Transmitted Human Immunodeficiency Virus Type 1 Carrying the D67N or K219Q/E Mutation Evolves Rapidly to Zidovudine Resistance In Vitro and Shows a High Replicative Fitness in the Presence of Zidovudine

Abstract

Drug-naive patients infected with drug-resistant human immunodeficiency virus type 1 (HIV-1) who initiate antiretroviral therapy show a shorter time to virologic failure than patients infected with wild-type (WT) viruses. Resistance-related HIV genotypes not commonly seen in treated patients, which likely result from reversion or loss of primary resistance mutations, have also been recognized in drug-naive persons. Little work has been done to characterize the patterns of mutations in these viruses and the frequency of occurrence, their association with phenotypic resistance, and their effect on fitness and evolution of resistance. Through the analysis of resistance mutations in 1082 newly diagnosed antiretroviral-naive persons from the United States, we found that 35 of 48 (72.9%) persons infected with HIV-1 containing thymidine analog mutations (TAMs) had viruses that lacked a primary mutation (T215Y/F, K70R, or Q151M). Of these viruses, 9 (25.7%) had only secondary TAMs (D67N, K219Q, M41L, or F77L), and all were found to be sensitive to zidovudine (AZT) and other drugs. To assess the impact of secondary TAMs on the evolution of AZT resistance, we generated recombinant viruses from cloned plasma-derived reverse transcriptase sequences. Two viruses had D67N, three had D67N and K219Q/E, and three were WT. Four site-directed mutants with D67N, K219Q, K219E, and D67N/K219Q were also made in HIV-1 HXB2 . In vitro selection of AZT resistance showed that viruses with D67N and/or K219Q/E acquired AZT resistance mutations more rapidly than WT viruses (36 days compared to 54 days; P ‫؍‬ 0.003). To investigate the factors associated with the rapid selection of AZT mutations in these viruses, we evaluated fitness differences among HXB2 WT and HXB2 D67N or HXB2 D67N/K219Q in the presence of AZT. Both HXB2 D67N/K219Q and HXB2 D67N were more fit than HXB2 WT in the presence of either low or high AZT concentrations, likely reflecting low-level resistance to AZT that is not detectable by phenotypic testing. In the absence of AZT, the fitness cost conferred by D67N or K219Q was modest. Our results demonstrate that viruses with unique patterns of TAMs, including D67N and/or K219Q/E, are commonly found among newly diagnosed persons and illustrate the expanding diversity of revertant viruses in this population. The modest fitness cost conferred by D67N and K219Q supports persistence of these mutants in the untreated population and highlights the potential for secondary transmission. The faster evolution of these mutants toward AZT resistance is consistent with the higher viral fitness in the presence of AZT and shows that these viruses are phenotypically different from WT HIV-1. Our study emphasizes the need for clinical studies to better define the impact of these mutants on treatment responses and evolution of resistance.

Universal Amplification, Next-Generation Sequencing, and Assembly of HIV-1 Genomes

“…In contrast, HIV-1 strain 92UG037, which was included as a control, exhibited only minority D67N or K219Q/E mutations. These are secondary thymidine analogue mutations that occur in untreated populations (5). We expect that the costeffective monitoring of complex drug resistance genotypes will be most efficiently achieved by NGS of complete HIV-1 genomes, as current antivirals target the HIV-1 protease, reverse transcriptase, integrase, and envelope (gp41), and CCR5/CXCR4 tropism needs to be considered as well.…”

HIV Drug Resistance in Sub-Saharan Africa – Implications for Testing and Treatment

“…Drug toxicity is another major health concern (Carvajal-Rodriguez, et al , Larder, et al 1993, Larder, et al 1995. Although some drug escape mutants are impaired in replication fitness, others seem unaffected or may even have increased fitness (Armstrong, et al 2009, Garcia-Lerma, et al 2004, Prado, et al 2002. Similar to escape seen in natural selection, reversion to wild type virus occurs rapidly following treatment cessation.…”

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.…”