title: T20-insensitive HIV-1 from naive patients exhibits high viral fitness in a novel dual-color competition assay on primary cells.
abstract: The relationship between sensitivity to antiviral drugs and viral fitness is of paramount importance in understanding the long-term implications of clinical resistance. Here we report the development of a novel recombinant virus assay to study entry inhibitor-resistant HIV variants using a biologically relevant cell type, primary CD4 T-cells. We have modified the replication-competent molecular clone HIV(NL4-3) to express a reporter protein (Renilla luciferase), Green Fluorescent Protein (EGFP), or Red Fluorescent Protein (DsRed2) upon infection, thus allowing quantification of replication. Luciferase-expressing virus was used to evaluate drug sensitivity, while co-infection with viruses carrying the green and red fluorescent proteins was employed in the competitive fitness assay. Using envelope proteins from three T20 insensitive variants, lower levels of resistance were observed in primary CD4 T-cells than had been previously reported for cell lines. Importantly, dual-color competition assays demonstrated comparable or higher fitness for these variants despite their reduced T20 sensitivity. We conclude that reduced sensitivity to T20 is compatible with high viral fitness in the absence of selection pressure. Thus, simultaneously measuring both resistance and viral fitness using this newly described dual-color competition assay will likely provide important information about resistant viral variants that emerge during therapy with entry inhibitors.
authors.affiliation: Department of Virology, Hygiene-Institute, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Neumann
authors.forename: Isabel
authors.initials: I
authors.lastname: Hagmann
authors.forename: Sabine
authors.initials: S
authors.lastname: Lohrengel
authors.forename: Marintha L
authors.initials: ML
authors.lastname: Heil
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Derdeyn
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Kräusslich
authors.forename: Matthias T
authors.initials: MT
authors.lastname: Dittmar
mesh_terms: D000595:Amino Acid Sequence; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000821:virology; D002460:Cell Line; D024882:Drug Resistance, Viral; D000077560:Enfuvirtide; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000494:pharmacology*; D023581:HIV Fusion Inhibitors / Q000494:pharmacology*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000302:isolation & purification / Q000472:pathogenicity / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008826:Microbial Sensitivity Tests / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D014779:Virus Replication / Q000187:drug effects
chemical_list: D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D010446:Peptide Fragments; D000077560:Enfuvirtide