C-terminal domain of SARS-CoV-2 nucleocapsid protein binds transcriptional regulatory sequence RNA with high affinity and forms higher-order assemblies
Year: 2026
Type: preprint
Abstract: The SARS-CoV-2 nucleocapsid (N) protein is a 419-amino-acid modular protein essential for viral RNA replication, genome packaging, and host-cell modulation. It contains two structured domains, the N-terminal RNA-binding domain (NTD) and C-terminal dimerization domain (CTD), separated and flanked by three intrinsically disordered regions. Although the CTD mediates N-protein dimerization and contributes to RNA binding, the molecular basis of its interaction with RNA remains poorly understood. Here, we characterized CTD binding to transcriptional regulatory sequence (TRS) RNAs from the SARS-CoV-2 genome using NMR spectroscopy, isothermal titration calorimetry, size-exclusion chromatography, and in vitro phase-separation assays. The CTD bound structured stem-loop TRS RNA with higher affinity than the canonical RNA-binding NTD. NMR chemical-shift perturbation experiments indicated formation of higher-order molecular assemblies upon titration of the CTD with longer, structured stem-loop TRS RNA, consistent with size-exclusion chromatography results. The CTD also underwent RNA-induced phase separation in vitro , with stem-loop TRS RNA promoting markedly more robust condensation than single-stranded RNA sequences. Together, these findings establish the CTD as a high-affinity, structure-selective RNA-binding domain with a preference for stem-loop TRS RNA. The results further show that CTD-TRS interactions promote higher-order assembly and phase separation, providing mechanistic insight into N-protein-mediated viral genome packaging and nucleocapsid assembly.
Authors: Parul Gupta, Mahavir Singh
Institution: Indian Institute of Science Bangalore
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
Funders: Arthritis National Research Foundation, Indian Institute of Science, National Research Foundation, Department of Biotechnology, Ministry of Science and Technology, India, Department of Science and Technology, Ministry of Science and Technology, India, Indian Council of Medical Research, Ministry of Education, India
OpenAlex ID: W7220473782