FIG.4.
Combination of amino acid substitutions in viral protease selected with ritonavir on day 12 after infection. Protease sequences of selected HIV-1NG-PRL isolates in the absence (A) or presence (B) of 0.03, 0.10 (C), and 0.30 (D) μM ritonavir are shown. WT, wild type. For DNA sequencing, InstaGene (Bio-Rad Laboratories, Hercules, Calif.) was used for preparation of DNA from HIV-1-infected MT-2 cells. A 706-bp fragment containing a viral protease-encoding sequence was amplified by PCR with primers PR5 (5′-GTT AAG TGT TTC AAT TGT GGC AAA GAA GGG C-3′) and PR12 (5′-CTC TGT ACA AAT TTC TAC TAA TGC-3′). The PCR products, including protease-encoding sequences and the cleavage sites NC/p1, p1/p6gag, TFP/p6pol, and PR/RT (protease/reverse transcriptase), were purified and subcloned in pCRΔZ vectors as described in Fig. 1A.