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Principal Investigator: PETER SCHUCK
Organization: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
Fiscal Year: 2022
Award: $985,108
Funding agency: National Institute of Biomedical Imaging and Bioengineering
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 nucleic acid (NA) -- is the highly stable N-protein dimer. Only ultra-weak higher oligomerization is observable in the absence of NA. However, occupation of the nucleic acid binding sites of N-protein 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 discovered that the G215C mutant of N-protein can form both covalent and non-covalent higher-order oligomers. The G215C mutation has become dominant in the Delta variant of SARS-CoV-2, outcompeting G215 variants without further spike or N-protein substitutions. This suggests the hypothesis that enhanced N-protein self-association may promote viral replication and infectivity.
Studying the G215C mutation in more detail, we were able to show that the position 215 is close to a transient helix in the leucine-rich region of the central disordered linker, and that the introduced cysteine poises the helix favoring protein-protein interactions. As a result, the originally weak higher oligomer formation of N-protein is enhanced by 100-fold by 215C. Interestingly, inspection of the mutational landscape of N-protein reveals the transient helix region to be highly conserved, indicating that it is an essential feature of RNP assembly.
In order to improve our capabilities of studying N-protein mutants, we have refined our protein expression protocol to a yield and purity sufficient for biophysical experiments. This allowed us to embark on the characterization of several other N-protein constructs to probe the protein interaction interface in the disordered linker in more detail. We expect to publish these results within the next several months.
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