Discovery of an Antiviral Electron Transfer Process to Create Catalytically Self‐Sufficient Viral Restriction Factors

Engineering immune-silent, catalytically self-sufficient antiviral restriction factor enzymes (iCAREs) provides a proof-of-concept for developing autonomous enzymes for future antiviral applications. To rationally design these enzymes, we sought to identify and engineer components of the intrinsic immune system. Here, we identified the endoplasmic reticulum (ER)-anchored cytochrome b5 reductase 3 (CYB5R3) as a putative electron donor to the ER-anchored and interferon-stimulated antiviral radical S-adenosylmethionine (SAM)-dependent nucleotide dehydratase (SAND), or RSAD2 (viperin) in humans. We demonstrate that their functional partnership depends on co-localisation. We obtained insights into the structure of the RSAD2:CYB5R3 complex and, using mutagenesis, identified the structural elements required for electron transfer. Based on this discovery, we engineered iCAREs that autonomously generate an antiviral nucleotide analogue to chain-terminate viral RNA polymerase activity. Because iCAREs are self-sufficient and do not require recruitment of endogenous redox partners, they are predicted to avoid interference with numerous biological processes associated with single-domain redox partners such as CYB5R3, including electron delivery to the oncogenic enzyme stearoyl-CoA desaturase (SCD1). This work solves a long-standing question of why ER localisation is critical for RSAD2 activity, resolves a key mechanistic question in radical-SAM enzymology, and establishes a general strategy to engineer self-sufficient radical-SAM enzymes for biotechnological applications.