English   español  
Por favor, use este identificador para citar o enlazar a este item: http://hdl.handle.net/10261/111471
Título

A non-infectious cell-based phenotypic assay for the assessment of HIV-1 susceptibility to protease inhibitors

AutorBuzón, María José; Castelló, Alfredo; Martínez-Picado, Javier
Palabras claveDrug resistance
PABP
Phenotypic susceptibility
eIF4GI
Protease inhibitors
Fecha de publicación2012
EditorOxford University Press
CitaciónJournal of Antimicrobial Chemotherapy 67: 32- 38 (2012)
ResumenObjectives: 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 andvalidate 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. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
URIhttp://hdl.handle.net/10261/111471
DOI10.1093/jac/dkr433
Identificadoresdoi: 10.1093/jac/dkr433
issn: 0305-7453
Aparece en las colecciones: (CBM) Artículos
Ficheros en este ítem:
Fichero Descripción Tamaño Formato  
accesoRestringido.pdf15,38 kBAdobe PDFVista previa
Visualizar/Abrir
Mostrar el registro completo
 

Artículos relacionados:


NOTA: Los ítems de Digital.CSIC están protegidos por copyright, con todos los derechos reservados, a menos que se indique lo contrario.