Application of restriction enzyme analysis to screen drug-resistant mutations in HIV-protease and in proteolytic cleavage sites in the gag region

Several mutations have been identified in HIV-1 isolates that are resistant to protease inhibitors. While some are directly involved in drug-resistance, others are compensatory changes which confer growth advantage to these mutant viruses. The amino acid substitutions at positions 82 and 90 that occur in many protease drug resistant isolates also cause the loss of two HindII restriction sites and these compositional changes provide a basis to rapidly screen for mutations at these sites. Using other restriction enzymes, mutations at positions 8, 10, 32, 46, 50, 71, 75, 84, 88 and 93 can also be detected by this method. While two important amino acid substitutions at positions 48 and 54 do not overlap with restriction enzyme sites, in the present study we show that by utilizing appropriate PCR primers to introduce new restriction enzyme sites, we can also screen isolates for substitutions at these positions. We have reported (Abst. Zhang et al.) that drug-resistant HIV variants contain mutations in the protease cleavage sites in gag in addition to amino acid substitutions in the protease gene. We have analyzed the changes in the p7/p1 and p1/p6 cleavage sites using MseI and Eco57I enzymes. In one indinavir treated patient, cleavage site mutations were detected as early as 6 weeks. The method for detection of mutations that is based on changes in the restriction enzyme sites offers a sensitive way to rapidly screen for virus isolates containing a wide variety of drug-resistant mutations.

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