scholarly journals A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yunkai Zhu ◽  
Fei Feng ◽  
Gaowei Hu ◽  
Yuyan Wang ◽  
Yin Yu ◽  
...  
Keyword(s):  
Critical Role ◽  
Surface Expression ◽  
Host Factors ◽  
Spike Protein ◽  
Hamster Model ◽  
Angiotensin Converting Enzyme 2 ◽  
Genome Wide ◽  
A Genome ◽  
Public Health Challenges ◽  
Crispr Screen

AbstractThe global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

scholarly journals The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

2020 ◽  
Author(s):  
Yunkai Zhu ◽  
Fei Feng ◽  
Gaowei Hu ◽  
Yuyan Wang ◽  
Yin Yu ◽  
...  
Keyword(s):  
Critical Role ◽  
Surface Expression ◽  
Spike Protein ◽  
Hamster Model ◽  
Entry Pathway ◽  
Genome Wide ◽  
A Genome ◽  
Model Animal ◽  
Public Health Challenges

SUMMARYThe global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.

scholarly journals A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Cell Reports ◽  
2021 ◽  
Vol 34 (11) ◽  
pp. 108859
Author(s):  
Jessie Kulsuptrakul ◽  
Ruofan Wang ◽  
Nathan L. Meyers ◽  
Melanie Ott ◽  
Andreas S. Puschnik
Keyword(s):  
Virus Infection ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001490
Author(s):  
Annika Kratzel ◽  
Jenna N. Kelly ◽  
Philip V’kovski ◽  
Jasmine Portmann ◽  
Yannick Brüggemann ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Huh7 Cell ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Dependent Inhibition ◽  
Crispr Screen ◽  
Human Coronaviruses ◽  
Approved Drugs ◽  
Huh7 Cell Line

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged—Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

scholarly journals A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

2021 ◽  
Author(s):  
Annika Kratzel ◽  
Jenna N. Kelly ◽  
Yannick Brueggemann ◽  
Jasmine Portmann ◽  
Philip V’kovski ◽  
...  
Keyword(s):  
Host Factors ◽  
The Novel ◽  
Genome Wide ◽  
A Genome ◽  
Dependent Inhibition ◽  
Crispr Screen ◽  
Dose Dependent Inhibition ◽  
Autophagy Pathway ◽  
Approved Drugs ◽  
Dose Dependent

SummaryOver the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) – SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 – has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

scholarly journals Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Bo Li ◽  
Sara M. Clohisey ◽  
Bing Shao Chia ◽  
Bo Wang ◽  
Ang Cui ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Meta Analysis ◽  
Host Factors ◽  
Validation Data ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Influenza A Virus Infection

AbstractHost dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.

scholarly journals A genome-wide haploid genetic screen for essential factors in vaccinia virus infection identifies TMED10 as regulator of macropinocytosis

2018 ◽  
Author(s):  
Rutger David Luteijn ◽  
Ferdy R van Diemen ◽  
Vincent A Blomen ◽  
Ingrid GJ Boer ◽  
Saravanan Manikam Sadasivam ◽  
...  
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Critical Role ◽  
Host Factors ◽  
Sequencing Analysis ◽  
Vaccinia Virus Infection ◽  
Genome Wide ◽  
A Genome ◽  
Viral Vaccine

Vaccinia virus is a promising viral vaccine and gene delivery candidate, and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to Modified Vaccinia Virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2 and TMED10 TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical role of EXT1 in heparan sulfate synthesis and vaccinia virus infection was confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, suggesting that TMED10 regulates actin cytoskeleton remodelling necessary for virus infection.

scholarly journals Genome-wide identification of cyclin-dependent kinase (CDK) genes affecting adipocyte differentiation in cattle

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Cuili Pan ◽  
Zhaoxiong Lei ◽  
Shuzhe Wang ◽  
Xingping Wang ◽  
Dawei Wei ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Analysis ◽  
Adipocyte Differentiation ◽  
Bos Taurus ◽  
Adipogenic Differentiation ◽  
Critical Role ◽  
Bos Indicus ◽  
Specific Expression ◽  
Genome Wide ◽  
A Genome

