Expression of functional Env clones. 293T cells were cotransfected with 8 μg of pSVIII-Env plasmid expressing control R5 Envs (A) or pSVIII-Env plasmid expressing functional Envs cloned from the cross sectional (PA-R5 viruses NB23, NB24, NB25, NB27 and A-R5 viruses NB2, NB6, NB7, NB8) (B) or longitudinal (PA- and A-R5 viruses from subject IK1) (C) primary R5 HIV-1 isolates and 2 μg pSVL-Tat, as described in the Methods. Env expression at 72 h post-transfection was measured by Western blot analysis of cell lysates using rabbit anti-gp120 polyclonal antisera. Positions of gp160 and gp120 are shown on the right. C1, C2, C3 and C4 refer to independent Envs cloned from each virus.