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Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

ABSTRACT: G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules.

SUBMITTER: Yang Z

PROVIDER: S-EPMC10327126 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

Yang Zemin Z Johnson Bryan A BA Meliopoulos Victoria A VA Ju Xiaohui X Zhang Peipei P Hughes Michael P MP Wu Jinjun J Koreski Kaitlin P KP Chang Ti-Cheng TC Wu Gang G Hixon Jeff J Duffner Jay J Wong Kathy K Lemieux Rene R Lokugamage Kumari G KG Alvardo Rojelio E RE Crocquet-Valdes Patricia A PA Walker David H DH Plante Kenneth S KS Plante Jessica A JA Weaver Scott C SC Kim Hong Joo HJ Meyers Rachel R Schultz-Cherry Stacey S Ding Qiang Q Menachery Vineet D VD Taylor J Paul JP

bioRxiv : the preprint server for biology 20230630

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by struct ...[more]

PMID: 37425880

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Project description:While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo . Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection.Author summarySince its emergence, SARS-CoV-2 has continued to adapt for human infection resulting in the emergence of variants with unique genetic profiles. Most studies of genetic variation have focused on spike, the target of currently available vaccines, leaving the importance of variation elsewhere understudied. Here, we characterize a highly variable motif at residues 203-205 in nucleocapsid. Recreating the prominent nucleocapsid R203K+G204R mutation in an early pandemic background, we show that this mutation is alone sufficient to enhance SARS-CoV-2 replication and pathogenesis. We also link augmentation of SARS-CoV-2 infection by the R203K+G204R mutation to its modulation of nucleocapsid phosphorylation. Finally, we characterize an analogous alanine double substitution at positions 203-204. This mutant was found to mimic R203K+G204R, suggesting augmentation of infection occurs by disrupting the ancestral sequence. Together, our findings illustrate that mutations outside of spike are key components of SARS-CoV-2's adaptation to human infection.

Dataset Information

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

Publications

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication.

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