Engineering viral protease-operated nanobodies for programmable and orthogonal control of protein function

Precise control of protein function in living cells is essential for engineering programmable biological systems and therapeutic applications. However, most current strategies act indirectly by altering protein stability, localization, or proximity rather than directly modulating binding activity. Here we present VIPbodies, nanobodies engineered with self-cleaving viral proteases that convert protease inhibition into drug-dependent antigen recognition. Using anti-mCherry nanobodies as prototypes, we create variants responsive to orthogonal viral protease–inhibitor pairs and extend the design to diverse nanobody scaffolds. Each VIPbody functions independently within the same cell, enabling multiplexed regulation of distinct targets. When incorporated into transcriptional circuits, VIPbodies mediate drug-tunable gene expression and execute all six canonical Boolean logic operations, providing a compact framework for programmable cellular computation. Beyond gene regulation, VIPbody circuits mediate bidirectional control of pyroptosis and selectively activate apoptotic or pyroptotic programs via caspase coupling, thereby enabling chemogenetic control of protein function and cell fate. Precise control of protein function in living cells is essential for engineering programmable biological systems. Here the authors present VIPbodies, nanobodies engineered with self-cleaving viral proteases that convert protease inhibition into drug-dependent antigen recognition.