Replication-dependent 65R→K reversion in human immunodeficiency virus type 1 reverse transcriptase double mutant K65R + L74V

Abstract

Understanding of the mechanisms of interaction among nucleoside reverse transcriptase inhibitor (NRTI)-selected mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) coding sequence is essential for the design of newer drugs and for enhancing our vision of the structure function relationship among amino acids of the polymerase domain of HIV-1. Although several nucleoside reverse transcriptase inhibitors select RT mutations K65R and L74V, the combination of 65R + 74V is rare in clinics. A novel NRTI (-)-beta-d-dioxolane-guanosine (DXG) is known to select in vitro either the 65R or 74V mutant virus. These mutations were not selected together during repeated passaging of the HIV-1 in the presence of this drug. To analyze the impact of these RT mutations on viral replication, a double mutant containing K65R + L74V was created by site-directed mutagenesis in a pNL4-3 background. Replication kinetic assays revealed that the mutant K65R + L74V is unstable, and 65R-->K reversion occurs during replication of virus in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication kinetic assays in MT-2 cells demonstrated that double mutant 65R + 74V is highly attenuated for replication and the initiation of reversion is related to the increase in RT activity. Additionally, the suppression of viral replication in the presence of DXG or under suboptimal human recombinant interleukin-2 leads to minimal or no 65R-->K reversion. These observations provide evidence that 65R-->K reversion in the double mutant 65R + 74V is dependent on a specific rate of viral replication in a pNL4-3 background. A similar phenomenon may occur in vivo, which may have implications for treatment management strategies.

Evaluation of a Multiple-Cycle, Recombinant Virus, Growth Competition Assay That Uses Flow Cytometry To Measure Replication Efficiency of Human Immunodeficiency Virus Type 1 in Cell Culture

“…In contrast, similar relative replication efficiencies were observed in cell lines and PBMC for some non-nucleoside inhibitor-and enfuvirtide-resistant HIV variants (15,23). Another potential problem that has been observed by others is the rapid reversion of a mutant over the course of a replication assay (37). To date, we have not observed reversion using direct sequence analysis (data not shown), but this could occur, particularly with poorly fit mutants, such as those studied by Sharma et al (37).…”

“…Another potential problem that has been observed by others is the rapid reversion of a mutant over the course of a replication assay (37). To date, we have not observed reversion using direct sequence analysis (data not shown), but this could occur, particularly with poorly fit mutants, such as those studied by Sharma et al (37). In addition, our assay is a recombinant virus assay, reflecting only determinants of replication efficiency that are present in the PR and RT regions of the viral genome.…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…Some examples of measuring virus directly include quantitation of a viral protein, such as p24, by enzyme-linked immunosorbent assay (106,156,163); measurement of reverse transcriptase activity (164, 175); and quantitation of proviral DNA (116). In addition, in growth competition assays, the relative amounts of test and reference strains can be quantified by sequence analysis of proviral DNA or viral RNA (65,69,78,86,117,154,155,169,174), heteroduplex tracking assays (HTAs) (140), or allele-specific real-time PCR (33,142,176). It should be noted that the latter methods do not provide information on the extent of viral replication.…”

“…Sequence analysis has also been used to quantify the relative amounts of the two variants in a growth competition assay either by direct sequence analysis of the bulk PCR product (65,69,78,86,117,154,155,174) or by analysis of individual clones derived from the PCR amplicon (119). One disadvantage of bulk sequencing is that differences in the surrounding sequence may influence the assay's sensitivity for detecting a minority variant at a given codon.…”

“…An assay used by several laboratories is a multiple-cycle recombinant-virus growth competition assay in which the relative proportion of test and reference strains is measured using either bulk or clonal sequence analysis (4,65,69,78,86,117,119,154,155,174). This type of assay is labor-intensive and difficult to apply to large numbers of clinical samples but has been commonly used to characterize the relative fitness of site-directed drug-resistant mutants of HIV-1 in protease and reverse transcriptase.…”