Figure 4. Expression and function of full-length Env clones in cell-cell fusion and infection assays.

(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.