In coronavirus (CoV) infection, polyproteins (pp1a/pp1ab) are processed into non-structural proteins (nsps), which largely form the replication/transcription complex (RTC). The polyprotein processing and complex formation is critical and offers potential therapeutic targets. However, the interplay of polyprotein processing and RTC-assembly remains poorly understood. Here, we study two key aspects: The order of polyprotein processing by viral main protease Mpro and its influence on complex formation with the methyltransferase nsp16. Moreover, we establish an approach to determine rate constants k from cleavage sites in structured CoV polyprotein based on native mass spectrometry (MS). The high sensitivity and precision of our method allow quantification of multi-reaction kinetics of nsp7-11 processing from four human pathogenic CoV species. The experimentally determined rate constants are put into perspective with a comprehensive analysis of primary sequences and structural models, revealing distinct cleavage mechanisms for each site based on their local structural environments. Our systematic approach provides a blueprint for kinetic analysis of complex multi-cleavage reactions.
© 2025. The Author(s).