title: Novel method for simultaneous quantification of phenotypic resistance to maturation, protease, reverse transcriptase, and integrase HIV inhibitors based on 3'Gag(p2/p7/p1/p6)/PR/RT/INT-recombinant viruses: a useful tool in the multitarget era of antiretroviral therapy.
abstract: Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458-467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens.
authors.affiliation: Diagnostic Hybrids, Inc., Cleveland, Ohio, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Weber
authors.forename: Ana C
authors.initials: AC
authors.lastname: Vazquez
authors.forename: Dane
authors.initials: D
authors.lastname: Winner
authors.forename: Justine D
authors.initials: JD
authors.lastname: Rose
authors.forename: Doug
authors.initials: D
authors.lastname: Wylie
authors.forename: Ariel M
authors.initials: AM
authors.lastname: Rhea
authors.forename: Kenneth
authors.initials: K
authors.lastname: Henry
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pappas
authors.forename: Alison
authors.initials: A
authors.lastname: Wright
authors.forename: Nizar
authors.initials: N
authors.lastname: Mohamed
authors.forename: Richard
authors.initials: R
authors.lastname: Gibson
authors.forename: Benigno
authors.initials: B
authors.lastname: Rodriguez
authors.forename: Vicente
authors.initials: V
authors.lastname: Soriano
authors.forename: Kevin
authors.initials: K
authors.lastname: King
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Arts
authors.forename: Paul D
authors.initials: PD
authors.lastname: Olivo
authors.forename: Miguel E
authors.initials: ME
authors.lastname: Quiñones-Mateu
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D001483:Base Sequence; D024921:Drug Resistance, Multiple, Viral; D015751:Genes, pol; D057809:HEK293 Cells; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D019428:HIV Integrase Inhibitors / Q000494:pharmacology; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D010641:Phenotype; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA
chemical_list: D019380:Anti-HIV Agents; D019428:HIV Integrase Inhibitors; D017320:HIV Protease Inhibitors; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H