{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T11:01:37Z","timestamp":1765278097412,"version":"3.46.0"},"reference-count":60,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T00:00:00Z","timestamp":1765238400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ruth and Fred Kunnes Fund"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Protein\u2013protein interactions (PPIs) are fundamental to viral replication, regulating transcription, assembly, and genome packaging. Despite their biological importance, few FDA-approved therapeutics directly target these complexes. The split luciferase complementation assay (SLCA) is a quantitative bioluminescence system to measure protein\u2013protein interactions in vitro after the proteins in question have been fused in-frame to N and C luciferase fragments. The SLCA can be performed both in vitro using purified protein components and in live cells, as the luciferase substrate luciferin is cell-permeable, allowing detection of protein interactions in intact cells. Assay performance, however, depends on the expression level and stability of the fusion proteins used. SLCA has been successfully applied to target Rev\u2013Rev interactions in human immunodeficiency virus type 1 (HIV-1) for high-throughput small-molecule screening, establishing a proof-of-concept to target other parts of the viral life cycle. The system can be extended to other pathogens that currently do not have specific antiviral therapies such as HIV-1 Tat\u2013cyclin T1, Capsid dimerization in Dengue virus, capsid interactions in equine encephalitis viruses, capsid assembly in Epstein\u2013Barr virus, and nucleoprotein oligomerization in rabies virus. These applications demonstrate how the assay\u2019s ability to quantify multimeric structural interactions is essential to viral replication, providing an avenue to identify small-molecule inhibitors that prevent viral replication and spread. Although there are challenges to protein stability and assay optimization, the sensitivity and adaptability of the SLCA has broader implications in virology to accelerate antiviral drug development.<\/jats:p>","DOI":"10.3390\/biom15121712","type":"journal-article","created":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T09:02:50Z","timestamp":1765270970000},"page":"1712","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Use of the Split Luciferase Complementation Assay to Identify Novel Small Molecules That Disrupt Essential Protein\u2013Protein Interactions of Viruses"],"prefix":"10.3390","volume":"15","author":[{"given":"Tisa","family":"Biswas","sequence":"first","affiliation":[{"name":"Section of Infectious Diseases, Department of Internal Medicine, Yale University, New Haven, CT 06520, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard E.","family":"Sutton","sequence":"additional","affiliation":[{"name":"Section of Infectious Diseases, Department of Internal Medicine, Yale University, New Haven, CT 06520, USA"},{"name":"Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT 06520, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Ulgheri, F.M., Bernardes, B.G., and Lancellotti, M. 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