Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

• Published in: Scientific reports Vol. 5; no. 1; p. 10323
• Main Authors: Zhu, Qinchang, Yu, Zhiqiang, Kabashima, Tsutomu, Yin, Sheng, Dragusha, Shpend, El-Mahdy, Ahmed F M, Ejupi, Valon, Shibata, Takayuki, Kai, Masaaki
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 19.05.2015
• Subjects: Anti-HIV Agents - pharmacology; Chromatography, High Pressure Liquid - methods; Drug development; Drug resistance; Drug Resistance, Viral - genetics; Fluorometry; High-performance liquid chromatography; HIV; HIV Infections - drug therapy; HIV Protease - genetics; HIV Protease Inhibitors - pharmacology; HIV-1 - drug effects; HIV-1 - genetics; Human immunodeficiency virus; Humans; Indinavir - pharmacology; Lopinavir - pharmacology; Microbial Sensitivity Tests - methods; Resistant mutant; Ritonavir - pharmacology; Saquinavir - pharmacology
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep10323

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance.