ABSTRACT
The SARS-CoV-2 nucleocapsid protein (N) is a multifunctional promiscuous nucleic acid-binding protein, which plays a major role in nucleocapsid assembly and discontinuous RNA transcription, facilitating the template switch of transcriptional regulatory sequences (TRS). Here, we dissect the structural features of the N protein N-terminal domain (N-NTD) and N-NTD plus the SR-rich motif (N-NTD-SR) upon binding to single and double-stranded TRS DNA, as well as their activities for dsTRS melting and TRS-induced liquid-liquid phase separation (LLPS). Our study gives insights on the specificity for N-NTD(-SR) interaction with TRS. We observed an approximation of the triple-thymidine (TTT) motif of the TRS to ß-sheet II, giving rise to an orientation difference of ~25° between dsTRS and non-specific sequence (dsNS). It led to a local unfavorable energetic contribution that might trigger the melting activity. The thermodynamic parameters of binding of ssTRSs and dsTRS suggested that the duplex dissociation of the dsTRS in the binding cleft is entropically favorable. We showed a preference for TRS in the formation of liquid condensates when compared to NS. Moreover, our results on DNA binding may serve as a starting point for the design of inhibitors, including aptamers, against N, a possible therapeutic target essential for the virus infectivity.
Subject(s)
COVID-19/virology , Nucleic Acids/metabolism , Nucleocapsid Proteins/metabolism , Protein Interaction Domains and Motifs , SARS-CoV-2/physiology , Binding Sites , DNA/chemistry , DNA/metabolism , Gene Expression Regulation, Viral , Host-Pathogen Interactions , Humans , Hydrogen Bonding , Models, Molecular , Nucleic Acids/chemistry , Nucleocapsid Proteins/chemistry , Protein Binding , RNA/chemistry , RNA/metabolism , Spectrum Analysis , Structure-Activity RelationshipABSTRACT
Coronaviruses have become of great medical and scientific interest because of the Covid-19 pandemic. The hCoV-HKU1 is an endemic betacoronavirus that causes mild respiratory symptoms, although the infection can progress to severe lung disease and death. During viral replication, a discontinuous transcription of the genome takes place, producing the subgenomic messenger RNAs. The nucleocapsid protein (N) plays a pivotal role in the regulation of this process, acting as an RNA chaperone and participating in the nucleocapsid assembly. The isolated N-terminal domain of protein N (N-NTD) specifically binds to the transcriptional regulatory sequences and control the melting of the double-stranded RNA. Here, we report the resonance assignments of the N-NTD of HKU1-CoV.