A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates

Abstract

To date, the recently discovered SARS-CoV-2 virus has afflicted >6.9 million people worldwide and disrupted the global economy. Development of effective vaccines or treatments for SARS-CoV-2 infection will be aided by a molecular-level understanding of SARS-CoV-2 proteins and their interactions with host cell proteins. The SARS-CoV-2 nucleocapsid (N) protein is highly homologous to the N protein of SARS-CoV, which is essential for viral RNA replication and packaging into new virions. Emerging models indicate that nucleocapsid proteins of other viruses can form biomolecular condensates to spatiotemporally regulate N protein localization and function. Our bioinformatic analyses, in combination with pre-existing experimental evidence, suggest that the SARS-CoV-2 N protein is capable of forming or regulating biomolecular condensates in vivo by interaction with RNA and key host cell proteins. We discuss multiple models, whereby the N protein of SARS-CoV-2 may harness this activity to regulate viral life cycle and host cell response to viral infection.

Keywords: enveloped viruses; liquid-liquid phase separation; low-complexity domain; stress granule; viral capsid.

FIGURE 1

The N protein of SARS‐CoV‐2 resembles the SARS‐CoV N protein and contains multiple LCDs. A, Multiple sequence alignment of N proteins from the seven coronavirus strains known to infect humans. B, Composition scan depicting the local S, R, and K content of the N protein sequence from SARS‐CoV‐2. The SARS‐CoV‐2 N protein sequence was scanned with a 20aa window and the percent composition of each amino acid was calculated at each position, similar to previous studies., A region was considered an LCD if any single amino acid constituted ≥40% of the window sequence

FIGURE 2

The SARS‐CoV‐2 N protein has an above‐average predicted phase separation propensity, with the SR‐domain being the highest‐scoring region. A, Phase separation scores for all SARS‐CoV‐2 proteins with both PSP and catGRANULE. A consensus sequence was built for each SARS‐CoV‐2 N protein by calculating the most frequent amino acid at each position from multiple sequence alignment of ~1900 sequences available on the NCBI Virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/; downloaded on 5/6/2020). ORF1ab sequences were parsed into separate sequences for the 16 nsp proteins in SARS‐CoV‐2 according to the cleavage sites in Chan et al and separately aligned. B, Phase separation score profile for the N protein from SARS‐CoV‐2 using PSP. C, Phase separation score profile for the N protein from SARS‐CoV‐2 using catGRANULE

FIGURE 3

Proposed models for the influence of the SARS‐CoV‐2 N protein on the formation and regulation of biomolecular condensates. A, Putative or directly observed interactions between the SARS‐CoV‐2 N protein and stress granule (SG) components. B, Three possible models for how the N protein of SARS‐CoV‐2 could affect stress granules in host cells: (1) N protein could be recruited to canonical host cell stress granules, which could have subtle or no effect on stress granules (“passive observer”), or could alter stress granule function by contributing to translation suppression, altering stress granule interactions, or remodeling stress granules; (2) N protein could recruit specific stress granule components to form unique, SARS‐specific stress granules that could serve as sites of viral translation or replication; or (3) N protein could inhibit the formation of canonical host cell stress granules by sequestering critical stress granule components. SARS‐CoV‐2 may inhibit the typical stress granule‐associated antiviral responses in host cells by suppressing downstream events that activate IFN‐β expression. Additionally, the features of the SARS‐CoV‐2 N protein that facilitate interaction with stress granules may also facilitate biomolecular condensation of N protein and genomic RNA during nascent virion formation at the ER‐Golgi intermediate compartment (ERGIC)