Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

ABSTRACTYersinia pestishas evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis,Y. pestisprevents phagolysosome maturation and establishes a modified compartment termed theYersinia-containing vacuole (YCV).Y. pestisactively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requiresY. pestisinteractions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required forY. pestissurvival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival ofY. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated thatY. pestisactively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV byY. pestisto resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered thatY. pestisdisrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence thatY. pestistargets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.IMPORTANCEYersinia pestiscan infect and survive within macrophages. However, the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. To provide a better understanding of these mechanisms, we used an RNAi approach to identify host factors required for intracellularY. pestissurvival. This approach revealed that the host endocytic recycling pathway is essential forY. pestisto avoid clearance by the macrophage. We further demonstrate thatY. pestisremodels the phagosome to resemble a recycling endosome, allowing the bacterium to avoid the normal phagolysosomal maturation pathway. Moreover, we show that infection withY. pestisdisrupts normal recycling in the macrophage and that disruption is required for bacterial replication. These findings provide the first evidence thatY. pestistargets the host endocytic recycling pathway in order to evade killing by macrophages.

Download Full-text

A Genome-Wide Small Interfering RNA Screen Identifies Host Factors Required for Vesicular Stomatitis Virus Infection

Journal of Virology ◽

10.1128/jvi.00642-14 ◽

2014 ◽

Vol 88 (15) ◽

pp. 8355-8360 ◽

Cited By ~ 19

Author(s):

A. S.-Y. Lee ◽

R. Burdeinick-Kerr ◽

S. P. J. Whelan

Keyword(s):

Virus Infection ◽

Vesicular Stomatitis Virus ◽

Small Interfering Rna ◽

Host Factors ◽

Genome Wide ◽

A Genome ◽

Vesicular Stomatitis ◽

Interfering Rna

Download Full-text

Genome-Wide RNAi Screening to Identify Human Host Factors Crucial for Influenza Virus Replication

Advances in Delivery Science and Technology - RNA Interference from Biology to Therapeutics ◽

10.1007/978-1-4614-4744-3_12 ◽

2012 ◽

pp. 243-257

Author(s):

Katharina Ahrens ◽

Alexander Karlas

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Host Factors ◽

Rnai Screening ◽

Human Host ◽

Genome Wide ◽

Influenza Virus Replication ◽

Human Host Factors

Download Full-text

Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

Scientific Reports ◽

10.1038/srep24242 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 38

Author(s):

Sebastian Virreira Winter ◽

Arturo Zychlinsky ◽

Bart W. Bardoel

Keyword(s):

Staphylococcus Aureus ◽

Host Factors ◽

Genome Wide ◽

Crispr Screen ◽

Novel Host

Download Full-text

Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening

mBio ◽

10.1128/mbio.00606-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 77

Author(s):

Justin A. McDonough ◽

Hayley J. Newton ◽

Scott Klum ◽

Rachel Swiss ◽

Hervé Agaisse ◽

...

Keyword(s):

Coxiella Burnetii ◽

Secretion System ◽

Effector Proteins ◽

Host Factors ◽

Content Type ◽

Host Determinants ◽

Genome Wide ◽

Type Ivb Secretion ◽

Type Ivb Secretion System ◽

Bacterial Effector Proteins

ABSTRACTCoxiella burnetiiis an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used byC. burnetiito translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required forC. burnetiiintracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication ofC. burnetiiwas measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogatedC. burnetiireplication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles withinC. burnetii-infected cells. Silencing of theSTX17gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containingC. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed forC. burnetiireplication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate thatC. burnetiivacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur duringC. burnetiiinfection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.IMPORTANCEQ fever in humans is caused by the bacteriumCoxiella burnetii. Infection withC. burnetiiis marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets ofC. burnetiiinfection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of theC. burnetiivacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.

Download Full-text

Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

10.1101/476440 ◽

2018 ◽

Cited By ~ 1

Author(s):

Michael F. Wells ◽

Max R. Salick ◽

Federica Piccioni ◽

Ellen J. Hill ◽

Jana M. Mitchell ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Zika Virus ◽

Large Scale ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

Autism Spectrum ◽

Host Factors ◽

Protein Markers ◽

Genome Wide

SUMMARYNeural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe humanStem cell-derivedNGN2-acceleratedProgenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum genePTEN.

Download Full-text

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

PLoS Biology ◽

10.1371/journal.pbio.3001490 ◽

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.

Download Full-text

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

10.1101/2021.02.24.432634 ◽

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.

Download Full-text