Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid

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 mutant proteins binding different nucleic acids in anin vitroassay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerizeviaa 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 multi-valent 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.

Modulation of Biophysical Properties of Nucleocapsid Protein in the Mutant Spectrum of SARS-CoV-2

“…We focused on Omicron mutations as these are obligatory an all currently circulating strains, and specifically on N-arm mutations, which have recently been implicated in altered intramolecular interactions with NA-occupied NTD (Cubuk et al, 2023). Even though SV-AUC showed no indication of self-association of N:P13L/Δ31-33 at low micromolar concentrations, weak interactions with K d > mM would not be detectable under these conditions yet could be highly relevant in the context of multi-valent complexes (Zhao et al, 2024). Following the roadmap used previously for the study of the weak self-association of the leucine-rich linker IDR (Zhao et al, 2023), we restricted the protein to the N-arm peptide such that it can be studied at much higher concentrations.…”

“…Indeed, these regions in the linker IDR have been recognized to play distinct functional roles: The SRregion provides a major hub for phosphorylation, aids in NA-binding, and mediates NA-binding induced allosteric interactions between NTD and the L-rich region (Pontoriero et al, 2022;Yaron et al, 2022;Zhao et al, 2023). This is distinct from the L-rich region, which has a propensity for the formation of transient helices that interact with NSP3 (Bessa et al, 2022), and can assemble via hydrophobic interactions to from coiled-coiled oligomers that contribute to the architecture of RNPs in viral assembly (Adly et al, 2023;Zhao et al, 2024Zhao et al, , 2023.…”

“…On the other hand, a very clear separation of physicochemical parameters far exceeding mutational dispersion is maintained between the L-rich and SR-rich region of the linker IDR, and the N3 and remaining region of the C-arm IDR. These distinctions are likely functionally important, with the polarity and charges of the SR-rich linker region aiding in nucleic acid binding (Pontoriero et al, 2022), the hydrophobicity of the L-rich region aiding in assembly functions (Bessa et al, 2022;Zhao et al, 2024Zhao et al, , 2023, and the acidic N3-region playing a role in NA-and M-protein interactions as suggested from analogy to MHV-and SARS-CoV-1 (Masters, 2019). These nonlocal features are also maintained in analogous consensus sequence regions of related coronaviruses, and thus provide further examples for nonlocal biophysical properties that are evolutionary conserved despite amino acid sequence divergence (Zarin et al 2017;Zarin et al 2021).…”

“…The eponymous structural function of N-protein is that of scaffolding genomic RNA for virion assembly. It proceeds via nucleic acid (NA)-binding induced conformational changes and oligomerization, leading to the formation of ribonucleoprotein (RNP) particles with as-of-yet unknown molecular architecture, ≈38 of which are arranged like beads-on-a-string in the viral particle (Carlson et al, 2022; Cubuk et al, 2021; Klein et al, 2020; Yao et al, 2020; Zhao et al, 2024, 2023, 2021), and are anchored through binding of N-protein to viral M-protein (Lu et al, 2021; Masters, 2019). Beyond this structural role, N-protein is highly multi-functional and binds to multiple host proteins to modulate or exploit different pathways, including stress granules (Biswal et al, 2022; Gordon et al, 2020; Savastano et al, 2020), the type 1 interferon signaling pathway (Chen et al, 2020; Li et al, 2020), the NLRP3 inflammasome (Pan et al, 2021), and others, as recently reviewed (Wu et al, 2023; Yu et al, 2023).…”

Bioinformatics Insights on Viral Gene Expression Transactivation: From HIV-1 to SARS-CoV-2

“…However, each domain uses different mechanisms to bind RNA, with N-NTD likely preferring unpaired single-stranded regions and N-CTD preferring double-stranded RNA. It has been suggested that optimal RNA binding requires contributions from both domains [ 160 , 161 ], creating a local high density of N [ 162 , 163 , 164 , 165 ].…”