biostudies-literatureSchamoni-Kast KEuropean Research Council8244https://www.ebi.ac.uk/biostudies/studies/S-EPMC12420816biostudies-literatureUnknown16(1)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 M^pro 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 kineticsNature communicationsThe kinetics of nsp7-11 polyprotein processing and impact on complexation with nsp16 among human coronaviruses.PMC12420816759661Damjanovic TSaid FAUetrecht CSchamoni-Kast KToker SKrichel BKierspel TfalseThe kinetics of nsp7-11 polyprotein processing and impact on complexation with nsp16 among human coronaviruses.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 M^pro 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 kinetics2025-01-01T00:00:00Z2025 Sep2026-06-02T20:21:13.83Z2026-04-20T03:10:02.198ZS-EPMC124208164092590710.1038/s41467-025-61554-y