Plasma viral RNA was purified from pelleted virus particles by centrifuging one milliliter of plasma at 20,000g × 60 min at 4 °C, removing 860 µl of cell-free supernatant and resuspending the pellet in the remaining 140 µl, to finally extract viral RNA using QIAamp Viral RNA Mini kit (Qiagen; Valencia, CA). Viral RNA was reverse-transcribed using AccuScript High Fidelity Reverse Transcriptase (Stratagene Agilent; Santa Clara, CA) and five previously described antisense external primers (Pan-HIV-1-1R [68 (link)], 1R, 2R, 3R, and 4R [69 (link)]) in 20 µl reaction mixtures containing 1 mM dNTPs, 10 mM DTT and 10 units of RNAse inhibitor. Six overlapping fragments, covering almost the entire HIV-1 genome, were amplified using a series of external and nested primers and Phusion High-Fidelity DNA Polymerase (NEB, Ipswich, MA) with defined cycling conditions as previously described [68 (link), 69 (link)], i.e., 5′LTR (675 bp; HXB2 coordinates 120 to 794), 5′LTR-gag/p7 (1269 bp; 658 to 1924), gag/p24-pol/RT (2327 bp; 1137 to 3463), pol/RT-vif (2259 bp; 2976 to 5234), pol/int-env/gp120 (2921 bp; 4602 to 7522), and env/gp120-3′LTR (2587 bp; 6858 to 9444).
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