Evolution of split-protein technologies in virology: From mechanistic discovery and diagnostics to therapeutic promise

The persistent challenge posed by viruses such as HIV, HBV, and SARS-CoV-2 necessitates the continuous evolution of molecular tools for their study and for advancing therapeutic research. Split-protein complementation assays (PCAs), where a reporter protein is divided into two inactive fragments, have evolved from simple reporters of biological events into an increasingly important tool in modern virology. This review traces the evolutionary trajectory of split-protein systems. We begin with their foundational use in mechanistic discovery, where they first visualized viral-host interactions in living cells. We then explore their translation into practical applications, such as high-throughput drug screening and rapid point-of-care diagnostics. A step in this evolution was the development of systematic engineering platforms, dramatically accelerating the creation of novel biosensors. Finally, we discuss the latest frontier: engineering therapeutically active "split effectors." By integrating principles from synthetic biology, these advanced systems can function as programmable logic gates that respond to specific viral signatures. While therapeutic translation remains preclinical, split-protein platforms are emerging as tangible tools for advanced research and potential therapeutic development.