Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhao D
PROVIDER: S-EPMC7994959 | biostudies-literature | 2021 Sep
REPOSITORIES: biostudies-literature
Publications
Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2.
Zhao Dan D Xu Weifan W Zhang Xiaofan X Wang Xiaoting X Ge Yiyue Y Yuan Enming E Xiong Yuanpeng Y Wu Shenyang S Li Shuya S Wu Nian N Tian Tingzhong T Feng Xiaolong X Shu Hantao H Lang Peng P Li Jingxin J Zhu Fengcai F Shen Xiaokun X Li Haitao H Li Pilong P Zeng Jianyang J
Protein & cell 20210326 9
Similar Datasets
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:The SARS-CoV-2 nucleocapsid (N) protein is crucial for virus replication and genome packaging. N protein forms biomolecular condensates both in vitro and in vivo in a process known as liquid-liquid phase separation (LLPS), but the exact factors regulating LLPS of N protein are not fully understood. Here, we show that pH and buffer choice have a profound impact on LLPS of N protein. The degree of phase separation is highly dependent on the pH of the solution, which is correlated with histidine protonation in N protein. Specifically, we demonstrate that protonation of H356 is essential for LLPS in phosphate buffer. Moreover, electrostatic interactions of buffer molecules with specific amino acid residues are able to alter the net charge of N protein, thus influencing its ability to undergo phase separation in the presence of RNA. Overall, these findings reveal that even subtle changes in amino acid protonation or surface charge caused by the pH and buffer system can strongly influence the LLPS behavior, and point to electrostatic interactions as the main driving forces of N protein phase separation. Further, our findings emphasize the importance of these experimental parameters when studying phase separation of biomolecules, especially in the context of viral infections where the intracellular milieu undergoes drastic changes and intracellular pH normally decreases.
| S-EPMC11151101 | biostudies-literature
Project description: Not available
| S-EPMC8368218 | biostudies-literature
Project description:Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.
| S-EPMC9371935 | 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:Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid
| PRJNA682223 | ENA
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