Interactions of SARS-CoV-2 N-protein

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

SARS-CoV-2 nucleocapsid (N-)protein is a 419 aa long protein with two folded domains (NTD and CTD), flanked and linked by intrinsically disordered regions (N-arm, linker, and C-arm). The major function of the NTD is nucleic acid (NA) binding, that of the CTD is high-affinity dimerization, but the functions of the disordered regions are less clear. In the SARS-CoV-2 virion, the viral genome is scaffolded by N-protein into regular ribonucleoprotein particles (RNPs), in an as-of-yet poorly understood process. In order to elucidate the mechanism, we have previously established several steps: The starting point -- the N-protein in solution without NA -- is the highly stable N-protein dimer. Only ultra-weak higher oligomerization is observable in the absence of NA. However, occupation of the NA binding sites of N-protein in the NTD causes a conformational change associated with reversible higher oligomer formation. For NAs longer than 20 nucleotides additional multi-valent cross-linking to NA can occur. Consistent with reports from other laboratories, in such mixtures we observe liquid-liquid phase separation producing macromolecular condensates with a concentrated mixed N-protein/NA phase. It is generally thought that RNP formation occurs in this concentrated phase. We observed additional conformational changes of N-protein upon phase separation consistent with increased helicity, possibly supporting RNP formation. In the reporting period, we have further explored protein-protein interactions of N-protein. We identified a transient helix in the leucine-rich region of the disordered linker, which can oligomerize into coiled-coil trimers, tetramers, and higher oligomers. Using structural models of the coiled-coil we identified point mutations that disrupt this oligomerization, and others that enhance this oligomerization. Using these mutants we were able to demonstrate that oligomerization process is allosterically linked to NA binding, in what we hypothesize is a switch to initiate the assembly process. Furthermore, we found that the formation of hydrophobic helices presents a constraint for mutations of viable viral genomes, and we identified analogous oligomerization sites in related coronaviruses. This suggests that this new oligomerization site is an essential step in viral assembly, and potentially a target for therapeutic intervention. To examine the assembly process in more detail, we extended our oligomerization studies to the interaction of N-protein with oligonucleotides that bind N-protein multivalently. This should provide information on the relative strengths of NA interactions and self-interactions of N-protein. To further explore the role and mechanism of this oligomerization site we have embarked on collaborations with the Ott laboratory and Yewdell laboratory to study viral assembly of mutants in vivo.