Extensive Biophysical Diversity in the Mutant Spectrum of SARS-CoV-2 Nucleocapsid Protein

Core Concepts

The SARS-CoV-2 nucleocapsid (N) protein exhibits significant biophysical diversity across its mutant spectrum, with mutations in the intrinsically disordered regions modulating thermodynamic stability, secondary structure, oligomeric state, particle formation, and liquid-liquid phase separation.

Abstract

The study examines the biophysical properties of the SARS-CoV-2 nucleocapsid (N) protein across its mutant spectrum, taking advantage of the extensive genomic database of SARS-CoV-2 sequences.
Key insights:

The intrinsically disordered regions (IDRs) of N-protein exhibit significant variation in physicochemical properties like polarity, hydrophobicity, and charges across the mutant spectrum, while the folded domains maintain more constrained properties.
Distinct biophysical features are conserved between the subregions of the IDRs (e.g., SR-rich vs. L-rich regions of the linker) across SARS-CoV-2 and related coronaviruses, suggesting functional constraints.
Experimental characterization of select N-protein mutants associated with variants of concern reveals that single point mutations can have nonlocal effects, modulating thermodynamic stability, secondary structure, oligomeric state, particle formation, and liquid-liquid phase separation.
The Omicron-defining mutations in the N-arm and linker IDRs exhibit compensatory effects, with the N-arm mutation P13L promoting self-association and phase separation, counteracting the inhibitory effects of the linker mutation R203K/G204R.
The results highlight the importance of IDRs in viral evolution, allowing significant sequence diversity while maintaining key biophysical constraints crucial for N-protein functions.

Stats

The N-protein amino acid sequences exhibit ≈43 million instances of mutations distributed across ≈92% of its residues.
The IDRs exhibit an average of 5.2 different possible amino acid mutations at each residue compared to 2.9 different mutations on average in the folded domains.
The defining mutations of the Delta and Omicron variants impact the hydrophobicity, polarity, and charges in all of the N-protein regions, but their values do not stand out from the clouds of values across the mutant spectrum.

Quotes

"Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success."
"Biophysical constraints implicit in the shape of such landscapes are key to understand the function and molecular evolution of viral proteins."
"A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs."

Key Insights Distilled From

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

by Nguyen,A., Z... at www.biorxiv.org 11-22-2023

https://www.biorxiv.org/content/10.1101/2023.11.21.568093v2

Deeper Inquiries

How do the biophysical properties of N-protein mutants impact the overall fitness and evolutionary dynamics of SARS-CoV-2?

The biophysical properties of N-protein mutants play a crucial role in determining the overall fitness and evolutionary dynamics of SARS-CoV-2. These properties, such as thermodynamic stability, secondary structure, oligomeric state, particle formation, and liquid-liquid phase separation, can be significantly altered by point mutations in the N-protein. These alterations can have nonlocal effects, impacting the protein's ability to function effectively in various cellular processes.
For example, mutations like N:G215C and Nδ can lead to changes in the protein's oligomeric state, affecting its interactions with other molecules and potentially influencing viral replication and assembly. The destabilizing effect of mutations like N:D63G and N:R203K/G204R on the thermodynamic stability of the folded domains can impact the overall structure and function of the protein. Additionally, mutations like N:P13L/Δ31-33 can enhance particle formation and liquid-liquid phase separation, potentially affecting viral assembly and infectivity.
These changes in biophysical properties can influence the fitness of the virus by altering its ability to interact with host cells, evade the immune system, and replicate efficiently. Mutations that confer advantages in terms of stability, structure, and interactions may be selected for during viral evolution, leading to the emergence of new variants with improved fitness and transmissibility.

What are the potential implications of the observed biophysical diversity in N-protein for the development of therapeutics and diagnostics targeting this viral protein?

The observed biophysical diversity in N-protein has significant implications for the development of therapeutics and diagnostics targeting SARS-CoV-2. Understanding the range of biophysical properties exhibited by mutant N-proteins can help in the design of targeted therapies that specifically disrupt or modulate these properties to inhibit viral replication and infectivity.
For therapeutics, targeting specific biophysical features of N-protein mutants, such as their stability, oligomeric state, or interactions with host proteins, could lead to the development of novel antiviral drugs. By disrupting essential functions of the protein, such as nucleic acid binding, assembly, or interactions with host factors, these therapeutics could inhibit viral replication and reduce the severity of infection.
In terms of diagnostics, the biophysical diversity of N-protein mutants could be leveraged to develop more sensitive and specific diagnostic tools. By targeting unique biophysical properties of mutant N-proteins, such as changes in secondary structure or particle formation, diagnostic assays could be designed to detect specific variants of the virus with high accuracy.
Overall, the insights gained from studying the biophysical diversity of N-protein mutants could pave the way for the development of innovative therapeutics and diagnostics that target specific aspects of viral protein function to combat SARS-CoV-2 and its variants effectively.

Could the insights into the role of IDRs in viral evolution be extended to other RNA viruses, and what broader principles might emerge?

The insights into the role of Intrinsically Disordered Regions (IDRs) in viral evolution gained from studying SARS-CoV-2 N-protein could indeed be extended to other RNA viruses. IDRs are common features of many viral proteins across different RNA viruses, and their flexibility and adaptability play a crucial role in viral replication, assembly, and interactions with host cells.
By studying the biophysical properties and functional roles of IDRs in other RNA viruses, similar principles of sequence diversity, nonlocal physicochemical properties, and evolutionary constraints may emerge. These principles could include the importance of maintaining a balance between flexibility and stability in IDRs, the role of IDRs in mediating protein-protein interactions and host-virus interactions, and the potential for IDRs to evolve new functions with few mutations.
Understanding the evolutionary dynamics of IDRs in RNA viruses could provide valuable insights into how viruses adapt to changing environments, evade host immune responses, and develop resistance to antiviral therapies. By uncovering the common principles governing IDR function and evolution in RNA viruses, researchers may be able to develop more effective strategies for combating a wide range of viral infections.