1. Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid
- Author
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Zhao, Huaying, Syed, Abdullah M, Khalid, Mir M, Nguyen, Ai, Ciling, Alison, Wu, Di, Yau, Wai-Ming, Srinivasan, Sanjana, Esposito, Dominic, Doudna, Jennifer A, Piszczek, Grzegorz, Ott, Melanie, and Schuck, Peter
- Subjects
Biological Sciences ,Bioinformatics and Computational Biology ,Genetics ,Coronaviruses ,Infectious Diseases ,Emerging Infectious Diseases ,2.1 Biological and endogenous factors ,Infection ,SARS-CoV-2 ,Coronavirus Nucleocapsid Proteins ,RNA ,Viral ,Protein Multimerization ,Protein Binding ,Binding Sites ,Ribonucleoproteins ,Virus Assembly ,Humans ,Nucleocapsid Proteins ,Models ,Molecular ,Phosphoproteins ,COVID-19 ,Environmental Sciences ,Information and Computing Sciences ,Developmental Biology ,Biological sciences ,Chemical sciences ,Environmental sciences - Abstract
The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.
- Published
- 2024