PT  - JOURNAL ARTICLE
AU  - Ícaro P. Caruso
AU  - Karoline Sanches
AU  - Andrea T. Da Poian
AU  - Anderson S. Pinheiro
AU  - Fabio C. L. Almeida
TI  - Dynamics of the N-terminal domain of SARS-CoV-2 nucleocapsid protein drives dsRNA melting in a counterintuitive tweezer-like mechanism
AID  - 10.1101/2020.08.24.264465
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.08.24.264465
4099  - http://biorxiv.org/content/early/2020/09/06/2020.08.24.264465.short
4100  - http://biorxiv.org/content/early/2020/09/06/2020.08.24.264465.full
AB  - The N protein of betacoronaviruses is responsible for nucleocapsid assembly and other essential regulatory functions. Its N-terminal domain (NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process. Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs’ Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, initiating melting. N-NTD dsRNA destabilizing activity was more efficient for dsTRS than dsNS. N-NTD dynamics, especially a tweezer-like motion of β2-β3 and 2-β5 loops, played a key role in WC base-pairing destabilization. Based on experimental information available in the literature, we constructed kinetics models for N-NTD-mediated dsRNA melting. Our results support a 1:1 stoichiometry (N-NTD:dsRNA), matching MD simulations and raising different possibilities for N-NTD action: (i) two N-NTDs of dimeric N would act independently, increasing efficiency; (ii) two N-NTDs of dimeric N would bind to two different RNA sites, bridging distant regions of the genome; and (iii) monomeric N would be active, opening up the possibility of a regulatory dissociation event.IMPORTANCE Coronaviruses are among the largest positive-sense RNA viruses. They display a unique discontinous transcription mechanism, involving N protein as a major player. The N-NTD promote the dsRNA melting releasing the nascent sense negative strand via a poorly known mechanism of action. It specifically recognizes the body TRS conserved RNA motif located at the 5’ end of each ORF. N protein has the ability to transfer the nascent RNA strand to the leader TRS. The mechanism is essential and one single mutation at the RNA binding site of the N-NTD impairs the viral replication. Here, we describe a counterintuitive mechanism of action of N-NTD based on molecular dynamics simulation and kinetic modelling of the experimental melting activity of N-NTD. This data impacts directly in the understanding of the way N protein acts in the cell and will guide future experiments.Competing Interest StatementThe authors have declared no competing interest.