Fluorescent HIV-1 integrases for a suite of new user-friendly stability, nucleic acid binding and strand transfer activity assays

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.