Phenotypic analysis of the sensitivity of HIV-1 to inhibitors of the reverse transcriptase, protease, and integrase using a self-inactivating virus vector system

• Published in: Journal of medical virology 2001-07, Vol.64 (3), p.223-231
• Main Authors: Jármy, Gergely, Heinkelein, Martin, Weissbrich, Benedikt, Jassoy, Christian, Rethwilm, Axel
• Format: Article
• Language: English
• Subjects: Anti-HIV Agents - pharmacology; antiviral treatment; APR gene; ART gene; Biological and medical sciences; Cell Line; Dose-Response Relationship, Drug; Fundamental and applied biological sciences. Psychology; Genetic Vectors; HIV Infections - drug therapy; HIV Infections - virology; HIV Integrase Inhibitors - pharmacology; HIV Protease Inhibitors - pharmacology; HIV Reverse Transcriptase - antagonists & inhibitors; HIV-1 - drug effects; HIV-1 - enzymology; HIV-1 - genetics; Human immunodeficiency virus 1; Humans; integrase inhibitor; Integrase inhibitors; lentiviral vector; Microbial Sensitivity Tests - methods; Microbiology; Mutation; nucleoside analogues; Phenotype; protease inhibitor; Reproducibility of Results; reverse transcriptase inhibitor; Reverse Transcriptase Inhibitors - pharmacology; RNA-directed DNA polymerase inhibitors (non-nucleoside); Sensitivity and Specificity; Transduction, Genetic; Virus Replication - drug effects; Virus Replication - genetics
• ISSN: 0146-6615; 1096-9071
• DOI: 10.1002/jmv.1040

Conventional phenotypic analysis of resistance of the human immunodeficiency virus (HIV) to antiviral therapy is time‐consuming and requires culture of infectious virus. Although phenotypic analyses may be desirable, rapid generation of test results and decentralized availability of the test system will be important to achieve utility in the clinical practice. This study describes the design of an alternative phenotypic resistance test using replication incompetent viral vectors. Chimeric HIV vectors containing a marker gene were generated. The env and most of the regulatory and accessory genes of HIV were removed. In addition, the 3′U3 region was deleted to obtain a self‐inactivating construct. Cotransfection of the plasmid with a plasmid that provided the vesicular stomatitis virus glycoprotein resulted in the production of replication‐incompetent virus vectors. Infection of susceptible cells with the vectors led to marker gene expression. Vector production in the presence of protease (PR) inhibitors, or infection in the presence of reverse transcriptase (RT) or integrase (IN) inhibitors reduced marker gene expression in a dose‐dependent manner. Marker gene activity was preserved at higher drug levels if vectors contained RT and PR genes from resistant virus isolates. Sensitivity to nucleoside and non‐nucleoside RT inhibitors, protease and integrase inhibitors could be determined in 10 working days. The phenotypic drug resistance test using replication‐incompetent HIV vectors significantly speeds up drug resistance measurements and allows testing at reduced biosafety levels. This will make clinical use of phenotypic assessment of antiviral resistance more feasible. J. Med. Virol. 64:223–231, 2001. © 2001 Wiley‐Liss, Inc.