Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

Savastano, Adriana; Opakua, Alain Ibáñez de; Ranković, M.; Zweckstetter, Markus

doi:10.1101/2020.06.18.160648

Cited by 65 publications

(155 citation statements)

References 34 publications

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“…In Measles virus, these condensates have also been implicated in nucleocapsid assembly 67 . We also note that others have recently observed SARS-CoV-2 N protein condensates [68][69][70][71] .…”

Section: Discussionsupporting

confidence: 61%

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

Carlson

Asfaha

Ghent

et al. 2020

Preprint

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The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell1–3. The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder4. Phosphorylation of the central disordered region is required for normal viral genome transcription5,6, which occurs in a cytoplasmic structure called the replication transcription complex (RTC)7–11. It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates12–15. Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.

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Section: Discussionsupporting

confidence: 61%

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

Carlson

Asfaha

Ghent

et al. 2020

Preprint

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“…Additionally, MS identified native proteins that co-purified with the N protein in 1 M salt (Table S3). Consistent with the recruitment of N to stress granules in cells (8,16,36), two of the most frequently identified proteins were stress granule proteins G3BP1 and G3BP2, with R2 of the N protein interacting with G3BP1 ( Figure 2_figure supplement 1D).…”

Section: Cross-linking Mass Spectrometry Identifies N Protein Interacsupporting

confidence: 56%

“…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: 80%

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

Jack¹,

Ferro²,

Trnka³

et al. 2020

Preprint

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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.

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“…For example, the poliovirus 3C protease was shown to cleave G3BP1 53 . How the recent characterization of a phase-separation phenomenon associated with N and viral RNA [54][55][56][57] relates to our findings should be addressed in the context of viral infection, or at least viral RNA transfection.…”

Section: Discussionmentioning

confidence: 99%

A SARS-CoV-2 – host proximity interactome

Samavarchi-Tehrani

Abdouni

Knight

et al. 2020

Preprint

131

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Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

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Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

Cited by 65 publications

References 34 publications

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

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

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

A SARS-CoV-2 – host proximity interactome

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