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.

Olivier Petitjean,Vladimir Lupashin,Erika Girardi,Sébastien Pfeffer,Richard Patryk Ngondo,Urs F Greber

doi:10.1128/msphere.00914-20

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.

Highlights

Upon infection by a virus, numerous mechanisms are put in place at the cellular level to raise the alarm and get rid of, or at least limit, the invader
We identified and characterized COG4, a component of the Conserved Oligomeric Golgi (COG) complex, as a novel factor involved in susceptibility to Double stranded RNA (dsRNA) and viral induced cell death linked to the heparan sulfate biogenesis pathway
Genome-wide CRISPR/Cas9 screen based on cell survival upon dsRNA transfection identify factors of the heparan sulfate pathway

Summary

Introduction

Upon infection by a virus, numerous mechanisms are put in place at the cellular level to raise the alarm and get rid of, or at least limit, the invader. Glycosaminoglycans, and more precisely heparan sulfates are ubiquitously expressed in human cells. They possess a global negative charge that is able to interact electrostatically with the basic residues that are exposed by viral surface glycoproteins. This allows viruses to increase their concentration at cell surface and so the possibility to interact with their specific entry receptor [2]. Alphaviruses such as Semliki Forest virus (SFV) and Sindbis virus (SINV) are enveloped positive-strand RNA viruses that contain two glycoproteins at the envelope, the proteins E1 and E2. E2 is involved in the interaction of the virus particle to the cell surface [3, 4], while E1 serves in the fusion process [5]

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Conclusion