The mutational landscape of SARS-CoV-2 nucleocapsid protein

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

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Abstract

The COVID19 pandemic has spawned an unprecedented worldwide collaboration to collect SARS-CoV-2 genome sequences in the GISAID database. It is currently approaching a size of 10 million genomes and is rapidly growing. This genomic database has been instrumental for enhancing our understanding of viral evolution and geographic spread, such as identifying and predicting variants of concern. Less attention has been devoted to the fact that the database provides unique information on viral sequence diversity and on seemingly inconsequential fluctuations of viral protein sequences that do not result in fixed mutations. Because the database consists of consensus sequences from viral samples taken from infected patients, all sequences represent alternate viral species, each of which is successfully replicating. Charting the scope of observed amino acid substitutions along the protein sequence we obtain a mutational landscape that, we hypothesize, can be interpreted in the context of biophysical protein properties. We have focused on the SARS-CoV-2 nucleocapsid (N-) protein, which we have comprehensively characterized with regard to its biophysical properties. N-protein is critical for viral assembly and scaffolding of viral RNA in ribonucleoprotein particles, serves essential functions to suppress intracellular antiviral defense pathways, and presents as a major antigen. N-protein consists of large intrinsically disordered regions, which flank and link the two folded N-protein domains. To analyze the sequence diversity we have initially charted the N-protein mutational landscape from all GISAID database entries as of November 2021, which comprises sequences from Delta-variant and preceding strains. We have observed astounding sequence diversity, with 25,000 different unique N-protein amino acid sequences that show > 85% of protein positions are variable and can assume an average of four to five different amino acids. We found that the range of substitutions approached a limit after 1 million sequences. Supporting the hypothesis that the mutational landscape reflects biophysical constraints, we found that local variability generally tracks known structural features. For example, the majority of positions conserved across related betacoronaviruses did not show any substitutions. However, the landscape also has several unexpected features, including variability in some positions previously thought to be highly conserved, and islands of low variability in the disordered regions. Of particular interest is the highly protected leucine-rich region 210-249 in the disordered linker, which is proximal to a G215C mutation that is defining for the 21J clade of the Delta variant. The G215C mutation is associated with the dominance of the 21J clade, having outcompeted G215 variants without further spike or N-protein substitutions. Through experimental and computational biophysical studies we were able to show that the leucine-rich region allows transient formation of a helix that serves as a protein-protein interaction interface. Based on the sparsity of viable substitutions in the mutational landscape in the region 210-249, we hypothesize that this self-association process is an essential component of the viral assembly mechanism. After the initial analysis of the mutational landscape in late 2021, the first wave of Omicron SARS-CoV-2 variants occurred. This variant has a different set of defining mutations in the N-protein, not sharing the G215C mutation in the central disordered linker and instead exhibiting several mutations and deletions in the N-terminal disordered arm. In the summer 2022, a sufficient number of genomes had been sequenced and deposited to GISAID to examine its mutational landscape independently in comparison with that of Delta- and preceding variants. Examining the new dataset, we find that overall features of the Omicron landscape are closely replicating those of the preceding variants. This supports the notion that the landscape reflects biophysical constraints for functioning N-protein, and shows the absence of dominant epistasis from the strain-defining mutations. The detailed analysis is still ongoing. We expect to publish these results within the next several months. In order to better understand the relationship between genotype diversity and resulting variability of protein biophysical properties, we have embarked on the characterization of protein variants with select mutations with regard to protein conformation, self- and co-assembly properties with nucleic acids, protein-protein interactions, thermal stability, and phase separation. We observed that point mutations can greatly affect these attributes, demonstrating significant plasticity of N-protein. We expect to publish these results within the next several months.