Abstract Background Cyclin-dependent kinases (CDKs) are protein kinases regulating important cellular processes such as cell cycle and transcription. Many CDK genes also play a critical role during adipogenic differentiation, but the role of CDK gene family in regulating bovine adipocyte differentiation has not been studied. Therefore, the present study aims to characterize the CDK gene family in bovine and study their expression pattern during adipocyte differentiation. Results We performed a genome-wide analysis and identified a number of CDK genes in several bovine species. The CDK genes were classified into 8 subfamilies through phylogenetic analysis. We found that 25 bovine CDK genes were distributed in 16 different chromosomes. Collinearity analysis revealed that the CDK gene family in Bos taurus is homologous with Bos indicus, Hybrid-Bos taurus, Hybrid Bos indicus, Bos grunniens and Bubalus bubalis. Several CDK genes had higher expression levels in preadipocytes than in differentiated adipocytes, as shown by RNA-seq analysis and qPCR, suggesting a role in the growth of emerging lipid droplets. Conclusion In this research, 185 CDK genes were identified and grouped into eight distinct clades in Bovidae, showing extensively homology. Global expression analysis of different bovine tissues and specific expression analysis during adipocytes differentiation revealed CDK4, CDK7, CDK8, CDK9 and CDK14 may be involved in bovine adipocyte differentiation. The results provide a basis for further study to determine the roles of CDK gene family in regulating adipocyte differentiation, which is beneficial for beef quality improvement.

scholarly journals A Genome-Wide Screen in Saccharomyces cerevisiae Reveals a Critical Role for Oxidative Phosphorylation in Cellular Tolerance to Lithium Hexafluorophosphate

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 888
Author(s):  
Xuejiao Jin ◽  
Jie Zhang ◽  
Tingting An ◽  
Huihui Zhao ◽  
Wenhao Fu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxidative Phosphorylation ◽  
Cellular Response ◽  
Critical Role ◽  
Lithium Ion ◽  
Deletion Mutants ◽  
High Concentration ◽  
Genome Wide ◽  
A Genome ◽  
Lithium Hexafluorophosphate

Lithium hexafluorophosphate (LiPF6) is one of the leading electrolytes in lithium-ion batteries, and its usage has increased tremendously in the past few years. Little is known, however, about its potential environmental and biological impacts. In order to improve our understanding of the cytotoxicity of LiPF6 and the specific cellular response mechanisms to it, we performed a genome-wide screen using a yeast (Saccharomyces cerevisiae) deletion mutant collection and identified 75 gene deletion mutants that showed LiPF6 sensitivity. Among these, genes associated with mitochondria showed the most enrichment. We also found that LiPF6 is more toxic to yeast than lithium chloride (LiCl) or sodium hexafluorophosphate (NaPF6). Physiological analysis showed that a high concentration of LiPF6 caused mitochondrial damage, reactive oxygen species (ROS) accumulation, and ATP content changes. Compared with the results of previous genome-wide screening for LiCl-sensitive mutants, we found that oxidative phosphorylation-related mutants were specifically hypersensitive to LiPF6. In these deletion mutants, LiPF6 treatment resulted in higher ROS production and reduced ATP levels, suggesting that oxidative phosphorylation-related genes were important for counteracting LiPF6-induced toxicity. Taken together, our results identified genes specifically involved in LiPF6-modulated toxicity, and demonstrated that oxidative stress and ATP imbalance maybe the driving factors in governing LiPF6-induced toxicity.

scholarly journals A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors

2016 ◽  
Vol 62 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Sergio Ruiz ◽  
Cristina Mayor-Ruiz ◽  
Vanesa Lafarga ◽  
Matilde Murga ◽  
Maria Vega-Sendino ◽  
...  
Keyword(s):  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Rowena DeJesus ◽  
Francesca Moretti ◽  
Gregory McAllister ◽  
Zuncai Wang ◽  
Phil Bergman ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Er Stress Response ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Selection Step ◽  
A Cell ◽  
Cell Type Specific ◽  
Cellular Pathways ◽  
Mtor Signalling

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

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