Project C06 (associated)

Principal Investigators

Prof. Dr. Charlotte Uetrecht

CSSB – Center for Structural Systems Biology
& Universität zu Lübeck

C06

PhD candidate

Fatema-Aqila Said

C06

Project Summary

Revealing SARS-CoV-2 replication-transcription super-complex (RTC) architecture and dynamics via native MS

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiated a global pandemic in 2019 due to its high pathogenicity causing acute respiratory illness [1]. The biochemical driver of viral replication is an enzymatic network consisting of the coronaviral non-structural proteins nsp1-16, the majority of which organize into the replication-transcription complex (RTC) [2,3]. While high resolution structures of the nsps including a minimal RTC were experimentally determined or computationally modelled, the construct of the proposed super-complex remains unknown [3]. This also holds for the impact of posttranslational modifications (PTM) on dynamic regulation and functional switch of the RTC between replication and transcription [4]. The aim is assembling a full RTC from bacterially expressed nsp7-16 and comparing to an RTC assembled from eukaryotic expressed proteins using native MS. Thus, the overall set of PTMs will be revealed. The impact of individual PTMs will be probed by mutagenesis. All RTC assemblies will be probed with and without RNA triggering distinct functional states and monitor stochiometric changes. Lastly, different coronaviral RTCs will be compared to identify conserved PTMs, regulatory and functional mechanisms. The project will contribute to the identification of new pan-coronaviral drug targets that have the potential to also address yet unknown coronavirus species.

References

  1. Krichel B, Bylapudi G, Schmidt C, et al. Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes. Science Advances. 2021;7(10):eabf1004. doi: 10.1126/sciadv.abf1004
  2. Perry JK, Appleby TC, Bilello JP, et al. An atomistic model of the coronavirus replication-transcription complex as a hexamer assembled around nsp15. Journal of Biological Chemistry. 2021;297(4):101218. doi: 10.1016/j.jbc.2021.101218 
  3. Krichel B, Falke S, Hilgenfeld R, et al. Processing of the SARS-CoV pp1a/ab nsp7–10 region. Biochemical Journal. 2020;477(5):1009-1019. doi: 10.1042/BCJ20200029
  4. Fung TS, Liu DX. Post-translational modifications of coronavirus proteins: roles and function. Future Virol. 2018;13(6):405-430. doi: 10.2217/fvl-2018-0008