A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis 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.

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

10.1101/2019.12.15.877100 ◽

2019 ◽

Cited By ~ 4

Author(s):

Jessie Kulsuptrakul ◽

Ruofan Wang ◽

Nathan L. Meyers ◽

Melanie Ott ◽

Andreas S. Puschnik

Keyword(s):

Protein Modification ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Rna Virus ◽

Translation Initiation Factor ◽

Host Factors ◽

Initiation Factor ◽

A Genome ◽

Crispr Screen ◽

Liver Organoids

AbstractHepatitis A virus (HAV) is a positive-sense RNA virus causing acute inflammation of the liver. Here, using a genome-scale CRISPR screen in a human hepatocyte cell line, we provide a comprehensive picture of the cellular factors, which are exploited by HAV during replication. We identified genes involved in sialic acid biosynthesis and members of the eukaryotic translation initiation factor complex, corroborating their putative roles in HAV infection. Additionally, we uncovered all components of the cellular machinery for UFMylation, a ubiquitin-like protein modification. We showed that HAV translation specifically depends on UFM1 conjugation of the ribosomal protein RPL26. Furthermore, we found that components related to the yeast Trf4/5– Air1/2–Mtr4 polyadenylation (TRAMP) complex, are required for viral translation, independent of controlling HAV poly(A) tails. While the identified HAV host factors were largely distinct compared to other picornaviruses, we highlighted a surprising co-dependency of HAV and hepatitis B virus (HBV) on the TRAMP-like complex. Finally, we demonstrated that pharmacological inhibition of the TRAMP-like complex decreased HAV replication in hepatocyte cells and human liver organoids, thus providing a strategy for host-directed therapy of HAV infection.

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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

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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.

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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.

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A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection

Journal of Virology ◽

10.1128/jvi.02160-18 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 6

Author(s):

Rutger D. Luteijn ◽

Ferdy van Diemen ◽

Vincent A. Blomen ◽

Ingrid G. J. Boer ◽

Saravanan Manikam Sadasivam ◽

...

Keyword(s):

Virus Infection ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Host Cell ◽

Host Factors ◽

Virus Infectivity ◽

Sequencing Analysis ◽

Vaccinia Virus Infection ◽

Genome Wide ◽

A Genome

ABSTRACTVaccinia 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 roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were 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, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCEPoxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

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

Nature Communications ◽

10.1038/s41467-021-21213-4 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 9

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.

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

10.1101/493205 ◽

2018 ◽

Cited By ~ 1

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.

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