Non-infectious fluorimetric assay for phenotyping of drug-resistant HIV proteinase mutants
Language English Country Netherlands Media print-electronic
Document type Comparative Study, Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't
PubMed
16527535
DOI
10.1016/j.jcv.2006.01.014
PII: S1386-6532(06)00043-6
Knihovny.cz E-resources
- MeSH
- Biological Assay methods MeSH
- Phenotype * MeSH
- Microscopy, Fluorescence MeSH
- Fluorometry MeSH
- HeLa Cells MeSH
- HIV Protease drug effects genetics metabolism MeSH
- Evaluation Studies as Topic MeSH
- HIV Protease Inhibitors pharmacology MeSH
- Kinetics MeSH
- Cloning, Molecular MeSH
- Humans MeSH
- Antibodies, Monoclonal metabolism MeSH
- Mutation * MeSH
- Plasmids MeSH
- Flow Cytometry MeSH
- Recombinant Fusion Proteins metabolism MeSH
- Genes, Reporter MeSH
- Spectrophotometry MeSH
- Transfection MeSH
- Drug Resistance, Viral MeSH
- Genes, Viral MeSH
- Dose-Response Relationship, Drug MeSH
- Blotting, Western MeSH
- Green Fluorescent Proteins metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Names of Substances
- HIV Protease MeSH
- HIV Protease Inhibitors MeSH
- Antibodies, Monoclonal MeSH
- Recombinant Fusion Proteins MeSH
- Green Fluorescent Proteins MeSH
BACKGROUND: The introduction of HIV proteinase inhibitors (PIs) as anti-AIDS drugs resulted in decreased mortality and prolonged life expectancy of HIV-positive patients. However, rapid selection of drug-resistant HIV variants is a common complication in patients undergoing highly active anti-retroviral therapy (HAART). Thus, monitoring of clinical resistance development is indispensable for rational pharmacotherapy. OBJECTIVE: We present a non-infectious cell-based assay for drug resistance quantification of HIV proteinase (PR) - an important target of HAART. STUDY DESIGN: Previously, we showed [Lindsten K, Uhlikova T, Konvalinka J, Masucci MG, Dantuma NP. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity. Antimicrob Agents Chemother 2001;45:2616-22] that the expression of a fusion protein (GFP-PR), comprised of HIV-1 proteinase wild-type artificial precursor (PR) and green fluorescent protein (GFP), in transiently transfected tissue culture cells depends on the presence of PR-specific inhibitors (PIs). Here we show that in the GFP-PR reporter the HIV wild-type PR can be replaced by a drug-resistant HIV PR mutant, yielding a simple and biologically relevant tool for the quantitative analysis of drug-resistant HIV PR mutants susceptibility to HIV proteinase inhibitors. RESULTS: We cloned a set of GFP-PR reporters, some of which possess a simple, well-defined drug-resistant PR mutant (G48V L90M, V82A, A71V V82T I84V, D30N, K45I); another four complex PR mutants were obtained from patients undergoing HAART. The results were compared with genotyping and enzyme kinetics data. Furthermore, we designed a single inhibitor concentration experiment setup for easy evaluation of drug resistance profiles for mutants of interest. The resistance profiles clearly demonstrate the importance of succession of individual drugs during the treatment for drug resistance development. CONCLUSION: We show that the GFP-PR assay might serve as a non-infectious, rapid, cheap, and reliable alternative to the currently used phenotypic assays.
References provided by Crossref.org
The zymogenic form of SARS-CoV-2 main protease: A discrete target for drug discovery
Viral proteases as therapeutic targets
Precursors of Viral Proteases as Distinct Drug Targets
Inhibition of the precursor and mature forms of HIV-1 protease as a tool for drug evaluation
