Functional correlates of insertion mutations in the protease gene of human immunodeficiency virus type 1 isolates from patients

Abstract

Twenty-four of over 24,000 patients genotyped over the past 3 years were found to have human immunodeficiency virus (HIV) isolates that possess an insert in the protease gene. In this report, we evaluated the spectrum of protease gene insertion mutations in patient isolates and analyzed the effect of these various insertion mutations on viral phenotypes. The inserts were composed of 1, 2, 5, or 6 amino acids that mapped at or between codons 35 and 38, 17 and 18, 21 and 25, or 95 and 96. Reduced susceptibility to protease inhibitors was found in isolates which possess previously reported drug resistance mutations. Fitness assays, including replication and competition experiments, showed that most of the isolates with inserts grew somewhat better than their counterparts with a deletion of the insert. These experiments demonstrate that, rarely, insertion mutations can develop in the HIV type 1 protease gene, are no more resistant than any other sequences which have similar associated resistance mutations, and can provide a borderline advantage in replication.

FIG. 1

Replication kinetics of HIV-1 recombinant isolates. One thousand TCID₅₀ of each virus was used per 10⁶ PHA-stimulated PBMCs. Virus production was monitored every day by p24 antigen assay. Culture supernatants were collected every day until day 7, and p24 antigen production was monitored by enzyme-linked immunosorbent assay. Data are the means of results of three different tests. To serve as a related protease inhibitor-resistant control, Q058 insertion (Ins) and Q058 deletion (Del) isolates were cultured with the IC₅₀ of each protease inhibitor (PI) and p24 values were measured daily for 7 days.

FIG. 2

Competitive HIV-1 replication assay of insertion (INS) and deletion (DEL) isolate pairs of Q781 with and without the insertion (a) and Q058 with and without the insertion (b). Data were generated based on relative peak heights in electropherograms produced from DNA sequencing of the HIV-1 genome. In the absence of protease inhibitors, insertion and deletion pairs were combined at three different ratios, 80:20, 50:50, and 20:80 based on TCID₅₀. In the presence of protease inhibitions, insertion and deletion isolates were coinfected at the same ratios and cultured in the presence of two different concentrations of three protease inhibitors (IDV, SQV, and NFV).