Fluorescent HIV-1 integrases for a suite of new user-friendly stability, nucleic acid binding and strand transfer activity assays
Bourquin, Rebecca A., Sorenson, Alanna E., and Schaeffer, Patrick M. (2025) Fluorescent HIV-1 integrases for a suite of new user-friendly stability, nucleic acid binding and strand transfer activity assays. International Journal of Biological Macromolecules, 309 (3). 142859.
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Abstract
Human immunodeficiency virus (HIV) infects CD4+ T-cells, causing acquired immunodeficiency syndrome. Despite advances in antiretroviral therapy, drug resistance remains a critical issue. HIV integrase is a key therapeutic target. Resistance to integrase strand transfer inhibitors requires development of new drugs with distinct mechanisms. Integrases tethered with GFP (IN-GFP) and mCherry (IN-mCherry) were evaluated for the development of a comprehensive suite of user-friendly assays. A new fluorescent protein-based stability assay (FP-Basta) effectively assessed protein thermal stability, revealing aggregation midpoints of 45.0 °C for IN-GFP and 45.4 °C for IN-mCherry. FP-Basta showed that IN-mCherry was stabilized by a target DNA and viral LTR, confirming protein-DNA interactions. A new qPCR-based integrase activity assay demonstrated robust detection of strand transfer activity, with a ∼21,500-fold sensitivity over background. Manganese ions were essential, enhancing integrase activity 56-fold compared to magnesium ions, while Zn2+ impaired functionality. The integrase activity assay can distinguish 3′-processing and strand transfer activities and was validated for inhibitor screening. The combination of FP-Basta and qPCR-based integrase activity assay provides a comprehensive, cost-effective platform for evaluating IN function and inhibitor efficacy. These tools, leveraging GFP- and mCherry-tagged IN, offer potential for future high-throughput applications in HIV drug discovery and the development of therapies addressing resistance challenges.
| Item ID: | 89121 |
|---|---|
| Item Type: | Article (Research - C1) |
| ISSN: | 1879-0003 |
| Keywords: | DNA binding,GFP,HIV-1,Integrase activity,mCherry,qPCR,Thermal stability assay |
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| Copyright Information: | © 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
| Date Deposited: | 14 Oct 2025 02:05 |
| FoR Codes: | 31 BIOLOGICAL SCIENCES > 3101 Biochemistry and cell biology > 310109 Proteomics and intermolecular interactions (excl. medical proteomics) @ 100% |
| SEO Codes: | 20 HEALTH > 2001 Clinical health > 200105 Treatment of human diseases and conditions @ 100% |
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