Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein

Carlson, Christopher R.; Asfaha, Jonathan B.; Ghent, Chloe M.; Howard, Conor J; Hartooni, Nairi; Morgan, David

doi:10.1101/2020.06.28.176248

Cited by 30 publications

(66 citation statements)

References 79 publications

(110 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this study, we expressed the N protein using a mammalian expression system, which more closely resembles the N protein produced in infected human cells (9). Consistent with Carlson et al and Chen et al, we observed that the N protein requires unstructured RNA fragments to form liquid condensates in physiological salt (38,54), suggesting that the positively charged protein and polyanion RNA form complex coacervates. The N protein is also capable of forming a biomolecular condensate in the presence of 1-5 kb viral RNA; however, these structures appear closer to gel-like filaments (38) and not as temperature-sensitive, liquid-like droplets observed by Iserman et al using bacterially expressed N (28).…”

Section: Discussionsupporting

confidence: 90%

“…Consistent with Carlson et al and Chen et al, we observed that the N protein requires unstructured RNA fragments to form liquid condensates in physiological salt (38,54), suggesting that the positively charged protein and polyanion RNA form complex coacervates. The N protein is also capable of forming a biomolecular condensate in the presence of 1-5 kb viral RNA; however, these structures appear closer to gel-like filaments (38) and not as temperature-sensitive, liquid-like droplets observed by Iserman et al using bacterially expressed N (28). However, in agreement with Iserman et al, we found that the structure of the RNA itself affects N condensation; base-paired or G-quadruplex-containing RNA produce irregularly shaped, gel-like condensates, similar to viral RNA (28).…”

Section: Discussionsupporting

confidence: 90%

“…Recent studies proposed that phosphorylation state on the N protein regulates genome packaging in SARS-CoV and SARS-CoV-2 (36,(38)(39)(40)(41), and the mass spectrometry analysis of our N protein purified from mammalian cells identified several phosphorylation sites ( Figure 2E, Table S2). Treating the full-length N protein with casein kinase 2 did not affect its phase separation with polyC ( Figure 3C,D) or its migration on a denaturing gel (Figure 3_figure supplement 2A), suggesting that the N protein expressed in human cells is already phosphorylated.…”

Section: The C-terminal Disordered Region and Phosphorylation Modulatmentioning

confidence: 81%

“…Recent studies showed that SARS-CoV-2 N protein expressed in bacteria forms biomolecular condensates with RNA in vitro (17,28,36,38,40,54,55). In this study, we expressed the N protein using a mammalian expression system, which more closely resembles the N protein produced in infected human cells (9).…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the SR motif is highly phosphorylated in human cells (57), and this promotes interactions with host proteins, nuclear targeting, and transcription of the viral genome, and either enhances or inhibits oligomerization in SARS-CoV (16,39,57,58). In SARS-CoV-2, SR phosphorylation is reported to make N condensates more liquid (38). In contrast to these reports, we did not see evidence of the SR motif driving the phase separation of SARS-CoV-2 N expressed in human cells.…”

Section: Discussionmentioning

confidence: 99%

See 4 more Smart Citations

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

Jack¹,

Ferro²,

Trnka³

et al. 2020

Preprint

View full text Add to dashboard Cite

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes COVID-19, a pandemic that seriously threatens global health. SARS CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with unstructured RNA, it forms mesh like-structures with viral RNA strands that contain secondary structure elements. Cross-linking mass spectrometry identified an intrinsically-disordered region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. By screening 1,200 FDA approved drugs in vitro, we identified a kinase inhibitor nilotinib, which affects the morphology of N condensates in vitro and disrupts phase separation of the N protein in vivo. These results indicate that the N protein compartmentalizes viral RNA in infected cells through liquid-liquid phase separation, and this process can be disrupted by a possible drug candidate.

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Discussionsupporting

confidence: 90%

Section: The C-terminal Disordered Region and Phosphorylation Modulatmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

Jack¹,

Ferro²,

Trnka³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

A little less aggregation a little more replication: Viral manipulation of stress granules

Brownsword

Locker

2022

WIREs RNA

View full text Add to dashboard Cite

Recent exciting studies have uncovered how membrane‐less organelles, also known as biocondensates, are providing cells with rapid response pathways, allowing them to re‐organize their cellular contents and adapt to stressful conditions. Their assembly is driven by the phase separation of their RNAs and intrinsically disordered protein components into condensed foci. Among these, stress granules (SGs) are dynamic cytoplasmic biocondensates that form in response to many stresses, including activation of the integrated stress response or viral infections. SGs sit at the crossroads between antiviral signaling and translation because they concentrate signaling proteins and components of the innate immune response, in addition to translation machinery and stalled mRNAs. Consequently, they have been proposed to contribute to antiviral activities, and therefore are targeted by viral countermeasures. Equally, SGs components can be commandeered by viruses for their own efficient replication. Phase separation processes are an important component of the viral life cycle, for example, driving the assembly of replication factories or inclusion bodies. Therefore, in this review, we will outline the recent understanding of this complex interplay and tug of war between viruses, SGs, and their components. This article is categorized under: RNA in Disease and Development > RNA in Disease Translation > Regulation RNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes

show abstract

1H, 13C and 15N backbone chemical shift assignments of SARS-CoV-2 nsp3a

et al. 2021

View full text Add to dashboard Cite

The non-structural protein nsp3 from SARS-CoV-2 plays an essential role in the viral replication transcription complex. Nsp3a constitutes the N-terminal domain of nsp3, comprising a ubiquitin-like folded domain and a disordered acidic chain. This region of nsp3a has been linked to interactions with the viral nucleoprotein and the structure of double membrane vesicles. Here, we report the backbone resonance assignment of both domains of nsp3a. The study is carried out in the context of the international covid19-nmr consortium, which aims to characterize SARS-CoV-2 proteins and RNAs, providing for example NMR chemical shift assignments of the different viral components. Our assignment will provide the basis for the identification of inhibitors and further functional and interaction studies of this essential protein.

show abstract

Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein

Cited by 30 publications

References 79 publications

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

A little less aggregation a little more replication: Viral manipulation of stress granules

1H, 13C and 15N backbone chemical shift assignments of SARS-CoV-2 nsp3a

Contact Info

Product

Resources

About