Conditions for single genome PCR amplification of full-length gp160 (plus Rev, Vpu, and partial Nef coding sequences) from the genomic DNA of uncultured peripheral blood mononuclear cells and cDNA from plasma have been described previously [38, 44] . The viral env amplicons were directionally T/A cloned into the CMV-driven expression plasmid pcDNA3.1-V5HisTOPO-TA and screened for biological function as pseudoviruses following co-transfection with an Env-deficient subtype B proviral plasmid (SG3Denv) into 293T cells [45] . Seventy-two hours later, supernatant was collected and used to infect JC53-BL13 (Tzmbl) cells. At 48 hours post-infection, b-gal staining was performed and each well was scored positive or negative for blue foci.