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<p>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<sup>pro</sup> and its influence on complex formation with the methyltransferase nsp16. Moreover, we establish an approach to determine rate constants <i>k</i> 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.</p>

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The kinetics of nsp7-11 polyprotein processing and impact on complexation with nsp16 among human coronaviruses

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月9日

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  • Kira Schamoni-Kast,
  • Boris Krichel,
  • Tomislav Damjanović,
  • Fatema-Aqila Said,
  • Thomas Kierspel,
  • Sibel Toker,
  • Charlotte Uetrecht

摘要

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.