title: Agreement between an in-house replication competent and a reference replication defective recombinant virus assay for measuring phenotypic resistance to HIV-1 protease, reverse transcriptase, and integrase inhibitors.
abstract: BACKGROUND
  Although clinical management of drug resistance is routinely based on genotypic methods, phenotypic assays remain necessary for the characterization of novel HIV-1 inhibitors, particularly against common drug-resistant variants. We describe the development and assessment of the performance of a recombinant virus assay for measuring HIV-1 susceptibility to protease (PR), reverse transcriptase (RT), and integrase (IN) inhibitors.
  
  
  METHODS
  The system is based on the creation of replication-competent chimeric viruses through homologous recombination between patient or laboratory virus-derived PCR fragments and the corresponding NL4-3 vector where the whole Gag-PR, RT-RNaseH or IN coding regions has been deleted through inverse PCR. The susceptibility to nucleoside (NRTIs) and non-nucleoside (NNRTIs) RT inhibitors and to IN inhibitors (INIs) is calculated through a single-round infection assay in TZM-bl cells, while protease inhibitor (PI) activity is determined through a first round of infection in MT-2 cells followed by infection of TZM-bl cells with MT-2 supernatants.
  
  
  RESULTS
  The assay showed excellent reproducibility and accuracy when testing PI, NRTI, NNRTI, and INI susceptibility of drug-resistant clones previously characterized through the reference pseudoparticle-based Phenosense assay. The coefficient of interassay variation in fold change (FC) resistance was 12.0%-24.3% when assaying seven drug/clones pairs in three runs. FC values calculated by the Phenosense and in-house for 20 drug/clones pairs were in good agreement, with mean±SD ratio of 1.14±0.33 and no cases differing by more than twofold.
  
  
  CONCLUSIONS
  The described phenotypic assay can be adopted to evaluate the antiviral activity of licensed and investigational HIV-1 drugs targeting any of the three HIV-1 enzymes.
authors.affiliation: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Francesco
authors.identifier: http://orcid.org/0000-0002-9934-377X
authors.initials: F
authors.lastname: Saladini
authors.affiliation: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Giannini
authors.affiliation: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Boccuto
authors.affiliation: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Vicenti
authors.affiliation: Department of Medical Biotechnologies, University of Siena, Siena, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Zazzi
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000145:classification / Q000187:drug effects*; D006801:Humans; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D015203:Reproducibility of Results; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*
chemical_list: D019380:Anti-HIV Agents; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors
keywords: HIV-1; HIV-1 inhibitors; drug resistance; phenotypic assay