Human immunodeficiency virus type 1 protease inhibitor drug-resistant mutants give discordant results when compared in single-cycle and multiple-cycle fitness assays.

Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Journal of clinical microbiology (Impact Factor: 4.23). 11/2010; 48(11):4035-43. DOI: 10.1128/JCM.00605-10
Source: PubMed

ABSTRACT The replication fitness of HIV-1 drug-resistant mutants has been measured using either multiple-cycle or single-cycle assays (MCAs or SCAs); these assays have not been systematically compared. We developed an MCA and an SCA that utilized either intact or env-deleted recombinant viral vectors, respectively, in which virus-infected cells were detected by flow cytometry of a reporter gene product. Fitness was measured using each assay for 11 protease mutants, 9 reverse transcriptase mutants, and two mutants with mutations in gag p6, which is important for the release of virus particles from the cell membrane. In the SCA, fitness (replication capacity [RC]) was defined as the proportion of cells infected by the mutant compared to the wild type 40 h after infection. MCA fitness (1+s) was determined by comparing the changes in the relative proportions of cells infected by the mutant and the wild type between 3 and 5 days after infection. Five protease mutants showed statistically different fitness values by the MCA versus the SCA: the D30N, G48V, I50V, I54L, and I54M mutants. When all the mutants were ranked in order from most to least fit for both assays, 4 protease mutants moved more than 5 positions in rank: the D30N, I54L, I54M, and V82A mutants. There were no significant differences in fitness for the gag p6 or reverse transcriptase mutants. We propose that discordant results in the MCA and SCA are due to alterations in late events in the virus life cycle that are not captured in an SCA, such as burst size, cell-to-cell transmission, or infected-cell life span.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method. RESULTS: The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations, which restored replication fitness to CTL escape mutants, were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but T247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region. CONCLUSIONS: These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.
    Retrovirology 10/2012; 9(1):89. · 4.77 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E+G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E+G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E+G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E+G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E+Y188L and K101E+V106I virus, but not K101E+L100I, K101E+K103N, K101E+Y181C, or K101E+G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.
    Journal of Virology 08/2011; 85(20):10861-73. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To examine whether baseline clinical genotypes are equivalent to diagnostic serum genotypes for surveillance of HIV transmitted drug resistance (TDR). Current HIV TDR surveillance in Canada is conducted through genotyping remnant diagnostic sera from new HIV diagnoses. As part of routine care, baseline genotyping is now conducted on all newly diagnosed HIV infections, with TDR data being generated a second time on the same patients. Surveillance genotyping, on HIV diagnostic serum, was performed on newly diagnosed HIV cases from 2007 to 2010 in Alberta, Canada. All subjects with a baseline clinical genotype result on file, and no evidence of antiretroviral therapy, were studied further. The HIV sequences from diagnosis and from the first clinical genotype were compared according to elapsed time between testing and by evaluating timing of infection based on BED capture enzyme immunoassay (BED-CEIA, abbreviated as BED in this article). Eighty-seven genotype pairs were available for analysis, most of which were subtype B. The time between genotypes ranged from 0 to 755 days, with a median of 36 days and an interquartile range of 155.25 days. Genetic distance between genotypes varied between 0 and 0.03389 substitutions per site and did not correlate with sampling times. There was a tendency for the genotypes of infections classified as recent by BED to be more similar to their clinical genotypes but this effect was lost when adjusted for elapsed time between tests. There was no difference in the identified drug resistance. Baseline clinical genotypes from treatment-naive patients may be used for HIV TDR surveillance.
    JAIDS Journal of Acquired Immune Deficiency Syndromes 12/2013; 64(5):443-447. · 4.39 Impact Factor

Full-text (2 Sources)

Available from
Jun 2, 2014