HOW DOES THE PROMISCUOUS NUCLEOCAPSID PROTEIN EXERT SPECIFIC ACTIONS?

The N protein of human beta coronaviruses (hCoV) is responsible for nucleocapsid assembly and other essential regulatory functions. Its N-terminal domain (N-NTD) interacts and melts the double-stranded transcriptional regulatory sequences (dsTRS), regulating the discontinuous subgenome transcription process, an essential feature for the virus cycle. There is little structural information on the specific binding of dsTRS and single-stranded RNA (TRS+ and TRS-) to the N-NTD. Here, we use NMR and molecular dynamics (MD) simulations to study the binding of SARS-CoV-2, and other hCoV N-NTDs to TRS nucleic acid. We will present the structure of hCoV-HKU1 N-NTD and the chemical shift mapping of positive sense TRS (TRS+), negative sense TRS- and dsTRS binding to SARS-CoV-2 and hCoV-HKU1 N-NTD. We also measured the melting activity of the proteins and described the thermodynamics of binding. We produced experimental-based structural models to understand the specific features of the binding. For the SARS-CoV-2 N-NTD/RNA complex, we ran 25 replicas of 100 ns MD simulations that suggested a mechanism for the destabilization of dsRNAs’ Watson and Crick (WC) base-pairing based on a tweezer-like motion of the N-NTD. We will also show the results of virtual and NMR-based screening of potential drug candidates. These results serve as the basis for the development of drug therapy using the inhibition of the regulatory machinery of subgenome transcription of hCoVs.
Acknowledgments: FAPERJ, CNPq, Covid-19 NMR Consortium, and Rio BioNMR Network