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

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

Cell Reports ◽

10.1016/j.celrep.2021.108859 ◽

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

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

Nature Communications ◽

10.1038/s41467-019-13965-x ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 24

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.

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Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei.

Journal of Virology ◽

10.1128/jvi.15.2.305-315.1975 ◽

1975 ◽

Vol 15 (2) ◽

pp. 305-315 ◽

Cited By ~ 35

Author(s):

P LaColla ◽

A Weissbach

Keyword(s):

Virus Infection ◽

Vaccinia Virus ◽

Host Cell ◽

Hela Cells ◽

Cell Nuclei ◽

Vaccinia Virus Infection

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Cell Surface Proteoglycans Are Necessary for A27L Protein-Mediated Cell Fusion: Identification of the N-Terminal Region of A27L Protein as the Glycosaminoglycan-Binding Domain

Journal of Virology ◽

10.1128/jvi.72.10.8374-8379.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 8374-8379 ◽

Cited By ~ 72

Author(s):

Jye-Chian Hsiao ◽

Che-Sheng Chung ◽

Wen Chang

Keyword(s):

Virus Infection ◽

Cell Surface ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Cell Fusion ◽

Binding Domain ◽

Terminal Region ◽

Virus Binding ◽

Vaccinia Virus Infection

ABSTRACT We previously showed that vaccinia virus infection of BSC40 cells was blocked by soluble heparin, suggesting that cell surface heparan sulfate mediates vaccinia virus binding (C.-S. Chung, J.-C. Hsiao, Y.-S. Chang, and W. Chang, J. Virol. 72:1577–1585, 1998). In this study, we extended our previous work and demonstrated that soluble A27L protein bound to heparan sulfate on cells and interfered with vaccinia virus infection at a postbinding step. In addition, we investigated the structure of A27L protein that provides for its binding to heparan sulfate on cells. A mutant of A27L protein, named D-A27L, devoid of a cluster of 12 amino acids rich in basic residues, was constructed. In contrast to the soluble A27L protein, purified D-A27L protein was inactive in all of our assays, including binding to heparin in vitro, binding to heparan sulfate on cells, and the ability to block virus infection. These data demonstrated that the N-terminal region acts as a glycosaminoglycan (GAG)-binding domain critical for A27L protein binding to cells. Previously A27L protein was thought to be involved in fusion of virus-infected cells induced by acid treatment. When we investigated whether cell surface GAGs also participate in A27L-dependent fusion, our results indicated that soluble A27L protein blocked cell fusion, whereas D-A27L protein did not. Taken together, the results therefore demonstrated that A27L-mediated cell fusion is triggered by its interaction with cell surface GAGs through the N-terminal domain.

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A27L Protein Mediates Vaccinia Virus Interaction with Cell Surface Heparan Sulfate

Journal of Virology ◽

10.1128/jvi.72.2.1577-1585.1998 ◽

1998 ◽

Vol 72 (2) ◽

pp. 1577-1585 ◽

Cited By ~ 211

Author(s):

Che-Sheng Chung ◽

Jye-Chian Hsiao ◽

Yuan-Shau Chang ◽

Wen Chang

Keyword(s):

Virus Infection ◽

Cell Surface ◽

Vaccinia Virus ◽

Heparan Sulfate ◽

Mammalian Cells ◽

Myxoma Virus ◽

Cowpox Virus ◽

Virus Binding ◽

Vaccinia Virus Infection

ABSTRACT Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.

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