Sequencing-based detection of low-frequency human immunodeficiency virus type 1 drug-resistant mutants by an RNA/DNA heteroduplex generator-tracking assay

Abstract

Drug-resistant viruses may be present as minority variants during early treatment failures or following discontinuation of failed antiretroviral regimens. A limitation of the traditional direct PCR population sequencing method is its inability to detect human immunodeficiency virus type 1 (HIV-1) variants present at frequencies lower than 20%. A drug resistance genotyping assay based on the isolation and DNA sequencing of minority HIV protease variants is presented here. A multiple-codon-specific heteroduplex generator probe was constructed to improve the separation of HIV protease genes varying in sequence at 12 codons associated with resistance to protease inhibitors. Using an RNA molecule as probe allowed the simple sequencing of protease variants isolated as RNA/DNA heteroduplexes with different electrophoretic mobilities. The protease gene RNA heteroduplex generator-tracking assay (RNA-HTA) was tested on plasma quasispecies from 21 HIV-1-infected persons in whom one or more protease resistance mutations emerged during therapy or following initiation of salvage regimens. In 11 of 21 cases, RNA-HTA testing of virus from the first episode of virologic failure identified protease resistance mutations not seen by population-based PCR sequencing. In 8 of these 11 cases, all of the low-frequency drug resistance mutations detected exclusively by RNA-HTA during the first episode became detectable by population-based PCR sequencing at the later time point. Distinct sets of protease mutations could be linked on different genomes in patients with high-frequency protease gene lineages. The enhanced detection of minority drug resistance variants using a sequencing-based assay may improve the efficacy of genotype-assisted salvage therapies.

FIG. 1.

RNA-HTA pro1 probe design and its use with protease variants. (A) Mutations engineered in the protease subtype B consensus sequence. Targeted codons are shown in capital letters. Six primary and six accessory drug resistance mutations were targeted by nucleotide insertion and substitutions, respectively (underlined). (B) RNA-HTA using protease variants from three different isolates (pNL, NIHRF, and PCPXM variants). The protease variants used in each lane correspond to the sequence numbers in panel C. The gel is shown from the loading wells to the DNA homoduplexes. (C) Alignment of the subtype B consensus, UHG-pro1 probe, and protease variants of pNL, NIHRF, and PCPXM used in panel B. Drug resistance codons targeted by the UHG-pro1 probe are underlined.

FIG. 2.

Detection of low-frequency variants using RNA-HTA. (A) Mixtures of two protease variant PCRs (PCPXM002 and PCPXM003) were separated by RNA-HTA, and gel pieces (plugs) were taken from the gel. Plug holes are visible in the gel. (B) RNA-HTA of the PCR product generated from the gel plugs.

FIG. 3.

Viral load and ARV drug history of 21 patients analyzed by RNA-HTA. The baseline and follow-up time points are shown with vertical lines. ARV drug history is presented in each box.

FIG. 4.

RNA-HTA of clinical samples. Patient identifications are similar to those in Fig. 3. (A) RNA-HTA of samples collected at baseline and follow-up are labeled −01 and −02, respectively. (B) RNA-HTA of samples from patient baseline only. (C) RNA-HTA analysis of the PCR products derived from RNA-HTA gel pieces. A clonal PCR target (PCPXM010) and RNA probe-alone lanes are included.

FIG. 5.

Comparison of direct PCR population sequencing and RNA-HTA-enhanced sequencing. Representative electropherograms show the detection of drug resistance mutations (arrows) in PCR products derived from RNA-HTA-derived gel plugs, while such mutations were not detected in the directly sequenced PCR product (population sequence).

FIG. 6.

Mutation linkage analysis and quasispecies sampling reproducibility. (A) Analysis of samples with distinct RNA-HTA bands. RNA/DNA heteroduplex bands labeled by a white dot were separately purified and sequenced. (B) Independently generated PCR products were analyzed by RNA-HTA in neighboring gel lanes to test for reproducible viral population sampling.