RNA-Seq of SARS-CoV-2 Nucleocapsid protein expression in human embryonic kidney cell line HEK293
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB78832 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR13475000_1.fastq.gz | Fastqsanger.gz | |||
| ERR13475000_2.fastq.gz | Fastqsanger.gz | |||
| ERR13475001_1.fastq.gz | Fastqsanger.gz | |||
| ERR13475001_2.fastq.gz | Fastqsanger.gz | |||
| ERR13475002_1.fastq.gz | Fastqsanger.gz |
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Project description:The RNA genome of the SARS-CoV-2 virus encodes for four structural proteins, 16 non-structural proteins and nine putative accessory factors. A high throughput analysis of interactions between human and SARS-CoV-2 proteins identified multiple interactions of the structural Nucleocapsid (N) protein with RNA processing factors. The N-protein, which is responsible for packaging of the viral genomic RNA was found to interact with two RNA helicases, UPF1 and MOV10 that are involved in nonsense-mediated mRNA decay (NMD). NMD is a translation-coupled mechanism that targets mRNAs harboring a premature stop codon (PTC) for degradation, thereby serving as a quality control and gene regulatory pathway ensuring transcriptome integrity. Here, we wanted to explore the impact of transiently expressed N protein on the transcriptome of human embryonic kidney cell line HEK293 by RNA-Sequencing. To this end, the SARS-CoV2-N protein was transiently expressed from a pcDNA3.1-HA-N plasmid for 48 hours and the corresponding empty vector was used as a control.
2024-09-16 | E-MTAB-14342 | biostudies-arrayexpress
Project description:SARS-CoV-2, the causative agent of COVID-19, is a positive-sense, single-stranded RNA virus that causes a spectrum of disease severity, from asymptomatic infection to severe illness to long-term sequelae. Similar to other human coronaviruses, SARS-CoV-2 proteins modulate host genomic responses through epigenomic modifications, facilitating viral replication and immune evasion. While the nucleocapsid protein is well known for its role in RNA stability and immune modulation, its impact on host chromatin organization remains unclear. To investigate this, we generated stable human alveolar cell lines expressing nucleocapsid proteins from endemic and pandemic human coronaviruses. Our analysis revealed that nucleocapsid proteins from all tested coronaviruses induced changes in nucleosome positioning and occupancy at specific gene promoters involved in coagulation pathways, hormone signaling, and innate immune responses. Additionally, SARS-CoV-2-specific alterations were identified in genes dysregulated in severe infections, such as suggesting a direct role for epigenomic modifications in disease pathophysiology. We also observed extensive changes in nucleosome susceptibility to nuclease digestion in SARS-CoV and SARS-CoV-2 samples that were not observed in common cold cell lines. Promoters with altered sensitivity and resistance to nuclease were linked to innate immune, metabolic, olfactory, and signaling pathways known to be dysregulated in severe COVID-19 and post-acute sequelae (PASC). These findings demonstrate that nucleocapsid protein expression alters chromatin structure at specific loci, implicating viral proteins in host genome dysregulation. Furthermore, we identified both shared and unique chromatin targets of SARS-CoV-2 and common cold coronaviruses, highlighting pathways for further investigation and potential therapeutic intervention.
2025-02-26 | GSE290042 | GEO
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is highly expressed upon infection and is essential for viral replication, making it a promising target for antiviral drug and vaccine development. Here, starting from a functional proteomics workflow, we catalogued the protein-protein interactions of 28 SARS-CoV-2 proteins in HEK293 cells, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N protein localizes to stress granules and sequesters G3BP1 and G3BP2 away from their normal interaction partners, thus attenuating stress granule formation. N also binds directly to host mRNAs, with a preference for 3´ UTRs, and modulates target mRNA stability. We show that SARS-CoV-2 N protein rewires the G3BP1 mRNA-binding profile, thereby suppressing host gene expression changes induced in response to cellular stress.
2021-12-17 | GSE171009 | GEO
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein is highly expressed upon infection and is essential for viral replication, making it a promising target for antiviral drug and vaccine development. Here, starting from a functional proteomics workflow, we catalogued the protein-protein interactions of 28 SARS-CoV-2 proteins in HEK293 cells, including an evolutionarily conserved specific interaction of N with the stress granule resident proteins G3BP1 and G3BP2. N protein localizes to stress granules and sequesters G3BP1 and G3BP2 away from their normal interaction partners, thus attenuating stress granule formation. N also binds directly to host mRNAs, with a preference for 3´ UTRs, and modulates target mRNA stability. We show that SARS-CoV-2 N protein rewires the G3BP1 mRNA-binding profile, thereby suppressing host gene expression changes induced in response to cellular stress.
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Project description:We used endpoint PCR to verify the presence of a new subgenomic RNA in SARS-CoV-2 (termed N.iORF3) and verify using nanopore sequencing that this is expressed via a newly evolved transcription regulatory sequence (TRS). We further show that this encodes a truncated C-terminal portion of Nucleocapsid, which is an antagonist of type I interferon production. Using reverse genetics-derived viruses we show N.iORF3 contributes to viral fitness during infection and observe distinct phenotypes when the Nucleocapsid coding sequence is mutated compared to when the TRS alone is ablated.
2025-01-01 | E-MTAB-14681 | biostudies-arrayexpress
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:Many people experiencing long COVID symptoms, or post-acute sequelae of SARS-CoV-2 infection (PASC), suffer from debilitating neurologic symptoms (Neuro-PASC). Although symptoms of Neuro-PASC are widely documented, it is still unclear whether virus-specific adaptive immunity is affected in these individuals. We report that Neuro-PASC patients exhibit distinct immunological signatures composed of elevated CD4+ T cell responses toward the C-terminal region of SARS-CoV-2 Nucleocapsid protein when examined both functionally and using TCR sequencing. In contrast, Neuro-PASC patients also have diminished activation of CD8+ memory T cells and increased IL-6 production to Nucleocapsid. Elevated plasma immunoregulatory and reduced pro-inflammatory and antiviral response signatures were evident in Neuro-PASC patients compared with COVID convalescent controls without lasting symptoms, correlating with worse neurocognitive dysfunction. These data provide new insight into the impact of virus-specific cellular immunity on the pathogenesis of long COVID and pave the way for the rational design of predictive biomarkers and therapeutic interventions.
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Project description:Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5’-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.
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Project description:The COVID-19 pandemic is arguably the most important global threat to humankind that the world has seen in a century. SARS-CoV-2 has now reached 196 countries across all continents with over 159 million confirmed cases with 3.3 million deaths todate. Currently, our knowledge on how and why SARS-CoV-2 causes severe COVID-19 is continually evolving. Here, we identified three consecutive SNPs (G28881A, G28882A, G28883C) resulting in the Nucleocapsid (N) protein amino acid mutations R203K and G204R (simply termed KR mutation) by large-scale SARS-CoV-2 genome sequencing from clinical swab samples of patients. The nucleocapsid (N) protein of SARS-CoV-2, the highly abundant structure protein within the infected cells, serves multiple functions during viral infection, which besides RNA binding, playing essential roles in viral transcription, replication, and translation. We investigated the impact of this KR mutation on the functional properties of N protein in terms of host protein interaction and phosphorylation status by mass spectrometry.
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