Identification of required host factors for SARS-CoV-2 infection in human cells [ECCITE-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE159519 | GEO | 2020/10/24
REPOSITORIES: GEO
Similar Datasets
Project description:Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike’s interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.
2024-02-17 | GSE255644 | GEO
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Alongside investigations into the virology of SARS-CoV-2, understanding the host–virus dependencies are vital for the identification and rational design of effective antiviral therapy. Here, we report the dominant SARS-CoV-2 entry receptor, ACE2, conjugates with small ubiquitin-like modifier 3 (SUMO3) through a proteome-wide protein interaction analysis. We further demonstrate that E3 SUMO ligase PIAS4 prompts the SUMOylation and stabilization of ACE2, whereas deSUMOylation enzyme SENP3 reverses this process. Conjugation of SUMO3 with ACE2 at lysine (K) 187 hampers the K48-linked ubiquitination of ACE2, thus suppressing its subsequent cargo receptor TOLLIP-dependent autophagic degradation. Pharmacological intervention of ACE2 SUMOylation blocks the entry of SARS-CoV-2 and viral infection-triggered immune responses. Collectively, our findings suggest selective autophagic degradation of ACE2 orchestrated by SUMOylation and ubiquitination can be targeted to future antiviral therapy of SARS-CoV-2.
2022-07-18 | GSE171130 | GEO
Project description:Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor and didn’t express TMPRSS2 protease, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity to pursue efforts to uncover the full landscape of SARS-CoV-2 regulators. We performed genome-wide, bidirectional CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. As well as identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Additionally, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins (MUC1, MUC4 and MUC21), IL6R and CD44 were identified . We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A virus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.
2022-01-20 | GSE193834 | GEO
Project description:Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor and didn’t express TMPRSS2 protease, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity to pursue efforts to uncover the full landscape of SARS-CoV-2 regulators. We performed genome-wide, bidirectional CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. As well as identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Additionally, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins (MUC1, MUC4 and MUC21), IL6R and CD44 were identified . We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A virus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.
2021-05-28 | GSE175666 | GEO
Project description:The global spread of SARS-CoV-2 lead to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.
2022-03-27 | GSE197962 | GEO
Project description:The rapid and escalating spread of SARS coronavirus 2 (SARS-CoV-2) poses an immediate public health emergency, and no approved therapeutics or vaccines are currently available. The viral spike protein S binds membrane-tethered ACE2 on host cells in the lungs to initiate molecular events that ultimately release the viral genome intracellularly. The extracellular protease domain of ACE2 inhibits cell entry of both SARS and SARS-2 coronaviruses by acting as a soluble decoy for receptor binding sites on S, and is a promising candidate for therapeutic and prophylactic development. Using deep mutagenesis, variants of ACE2 are identified with increased binding to the receptor binding domain of S at a cell surface. Mutations are found across the protein-protein interface and also at buried sites where they are predicted to enhance folding and presentation of the interaction epitope. The mutational landscape offers a preliminary blueprint for engineering high affinity ACE2 receptors to meet this unprecedented challenge.
2020-03-19 | GSE147194 | GEO
Project description:Nanobodies are emerging as ideal instruments for drug design and several have recently been created to block SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not efficiently target Spike during viral entry. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for replication of the viral genome. To find out if this strategy efficiently blocked viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA-2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells subjected to LNP-mRNA-2NSP23 with multiple SARS-CoV-2 variants. Analysis of total RNA isolated form infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We propose that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.
2023-10-10 | GSE244714 | GEO
Project description:There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) which causes the disease COVID-19. SARS-CoV-2 spike (S)-protein binds ACE2, and in concert with host proteases, principally TMPRSS2, promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues, and the factors that regulate ACE2 expression, remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 amongst tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discover that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells, and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
2020-04-23 | GSE148829 | GEO
Project description:The Angiotensin-converting enzyme 2 (ACE2) receptor is the central entry point for SARS-Cov2. Several SAR-Cov2 substrains have developed mutations in their spike protein to maximize their use of ACE2, e.g. to strengthen ACE2 binding for increased uptake or adapt to specific amino acid properties of ACE2 to cross the species barrier. But little is known about the effect of host regulators on ACE2 and subsequently their impact on SARS-Cov2 infection. Here we identify the E3 ligase MDM2 as a ACE2 modulator. The knockout of MDM2 induced a strong pro-viral effect specific for SARS-Cov2 and we could see the increase of ACE2 levels. This effect is likely dependent on the ubiquitination site Lysine 788, which MDM2 uses to induce proteasomal degradation of ACE2. Substituting this amino acid led to increased ACE2 levels and increased SARS-CoV2 infection facilitated by enhanced SARS-Cov2 uptake.
2023-09-08 | PXD040891 | Pride
Project description:Nipah virus (NiV) is a highly lethal zoonotic virus with a potential large-scale outbreak, which poses a great threat to world health and security. Cell entry is the first step in viral infection, and the entry of NiV into host cells is inseparable from the attachment (G) and fusion (F) glycoproteins. To discover the host factors potentially involved in the entry of NiV into host cells, we systematically studied the interaction of NiV F and G proteins with host proteins using proximity labeling methods.
2023-12-22 | PXD048109 |