Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2

Tayeb-Fligelman, Einav; Cheng, Xinyi; Tai, Christen; Bowler, Jeannette T.; Griner, Sarah L.; Sawaya, M.R.; Seidler, Paul M.; Jiang, Yi; Lu, Jiahui; Rosenberg, Gregory M.; Salwiński, Łukasz; Abskharon, Romany; Zee, Chih-Te; Hou, Ke; Li, Yan; Boyer, David R.; Murray, Kevin; Falcon, Genesis; Anderson, D.; Cascio, Duilio; Saelices, Lorena; Damoiseaux, Robert; Guo, Feng; Eisenberg, David

doi:10.1101/2021.03.05.434000

Cited by 26 publications

(31 citation statements)

References 64 publications

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“…In this study, we showed that the SARS-CoV-2 N protein phase separates with nonspecific RNA sequences in vitro and the viscosity and shape of these condensates depend on the structure of the RNA substrate. Concurrent studies have shown that the SARS-CoV-2 N protein expressed in bacteria forms biomolecular condensates with RNA in vitro [8,31,36,38,46,54,56,63,[71][72][73][74]. We purified N protein from mammalian cells to recapitulate the posttranslational modifications that occur in infected human cells [10].…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (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 genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…Phase-separated condensates can nucleate amyloid-like fibrils, the nucleation process being enhanced in droplets or gels due to local concentration increase effects. A recent example is provided by the SARS-CoV2 nucleocapsid protein that forms amyloids in phase-separated droplets [ 106 ]. In line with this, IDRs are known to form amyloid-like structures via the formation of hydrogels [ 87 , 107 , 108 ].…”

Section: Discussionmentioning

confidence: 99%

Identification of a Region in the Common Amino-terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation

et al. 2021

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Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

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“…N‐NTR; 28–30, 35–184) had 34 matching structures, of which two were homotetramers, eight were homodimers, 14 were monomers, two showed binding to viral RNA, and one showed binding to an antibody. In addition, two structures matched to nine‐residue regions of N protein, showing these regions presented as epitopes by HLA (Szeto et al , 2021), while a final structure showed a six‐residue region assembled as an homo‐16‐mer that is implicated in amyloid formation (preprint: Tayeb‐Fligelman et al , 2021). The C‐terminal region (a.k.a.…”

Section: Resultsmentioning

confidence: 99%

“…The C‐terminal region (a.k.a. N‐CTR; 217–230, 243–365) had 22 matching structures, of which 16 were homodimers, four showed nine‐residue regions of N protein presented as epitopes by HLA (Szeto et al , 2021), and two showed six‐residue regions assembled as homo‐16‐mers that are implicated in amyloid formation (preprint: Tayeb‐Fligelman et al , 2021). These structures suggest the oligomerization and RNA‐binding activities of SARS‐CoV‐2 N protein may be disrupted by therapeutic strategies based on small molecule inhibitors developed for HCoV‐OC43 (human coronavirus OC43) and MERS‐CoV (Peng et al , 2020).…”

Section: Resultsmentioning

confidence: 99%

SARS‐CoV‐2 structural coverage map reveals viral protein assembly, mimicry, and hijacking mechanisms

O’Donoghue

Schafferhans

Sikta

et al. 2021

Molecular Systems Biology

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We modeled 3D structures of all SARS‐CoV‐2 proteins, generating 2,060 models that span 69% of the viral proteome and provide details not available elsewhere. We found that ˜6% of the proteome mimicked human proteins, while ˜7% was implicated in hijacking mechanisms that reverse post‐translational modifications, block host translation, and disable host defenses; a further ˜29% self‐assembled into heteromeric states that provided insight into how the viral replication and translation complex forms. To make these 3D models more accessible, we devised a structural coverage map, a novel visualization method to show what is—and is not—known about the 3D structure of the viral proteome. We integrated the coverage map into an accompanying online resource (https://aquaria.ws/covid) that can be used to find and explore models corresponding to the 79 structural states identified in this work. The resulting Aquaria‐COVID resource helps scientists use emerging structural data to understand the mechanisms underlying coronavirus infection and draws attention to the 31% of the viral proteome that remains structurally unknown or dark.

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Inhibition of amyloid formation of the Nucleoprotein of SARS-CoV-2

Cited by 26 publications

References 64 publications

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

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

Identification of a Region in the Common Amino-terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation

SARS‐CoV‐2 structural coverage map reveals viral protein assembly, mimicry, and hijacking mechanisms

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