Use of the Split Luciferase Complementation Assay to Identify Novel Small Molecules That Disrupt Essential Protein–Protein Interactions of Viruses

Protein-protein 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-protein 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-Rev 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-cyclin T1, Capsid dimerization in Dengue virus, capsid interactions in equine encephalitis viruses, capsid assembly in Epstein-Barr virus, and nucleoprotein oligomerization in rabies virus. These applications demonstrate how the assay's 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.