Journal Article

FRET-based assay to screen inhibitors of HIV-1 reverse transcriptase and nucleocapsid protein

Kamal K. Sharma, Frédéric Przybilla, Tobias Restle, Julien Godet and Yves Mély

in Nucleic Acids Research

Volume 44, issue 8, pages e74-e74
Published in print May 2016 | ISSN: 0305-1048
Published online January 2016 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gkv1532

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During HIV-1 reverse transcription, the single-stranded RNA genome is converted into proviral double stranded DNA by Reverse Transcriptase (RT) within a reverse transcription complex composed of the genomic RNA and a number of HIV-1 encoded proteins, including the nucleocapsid protein NCp7. Here, we developed a one-step and one-pot RT polymerization assay. In this in vitro assay, RT polymerization is monitored in real-time by Förster resonance energy transfer (FRET) using a commercially available doubly-labeled primer/template DNA. The assay can monitor and quantify RT polymerization activity as well as its promotion by NCp7. Z-factor values as high as 0.89 were obtained, indicating that the assay is suitable for high-throughput drug screening. Using Nevirapine and AZT as prototypical RT inhibitors, reliable IC50 values were obtained from the changes in the RT polymerization kinetics. Interestingly, the assay can also detect NCp7 inhibitors, making it suitable for high-throughput screening of drugs targeting RT, NCp7 or simultaneously, both proteins.

Journal Article.  6566 words.  Illustrated.

Subjects: Enzymology ; Molecular Biology and Genetics

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