Assembly of SARS-CoV-2 ribonucleosomes by truncated N∗ variant of the nucleocapsid protein

Adly, Armin N.; Bi, Maxine; Carlson, Christopher R.; Syed, Abdullah M.; Ciling, Alison; Doudna, Jennifer A.; Cheng, Yifan; Morgan, David O.

doi:10.1016/j.jbc.2023.105362

Cited by 16 publications

(18 citation statements)

References 72 publications

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“…Indeed, these regions in the linker IDR have been recognized to play distinct functional roles: The SRregion provides a major hub for phosphorylation, aids in NA-binding, and mediates NA-binding induced allosteric interactions between NTD and the L-rich region (Pontoriero et al, 2022;Yaron et al, 2022;Zhao et al, 2023). This is distinct from the L-rich region, which has a propensity for the formation of transient helices that interact with NSP3 (Bessa et al, 2022), and can assemble via hydrophobic interactions to from coiled-coiled oligomers that contribute to the architecture of RNPs in viral assembly (Adly et al, 2023;Zhao et al, 2024Zhao et al, , 2023.…”

Section: Resultsmentioning

confidence: 99%

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

Nguyen,

Zhao,

Myagmarsuren

et al. 2023

Preprint

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Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also exhibiting functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.

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Section: Resultsmentioning

confidence: 99%

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

Nguyen,

Zhao,

Myagmarsuren

et al. 2023

Preprint

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“…The level of phosphorylation of N is thought to act as a functional switch in the replication cycle, or in genome packaging or unpackaging (14,(55)(56)(57)(58). Phosphorylation has also been shown to affect the compactness of viral ribonucleoprotein (vRNP) complexes (59), and concurrent studies have shown that binding of the isolated N3 to long RNAs is modulated by phosphorylation (60), while fluorescence polarization measured significantly weaker binding to a 10-nucleotide RNA following hyperphosphorylation (46). A recent study proposed the sequential phosphorylation of N by host kinases SRPK1, GSK-3 and CK1 (58).…”

Section: Discussionmentioning

confidence: 99%

“…N4 forms a highly stable dimeric structure involving two domain-swapped b-hairpins. Although N2 is known to represent the main RNA binding domain, secondary RNA binding sites have been proposed to exist in both N3 (39,40,26,36) and N4 (39,(41)(42)(43)(44)(45), an observation supported by assembly of truncation mutants of N into large supramolecular complexes of the dimensions of RNPs (46). N5 has also been associated with assembly into higher order oligomeric states (47).…”

Section: Introductionmentioning

confidence: 99%

A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding

Botova,

Camacho-Zarco,

Tognetti

et al. 2024

Preprint

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The nucleoprotein (N) of SARS-CoV-2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich domain (SR) that is hyperphosphorylated in infected cells. This modification is thought to regulate function of N, although mechanistic details remain unknown. We use time-resolved NMR to follow local and long-range structural changes occurring during hyperphosphorylation by the kinases SRPK1/GSK-3/CK1, thereby identifying a conformational switch that abolishes interaction with RNA. When 8 approximately uniformly-distributed sites are phosphorylated, the SR domain competitively binds the same interface as single-stranded RNA, resulting in RNA binding inhibition. Phosphorylation by PKA does not prevent RNA binding, indicating that the pattern resulting from the physiologically-relevant kinases is specific for inhibition. Long-range contacts between the RNA-binding, linker and dimerization domains are also abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into recruitment of specific host kinases to regulate viral function.

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“…(14, 24, 27–29, 42, 43) Substantial published work indicates interactions via the leucine-rich region of the LKR IDR connecting the ordered NTD and CTD domains. (27, 28) Crystal contacts suggest CTD-CTD interaction. (44) Also the C-terminal IDR has been implicated a site of interaction.…”

Section: Discussionmentioning

confidence: 99%

“…(25,26) The IDR linking the NTD and CTD (LKR) incorporates a leucine-rich helix that appears to participate in protein-protein interactions and a serine/arginine-rich region that can be phosphorylated to modulate LLPS formation. (11,24,(27)(28)(29) Proteins with IDR's tend to undergo LLPS because they can interact with each other or with substrates such as RNA in a fluid manner. (30) These multivalent interactions lead to oligomerization and phase separation from bulk solution into membraneless organelles, whose formation must be reversible and responsive to their environment.…”

Section: Introductionmentioning

confidence: 99%

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation

Laughlin,

Young,

Gonzalez-Gutierrez

et al. 2024

Preprint

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SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a ~1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 micromolar. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

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Assembly of SARS-CoV-2 ribonucleosomes by truncated N∗ variant of the nucleocapsid protein

Cited by 16 publications

References 72 publications

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

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

A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation

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