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

Cascarina, Sean M.; Ross, Eric D.

doi:10.1096/fj.202001351

Cited by 109 publications

(95 citation statements)

References 102 publications

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“…Previous studies have reported that the GSK-3 and SRPK families are involved in the phosphorylation of the SR-rich domain of the nucleocapsid protein of SARS-CoV ( 5, 15 ). Recent reports have suggested these kinase families to be involved in the phosphorylation of SARS-CoV-2 N protein phosphorylation as well ( 32–34 ), however an exact phosphorylation model has never been validated experimentally. To that end, we empirically characterized the biochemical substrate specificities of GSK-3α/β and SRPK1/2/3 ( Figure 2A and Figure S1 ).…”

Section: Resultsmentioning

confidence: 99%

The FDA-approved drug Alectinib compromises SARS-CoV-2 nucleocapsid phosphorylation and inhibits viral infection in vitro

Yaron

Heaton

Levy

et al. 2020

Preprint

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Antiviral therapeutics against SARS-CoV-2 are needed to treat the pandemic disease COVID-19. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. Here, we used a genome-wide loss of function CRISPR/Cas9 screen in human lung epithelial cells to identify potential host therapeutic targets. Validation of our screening hits revealed that the kinase SRPK1, together with the closely related SRPK2, were jointly essential for SARS-CoV-2 replication; inhibition of SRPK1/2 with small molecules led to a dramatic decrease (more than 100,000-fold) in SARS-CoV-2 virus production in immortalized and primary human lung cells. Subsequent biochemical studies revealed that SPRK1/2 phosphorylate the viral nucleocapsid (N) protein at sites highly conserved across human coronaviruses and, due to this conservation, even a distantly related coronavirus was highly sensitive to an SPRK1/2 inhibitor. Together, these data suggest that SRPK1/2-targeted therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.

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Section: Resultsmentioning

confidence: 99%

The FDA-approved drug Alectinib compromises SARS-CoV-2 nucleocapsid phosphorylation and inhibits viral infection in vitro

Yaron

Heaton

Levy

et al. 2020

Preprint

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“…Among the top-enriched interactors is the SARS-CoV-2 N protein. Multiple computational and experimental analyses provided compelling evidences that the N protein has a high propensity to undergo liquid-liquid phase separation (LLPS), that is stimulated by interaction with the viral gRNA [78][79][80][81]. This interaction preferentially occurs with single-stranded RNA regions flanked by stably structured elements [82], further suggesting that LLPS might be exploited by SARS-CoV-2 for efficient genome packaging.…”

Section: Additional Insights From Transcriptome-wide Studiesmentioning

confidence: 99%

Structure and regulation of coronavirus genomes: state-of-the-art and novel insights from SARS-CoV-2 studies

Manfredonia

Incarnato

2020

Biochemical Society Transactions

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Coronaviruses (CoV) are positive-sense single-stranded RNA viruses, harboring the largest viral RNA genomes known to date. Apart from the primary sequence encoding for all the viral proteins needed for the generation of new viral particles, certain regions of CoV genomes are known to fold into stable structures, controlling several aspects of CoV life cycle, from the regulation of the discontinuous transcription of subgenomic mRNAs, to the packaging of the genome into new virions. Here we review the current knowledge on CoV RNA structures, discussing it in light of the most recent discoveries made possible by analyses of the SARS-CoV-2 genome.

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“…Indeed, the crystal structure of the N-terminal domain (NTD) reveals an RNA binding groove [25][26][27], while crystal structures of the Cterminal domain (CTD) show a highly interlaced dimer with additional nucleic acid binding capacity [28,29]. The N protein shows unusual properties in the presence of RNA, displaying concentration-dependent liquid-liquid phase separation [22,23,30,31] that is pertinent to the viral genome packaging mechanism [32,33]. In human cells, the assembly of condensates is down-regulated by phosphorylation of the SR-rich region [30,34].…”

Section: Introductionmentioning

confidence: 99%

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

Tugaeva

Hawkins

Smith

et al. 2020

Preprint

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The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.HighlightsSARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a μM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

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

Cited by 109 publications

References 102 publications

The FDA-approved drug Alectinib compromises SARS-CoV-2 nucleocapsid phosphorylation and inhibits viral infection in vitro

The FDA-approved drug Alectinib compromises SARS-CoV-2 nucleocapsid phosphorylation and inhibits viral infection in vitro

Structure and regulation of coronavirus genomes: state-of-the-art and novel insights from SARS-CoV-2 studies

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

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