Revealing the mechanism of nucleotide selection, addition and proofreading of the SARS-coronavirus replication transcription complex at the single molecule level

• Applicant: David Dulin, Ph.D.
• Subject Area: Virology ; Biophysics
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 447835095

• Final Report Year: 2024

Final Report Abstract

One of the key antiviral targets against coronaviruses (CoVs), such as SARS-CoV-1 and SARS-CoV-2, is their replication-transcription complex (RTC), a large complex made of several non-structural proteins (nsp) encoded in the coronavirus genome that synthesizes all the viral RNA in the infected cell. The RTC is made of core, constituted of nsp12-polymerase with its associated co-factors nsp7 and nsp8 in a 1:1:2 stoichiometry, to which associates e.g. nsp9, the nsp13-helicase, the exonuclease (ExoN)-methyltransferase nsp14. When this proposal was submitted, in February 2020, little was known about the structure and the functional role of each factor composing the CoV RTC. The goal of this proposal was to determine the nucleotide addition cycle of the core RTC, the mechanism of action of different nucleotide analogs on the RTC elongation dynamics, and to decipher how nsp14-ExoN assists the RTC in proofreading, which protects CoV replication in infected cell against many existing approved nucleotide analogs. Structural advances in the knowledge related to the complex assembly after the proposal submission directed my research towards the RTC assembly and revealing the role of nsp13-helicase during viral RNA synthesis.

Publications

• Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective. eLife, 10.
  Seifert, Mona; Bera, Subhas C.; van Nies, Pauline; Kirchdoerfer, Robert N.; Shannon, Ashleigh; Le, Thi-Tuyet-Nhung; Meng, Xiangzhi; Xia, Hongjie; Wood, James M.; Harris, Lawrence D.; Papini, Flavia S.; Arnold, Jamie J.; Almo, Steven; Grove, Tyler L.; Shi, Pei-Yong; Xiang, Yan; Canard, Bruno; Depken, Martin; Cameron, Craig E. & Dulin, David
  
• The nucleotide addition cycle of the SARS-CoV-2 polymerase. Cell Reports, 36(9), 109650.
  Bera, Subhas Chandra; Seifert, Mona; Kirchdoerfer, Robert N.; van Nies, Pauline; Wubulikasimu, Yibulayin; Quack, Salina; Papini, Flávia S.; Arnold, Jamie J.; Canard, Bruno; Cameron, Craig E.; Depken, Martin & Dulin, David
  
• Interfering with nucleotide excision by the coronavirus 3′-to-5′ exoribonuclease. Nucleic Acids Research, 51(1), 315-336.
  Chinthapatla, Rukesh; Sotoudegan, Mohamad; Srivastava, Pankaj; Anderson, Thomas K.; Moustafa, Ibrahim M.; Passow, Kellan T.; Kennelly, Samantha A.; Moorthy, Ramkumar; Dulin, David; Feng, Joy Y.; Harki, Daniel A.; Kirchdoerfer, Robert N.; Cameron, Craig E. & Arnold, Jamie J.
  
• Mechanism of assembly of an elongation-competent SARS-CoV-2 replication transcription complex, Biophysical Society conference abstract, Volume 122, ISSUE 3, SUPPLEMENT 1, 171a, February 10, 2023
  Klein M. et al.
  
• Temperature-Dependent Twist of Double-Stranded RNA Probed by Magnetic Tweezer Experiments and Molecular Dynamics Simulations. The Journal of Physical Chemistry B, 128(3), 664-675.
  Dohnalová, Hana; Seifert, Mona; Matoušková, Eva; Klein, Misha; Papini, Flávia S.; Lipfert, Jan; Dulin, David & Lankaš, Filip