Journal Article

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

Maria José Buzon, Itziar Erkizia, Christian Pou, Gerard Minuesa, Maria Carmen Puertas, Anna Esteve, Alfredo Castello, Jose Ramón Santos, Julia G. Prado, Nuria Izquierdo-Useros, Theresa Pattery, Margriet Van Houtte, Luis Carrasco, Bonaventura Clotet, Lidia Ruiz and Javier Martinez-Picado

in Journal of Antimicrobial Chemotherapy

Published on behalf of British Society for Antimicrobial Chemotherapy

Volume 67, issue 1, pages 32-38
Published in print January 2012 | ISSN: 0305-7453
Published online October 2011 | e-ISSN: 1460-2091 | DOI:
A non-infectious cell-based phenotypic assay for the assessment of HIV-1 susceptibility to protease inhibitors

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  • Medical Oncology
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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.


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.


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).


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.

Keywords: drug resistance; protease inhibitors; phenotypic susceptibility; eIF4GI; PABP

Journal Article.  4156 words.  Illustrated.

Subjects: Medical Oncology ; Critical Care

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