title: A non-infectious cell-based phenotypic assay for the assessment of HIV-1 susceptibility to protease inhibitors.
abstract: OBJECTIVE
  HIV-1 genotyping is widely accepted as a diagnostic tool to optimize therapy changes in patients whose antiretroviral regimen is failing. Phenotyping can substantially complement the information obtained from genotyping, especially in the presence of complex mutational patterns. However, drug susceptibility tests are laborious and require biosafety facilities. We describe the molecular mechanism of a non-infectious HIV-1 protease phenotypic assay in eukaryotic cells and validate its applicability as a tool for monitoring drug resistance.
  
  
  METHODS
  A cloning vector containing the fusion protein green fluorescent protein-HIV-1 protease (GFP-PR) was modified to facilitate the insertion of HIV-1 protease from infected subjects. Real-time quantitative PCR and western blot analysis were used to establish the molecular mechanism of the new phenotypic assay. The method was validated by analysing HIV-1 protease from 46 clinical isolates. Statistical comparisons were made between values obtained using our assay and those reported from alternative standardized phenotypic assays.
  
  
  RESULTS
  The capacity of HIV-1 protease to cleave cellular translation factors, such as the eukaryotic translation initiation factor 4 (eIF4GI) and the poly(A)-binding protein (PABP), led to cyclical accumulation of GFP that varied with the dose of protease inhibitors. Validation and comparison revealed a significant correlation with the Virco TYPE HIV-1 test (P < 0.0001, Spearman's ρ = 0.60), the Antivirogram test (P = 0.0001, Spearman's ρ = 0.60) and the Stanford HIVdb (P < 0.0001, Spearman's ρ = 0.69).
  
  
  CONCLUSIONS
  This cell-based non-infectious phenotypic method with a well-understood molecular mechanism was highly reliable and comparable to other widely used assays. The method can be used for both phenotyping of HIV-1 viral isolates resistant to protease inhibitors and screening of new protease inhibitors.
authors: Institut de Recerca de SIDA (IrsiCaixa), Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain.
authors: Maria José
authors: MJ
authors: Buzon
authors: Itziar
authors: I
authors: Erkizia
authors: Christian
authors: C
authors: Pou
authors: Gerard
authors: G
authors: Minuesa
authors: Maria Carmen
authors: MC
authors: Puertas
authors: Anna
authors: A
authors: Esteve
authors: Alfredo
authors: A
authors: Castello
authors: Jose Ramón
authors: JR
authors: Santos
authors: Julia G
authors: JG
authors: Prado
authors: Nuria
authors: N
authors: Izquierdo-Useros
authors: Theresa
authors: T
authors: Pattery
authors: Margriet
authors: M
authors: Van Houtte
authors: Luis
authors: L
authors: Carrasco
authors: Bonaventura
authors: B
authors: Clotet
authors: Lidia
authors: L
authors: Ruiz
authors: Javier
authors: J
authors: Martinez-Picado
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1