Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly

Guseva, Serafima; Milles, Sigrid; Jensen, Malene Ringkjøbing; Salvi, Nicola; Kleman, Jean-Philippe; Maurin, Damien; Ruigrok, Rob W.H.; Blackledge, Martin

doi:10.1126/sciadv.aaz7095

Cited by 179 publications

(202 citation statements)

References 52 publications

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“…Our study shows that the nucleocapsid protein of the SARS-CoV-2 virus undergoes RNA-induced liquid-liquid phase separation. Although nucleocapsid assembly can occur outside of liquid-like compartments, the rate of assembly is increased when the nucleocapsid protein is concentrated through LLPS (Guseva et al, 2020). In addition, N SARS-CoV-2 interacts with human ribonucleoproteins (Gordon et al, 2020), which are found in several LLPS-driven cytosolic protein/RNA granules, indicating that N SARS-CoV-2 modulates protein/RNA granule formation in order to maximize viral replication.…”

Section: Discussionmentioning

confidence: 99%

“…Recent evidence indicates that negative-sense RNA viruses, which replicate in the cytoplasm of infected cells (Novoa et al, 2005), concentrate their replication machinery in dynamic compartments formed by LLPS of the viral structural proteins L, phosphoprotein (P) and nucleocapsid (N) protein (Nikolic et al, 2017;Heinrich et al, 2018). The genomes of positive-sense RNA viruses such as SARS-CoV-2, however, lack the genetic code for the phosphoprotein P, which is essential for LLPS in negative-sense RNA viruses (Nikolic et al, 2017;Heinrich et al, 2018;Guseva et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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

Savastano

Opakua

Ranković

et al. 2020

Preprint

136

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The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription and is essential for viral replication. Here we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Savastano

Opakua

Ranković

et al. 2020

Preprint

136

View full text Add to dashboard Cite

show abstract

“…Similar mechanisms are likely to exist in negative-sense RNA viruses, where replication is focused in dynamic biomolecular condensates [64][65][66] . 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: Discussionmentioning

confidence: 99%

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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“…In the measles virus, nucleoprotein (N) contains an RNA binding domain while phosphoprotein (P) contains an oligomerization domain. N and P form condensates when mixed together (59). In coronaviruses, the N protein possesses both an RNA-binding and oligomerization domain in a single peptide chain.…”

Section: Discussionmentioning

confidence: 99%

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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Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly

Cited by 179 publications

References 52 publications

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

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

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

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

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