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. We have previously established several steps of the assembly mechanism, exploiting the synergistic capabilities to measure macromolecular size-distributions by sedimentation velocity analytical ultracentrifugation and mass photometry: 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. Recently we have identified the protein-protein interface underlying this self-association process as a transient helix in the leucine-rich sequence (LRS) of the disordered linker. It 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 this LRS oligomerization process is allosterically linked to NA binding. Furthermore, we found that the formation of transient helices presents a constraint for mutations of viable viral genomes, and we identified analogous conserved oligomerization sites in related coronaviruses. Our studies showed that this new oligomerization site is an essential step in viral assembly, and potentially presents a pan-coronavirus target for therapeutic intervention. After discovery of this key protein-protein interface, we have developed a coarse-grained model for RNP assembly based on biophysical data on additional protein-protein and protein-nucleic acid binding interfaces. In order to explore further the assembly process, we have now embarked on the expression of viral M-protein, which is a structural viral membrane protein critical for packaging of the virion. Based on the literature of SARS-CoV-2 and related coronaviruses, the interaction of M-protein with N-protein is essential for viral assembly, although the biophysical mechanisms are unclear. We aim to study the interaction between N-protein and pre-assembled RNPs with M-protein in detergent solutions, with M-protein embedded in nanodiscs or in lipid vesicles as model systems. To this end, we have established that N-protein and RNP assemblies are stable in detergent solutions. For the expression of M-protein, we have started to implement a recently published bacterial expression protocol, while in parallel pursuing mammalian expression, with the goal to compare the yields and biophysical properties of the M-protein preparations. We envision the study of ternary interactions between N, M, and different NA ligands. In the reporting period, we have also embarked on studying OC43 N-protein as a second model system for viral assembly. We hypothesize that the mechanism we have identified in SARS-CoV-2 are conserved and present similarly in OC43. Importantly, OC43 can be studied in laboratories with biosafety level 2, which facilitates bridging the gap between in vitro biophysical experiments and virus replication in cells. To this end, we have identified equivalent N-protein linker regions in OC43 that provide transient self-association interfaces, verified their interactions in a peptide model, and designed abrogating mutants. As a next step, we have developed a protocol to express full-length OC43 N-protein. This will provide the basis for exploring the viral assembly mechanism, with the working hypothesis that assembly is similarly initiated by NA binding causing allosteric linker self-association followed by higher-order oligomerization with suitable NA ligands.