Rapid phenotypic assay for human immunodeficiency virus type 1 protease using in vitro translation.
A rapid in vitro phenotyping method for human immunodeficiency virus type 1 (HIV-1) protease was developed. In this system, both HIV-1 protease and substrates are prepared using a rabbit reticulocyte based coupled in vitro transcription/translation system. The activity of protease is evaluated by the amount of cleaved substrate measured by ELISA. In this system, wild-type protease derived from strain HXB2 was specifically inhibited in a dose-dependent manner by the protease inhibitors, indinavir and nelfinavir. Three drug-resistant proteases carrying a single mutation, D30N, L90M, and V82F, were analyzed in the absence of the inhibitors. Reflecting their impaired fitness, they exhibited decreased protease activity compared with the wild type. The apparent protease activity was greater for a Gag-Pol substrate encompassing the Gag-protease-reverse transcriptase junctions than for a substrate only covering the Gag region. Using the Gag-Pol substrate as the target, the indinavir-resistant mutant V82F was evaluated further. V82F showed 9-fold resistance to its cognitive protease inhibitor, indinavir; however, it manifested only moderate (2-fold) resistance to a non-cognitive inhibitor, nelfinavir. This simple and rapid method may be useful for phenotyping of drug-resistant HIV-1 protease as well as for screening new inhibitors of HIV-1 protease.
D000818:Animals; D024882:Drug Resistance, Viral; D004797:Enzyme-Linked Immunosorbent Assay; D015685:Fusion Proteins, gag-pol / Q000378:metabolism; D016333:HIV Protease / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D019469:Indinavir / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D008826:Microbial Sensitivity Tests; D019888:Nelfinavir / Q000494:pharmacology*; D010641:Phenotype; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes; D013997:Time Factors; D014158:Transcription, Genetic
D015685:Fusion Proteins, gag-pol; D017320:HIV Protease Inhibitors; D019469:Indinavir; D016333:HIV Protease; D019888:Nelfinavir