Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2.
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ABSTRACT:
SUBMITTER: Zhao D
PROVIDER: S-EPMC7994959 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.
2020-12-17 | GSE162569 | GEO
Project description: Not available
| S-EPMC7477871 | biostudies-literature
Project description:We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.
| S-EPMC7691212 | biostudies-literature
Project description:The current work identified the protein interactors of SARS-CoV-2 Nucleocapsid RNA binding protein (NCAP or N protein) in lung cancer A-549 cells, using a co-immunoprecipitation strategy, which is coupled with SILAC-based mass spec. A significant proportion of the identified NCAP interacting proteins included stress granule (SG) and immunoregulators. SG nucleator G3BP1 showed specific interaction wit NCAP in unstressed and oxidative- or heat-stressed cells. Following stress treatment, NCAP showed enhanced association with SGs. Recombinant NCAP exhibited in vitro liquid-liquid phase separation (LLPS)to form liquid droplets. RNA stimulated NCAP to develop droplets in vitro and SG assembly in cells, and RNase A treatment completely blocked both of these functions. An RNA intercalating mitoxantrone disrupted NCAP assembly in SGs invitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS-CoV-2 NCAP and identifies a candidate way to target this protein.
2021-11-25 | PXD023989 | Pride
Project description: Not available
| S-EPMC8368218 | biostudies-literature
Project description:The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.
| S-EPMC11354357 | biostudies-literature
Project description:The ongoing 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has posed a worldwide pandemic and a major global public health threat. The severity and mortality of COVID-19 are associated with virus-induced dysfunctional inflammatory responses and cytokine storms. However, the interplay between host inflammatory responses and SARS-CoV-2 infection remains largely unknown. Here, we demonstrate that SARS-CoV-2 nucleocapsid (N) protein, the major structural protein of the virion, promotes the virus-triggered activation of NF-κB signaling. After binding to viral RNA, N protein robustly undergoes liquid-liquid phase separation (LLPS), which recruits TAK1 and IKK complex, the key kinases of NF-κB signaling, to enhance NF-κB activation. Moreover, 1,6-hexanediol, the inhibitor of LLPS, can attenuate the phase separation of N protein and restrict its regulatory functions in NF-κB activation. These results suggest that LLPS of N protein provides a platform to induce NF-κB hyper-activation, which could be a potential therapeutic target against COVID-19 severe pneumonia.
| S-EPMC8065320 | biostudies-literature
Project description:Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid
| PRJNA682223 | ENA
Project description:SARS-CoV-2 is an emerging coronavirus that causes dysfunctions in multiple human cells and tissues. Studies have looked at the entry of SARS-CoV-2 into host cells mediated by the viral spike protein and human receptor ACE2. However, less is known about the cellular immune responses triggered by SARS-CoV-2 viral proteins. Here, we show that the nucleocapsid of SARS-CoV-2 inhibits host pyroptosis by blocking Gasdermin D (GSDMD) cleavage. SARS-CoV-2-infected monocytes show enhanced cellular interleukin 1b (IL-1b) expression, but reduced IL-1b secretion. While SARS-CoV-2 infection promotes activation of the NLRP3 inflammasome and caspase-1, GSDMD cleavage and pyroptosis are inhibited in infected human monocytes. SARS-CoV-2 nucleocapsid protein associates with GSDMD in cells and inhibits GSDMD cleavage in vitro and in vivo. The nucleocapsid binds the GSDMD linker region and hinders GSDMD processing by caspase-1. These insights into how SARS-CoV-2 antagonizes cellular inflammatory responses may open new avenues for treating COVID-19 in the future.
| S-SCDT-EMBOJ-2021-108249 | biostudies-other
Project description:Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.
| S-EPMC7302208 | biostudies-literature