed for luciferase activity. Data are represented as means from duplicate wells in one experiment. Error bars represent standard deviations. Results are representative of two independent experiments, each performed in duplicate.

Swiss PDB viewer was used to model amino acid changes at positions 308, 317, and 321 on the V3 region of the JRFL gp120-CD4-X5 CD4i Ab crystal structure (2B4C) (Huang et al., 2005). JRFL V3 has a single amino acid change relative to the clade B consensus (N301Q). Red, position 308. Gray, position 317. Cyan, position 321. (A) V3 region of JRFL with H308, F317, and G321. (B) A Y308 change in JRFL results in steric clashing with F317 (dotted lines). (C) Changes Q301N, K305R, S306G, Y308H, F317I, G321D, E322K, I324V, Q328K, H330Y, and N332T were introduced in JRFL to produce a model of the V3 region of Env-30. Positions Y308, I317, and D321 are indicated.