Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection

ABSTRACT Double-stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this...

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Autores principales:	Olivier Petitjean, Erika Girardi, Richard Patryk Ngondo, Vladimir Lupashin, Sébastien Pfeffer
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	CRISPR-Cas9 screen complex oligomeric Golgi complex double-stranded RNA heparan-sulfate transfection virus Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/ce2e1b6b2c6d4e95b2cdb7888fed112e
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spelling	oai:doaj.org-article:ce2e1b6b2c6d4e95b2cdb7888fed112e2021-11-15T15:31:13ZGenome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection10.1128/mSphere.00914-202379-5042https://doaj.org/article/ce2e1b6b2c6d4e95b2cdb7888fed112e2020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00914-20https://doaj.org/toc/2379-5042ABSTRACT Double-stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this study, we employed a genome-wide CRISPR-Cas9 loss-of-function screen based on cell survival in order to identify genes implicated in the host response to dsRNA. By challenging HCT116 human cells with either synthetic dsRNA or Sindbis virus (SINV), we identified the heparan sulfate (HS) pathway as a crucial factor for dsRNA entry, and we validated SINV dependency on HS. Interestingly, we uncovered a novel role for COG4, a component of the conserved oligomeric Golgi (COG) complex, as a factor involved in cell survival to both dsRNA and SINV in human cells. We showed that COG4 knockout led to a decrease of extracellular HS that specifically affected dsRNA transfection efficiency and reduced viral production, which explains the increased cell survival of these mutants. IMPORTANCE When facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded RNA (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing, and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the conserved oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.Olivier PetitjeanErika GirardiRichard Patryk NgondoVladimir LupashinSébastien PfefferAmerican Society for MicrobiologyarticleCRISPR-Cas9 screencomplex oligomeric Golgi complexdouble-stranded RNAheparan-sulfatetransfectionvirusMicrobiologyQR1-502ENmSphere, Vol 5, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR-Cas9 screen complex oligomeric Golgi complex double-stranded RNA heparan-sulfate transfection virus Microbiology QR1-502
spellingShingle	CRISPR-Cas9 screen complex oligomeric Golgi complex double-stranded RNA heparan-sulfate transfection virus Microbiology QR1-502 Olivier Petitjean Erika Girardi Richard Patryk Ngondo Vladimir Lupashin Sébastien Pfeffer Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection
description	ABSTRACT Double-stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this study, we employed a genome-wide CRISPR-Cas9 loss-of-function screen based on cell survival in order to identify genes implicated in the host response to dsRNA. By challenging HCT116 human cells with either synthetic dsRNA or Sindbis virus (SINV), we identified the heparan sulfate (HS) pathway as a crucial factor for dsRNA entry, and we validated SINV dependency on HS. Interestingly, we uncovered a novel role for COG4, a component of the conserved oligomeric Golgi (COG) complex, as a factor involved in cell survival to both dsRNA and SINV in human cells. We showed that COG4 knockout led to a decrease of extracellular HS that specifically affected dsRNA transfection efficiency and reduced viral production, which explains the increased cell survival of these mutants. IMPORTANCE When facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded RNA (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing, and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the conserved oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.
format	article
author	Olivier Petitjean Erika Girardi Richard Patryk Ngondo Vladimir Lupashin Sébastien Pfeffer
author_facet	Olivier Petitjean Erika Girardi Richard Patryk Ngondo Vladimir Lupashin Sébastien Pfeffer
author_sort	Olivier Petitjean
title	Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection
title_short	Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection
title_full	Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection
title_fullStr	Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection
title_full_unstemmed	Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection
title_sort	genome-wide crispr-cas9 screen reveals the importance of the heparan sulfate pathway and the conserved oligomeric golgi complex for synthetic double-stranded rna uptake and sindbis virus infection
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/ce2e1b6b2c6d4e95b2cdb7888fed112e
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Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection

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