DWV 3C Protease Uncovers the Diverse Catalytic Triad in Insect RNA Viruses

Deformed wing virus (DWV) is the most prevalent Iflavirus that is infecting honey bees worldwide. However, the mechanisms of its infection and replication in host cells are poorly understood. In this study, we analyzed the structure and function of DWV 3C protease (3C), which is necessary for the cleavage of the polyprotein to synthesize mature viral proteins. Thus, it is one of the nonstructural viral proteins essential for the replication. We found that the 3Cs of DWV and picornaviruses share common enzymatic properties, including sensitivity to the same inhibitors, such as rupintrivir. The predicted structure of DWV 3C by AlphaFold2, the predicted rupintrivir binding domain, and the protease activities of mutant proteins revealed that it has a Cys-His-Asn catalytic triad. Moreover, 3Cs of other Iflaviruses and Dicistrovirus appear to contain Asn, Ser, Asp, or Glu as the third residue of the catalytic triad, suggesting diversity in insect RNA viruses. Both precursor 3C with RNA-dependent RNA polymerase and mature 3C are present in DWV-infected cells, suggesting that they may have different enzymatic properties and functions. DWV 3C is the first 3C characterized among insect RNA viruses, and our study uncovered both the common and unique characteristics among 3Cs of . Furthermore, it would be possible to use the specific inhibitors of DWV 3C to control DWV infection in honey bees in future. The number of managed honey bee (Apis mellifera) colonies has considerably declined in many developed countries in the recent years. Deformed wing virus (DWV) vectored by the mites is the major threat to honey bee colonies and health. To give insight into the mechanism of DWV replication in the host cells, we studied the structure-function relationship of 3C protease (3C), which is necessary to cleave a viral polyprotein at the specific sites to produce the mature proteins. We found that the overall structure, some inhibitors, and processing of 3C are shared between ; however, there is diversity in the catalytic triad. DWV 3C is the first viral protease characterized among insect RNA viruses and reveals the evolutionary history of 3C among . Furthermore, DWV 3C inhibitors identified in our study could also be applied to control DWV in honey bees in future.