title: Performance of a clonal-based HIV-1 tropism phenotypic assay.
abstract: Adequate determination of HIV-1 tropism is important in clinical and research settings. Genotypic and phenotypic approaches to evaluate tropism have been described. Phenotypic assays are widely used to determine HIV-1 tropism because of their sensitivity to detect minor CXCR4-using variants (X4). However they cannot differentiate mixed quasi-species of R5 and X4 viruses from dual-tropic viruses. We describe here a clonal-based HIV-1 tropism phenotypic assay. Env-pseudo-typed viruses were produced by co-transfection of the env expression plasmid pcDNA3.1/V5HisTOPO and a backbone vector pNL4-3.Luc.E-R- that expresses the entire HIV-1 genome except for env and vpr in 293T cell cultures. Co-receptor use was tested by infecting U87.CD4.CCR5+ and U87.CD4.CXCR4+ cells in the presence or absence of co-receptor inhibitors, using 10 clones from each sample. The ability of the assay to detect minor variants in a viral population was assessed by mixing X4 and R5 clones using different ratios. Both R5 and X4 minority variants were detected when present at greater than 0.4% in a mixture of envelope populations. This assay can be useful in both clinical and research laboratories.
authors.affiliation: Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada.
authors.forename: Odalis
authors.initials: O
authors.lastname: Asin-Milan
authors.affiliation: Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Sylla
authors.affiliation: Department of Pharmacology, McGill University, Canada.
authors.forename: Farida
authors.initials: F
authors.lastname: Vaisheva
authors.affiliation: Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada.
authors.forename: Annie
authors.initials: A
authors.lastname: Chamberland
authors.affiliation: Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montréal, Canada; Department of Microbiology, Infectiology and Immunology, Faculty of Medicine, University of Montreal, Canada; Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Canada. Electronic address: c.tremblay@umontreal.ca.
authors.forename: Cécile L
authors.initials: CL
authors.lastname: Tremblay
mesh_terms: D002460:Cell Line; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D012680:Sensitivity and Specificity; D056189:Viral Tropism; D014773:Virology / Q000379:methods*
keywords: CCR5; CXCR4; Coreceptor usage; HIV; Luciferase assay; Pseudovirus; Tropism; Viral tropism