HIV-1 protease codon 36 polymorphisms and differential development of resistance to nelfinavir, lopinavir, and atazanavir in different HIV-1 subtypes

Abstract

The amino acid at position 36 of the HIV-1 protease differs among various viral subtypes, in that methionine is usually found in subtype B viruses but isoleucine is common in other subtypes. This polymorphism is associated with higher rates of treatment failure involving protease inhibitors (PIs) in non-subtype B-infected patients. To investigate this, we generated genetically homogeneous wild-type viruses from subtype B, subtype C, and CRF02_AG full-length molecular clones and showed that subtype C and CRF02_AG I36 viruses exhibited higher levels of resistance to various PIs than their respective M36 counterparts, while the opposite was observed for subtype B viruses. Selections for resistance with each variant were performed with nelfinavir (NFV), lopinavir (LPV), and atazanavir (ATV). Sequence analysis of the protease gene at week 35 revealed that the major NFV resistance mutation D30N emerged in NFV-selected subtype B viruses and in I36 subtype C viruses, despite polymorphic variation. A unique mutational pattern developed in subtype C M36 viruses selected with NFV or ATV. The presence of I47A in LPV-selected I36 CRF02_AG virus conferred higher-level resistance than L76V in LPV-selected M36 CRF02_AG virus. Phenotypic analysis revealed a >1,000-fold increase in NFV resistance in I36 subtype C NFV-selected virus with no apparent impact on viral replication capacity. Thus, the position 36 polymorphism in the HIV-1 protease appears to have a differential effect on both drug susceptibility and the viral replication capacity, depending on both the viral subtype and the drug being evaluated.

FIG. 1.

Alignment of deduced amino acid sequences of protease from parental HIV-1. Shown are the predicted protease amino acid sequences from the six different parental HIV-1 variants generated from subtype-specific full-length molecular clones: subtype B (B 36M and B 36I), CRF02_AG (AG 36M and AG 36I), and subtype C (C 36M and C 36I). Protease amino acid sequences were ultimately determined by RT-PCR amplification of genomic HIV-1 RNA isolated from transfected 293T cell supernatants and the subsequent sequencing of the amplification products. Data analyses and alignments were completed using SeqScape and BioEdit software, respectively. Vertical dashes and numbers indicate amino acid position. Dots indicate identical residues using subtype B (B 36M) as the reference sequence.

FIG. 2.

Replication capacity of parental HIV-1 variants compared to that of drug-selected progeny HIV-1. The results for parental isolates and subtype B, subtype C, and CRF02_AG HIV-1 isolates selected with NFV (a, b, and c, respectively), LPV (d, e, and f, respectively), and ATV (g, h, and i, respectively) are shown. Percent replication of preselected (---) and drug-selected (—) M36 (▴) and I36 (•) HIV-1 in TZM-bl cells are expressed as a function of HIV-1 RT activity (cpm). Data points depict the averages (mean ± standard deviation) of three independent experiments performed in duplicate.

FIG. 3.

Growth of subtype B M/I36 HIV-1 in the presence of drug. CBMCs were infected and cultured over 35 weeks with subtype B M36 (a to c) or I36 (d to f) HIV-1 in the presence of NFV (a and d), LPV (b and e), or ATV (c and f). Drug (- - × - -) concentrations (μM for NFV and LPV and nM for ATV, right vertical axis) were adjusted every week, according to virus replication (- - ○ - -), measured as RT activity (cpm, left vertical axis) in the cell culture supernatants. Data represent means ± standard deviations (n = 3).

FIG. 4.

Growth of M/I36 subtype C HIV-1 in the presence of drug. CBMCs were infected and cultured over 35 weeks with M36 subtype C (a to c) or I36 (d to f) HIV-1 in the presence of NFV (a and d), LPV (b and e), or ATV (c and f). Drug (- - × - -) concentrations (μM for NFV and LPV and nM for ATV, right vertical axis) were adjusted every week, according to virus replication (- - ○ - -), measured as RT activity (cpm, left vertical axis) in the cell culture supernatants. Data represent means ± standard deviations (n = 3).

FIG. 5.

Growth of CRF02_AG M/I36 HIV-1 in the presence of drug. CBMCs were infected and cultured over 35 weeks with CRF02_AG M36 (a to c) or I36 (d to f) HIV-1 in the presence of NFV (a and d), LPV (b and e), or ATV (c and f). Drug (- - × - -) concentrations (μM for NFV and LPV and nM for ATV, right vertical axis) were adjusted every week according to virus replication (- - ○ - -) measured as RT activity (cpm, left vertical axis) in the cell culture supernatants. Data represent means ± standard deviations (n = 3).