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. 2020 Nov 11;5(6):e00914-20.
doi: 10.1128/mSphere.00914-20.

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 #  1 , Erika Girardi #  1 , Richard Patryk Ngondo  2 , Vladimir Lupashin  3 , Sébastien Pfeffer  4
Affiliations

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 et al. mSphere. .

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.

Keywords: CRISPR-Cas9 screen; complex oligomeric Golgi complex; double-stranded RNA; heparan-sulfate; transfection; virus.

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Figures

FIG 1
FIG 1
CRISPR-Cas9 survival screen of long dsRNA identifies the extracellular heparan sulfates as necessary for nucleic acid internalization and cell death induction. (A) Schematic representation of the CRISPR-Cas9 approach. HCT116 cells stably expressing a human codon-optimized S. pyogenes Cas9 protein were transduced with the lentiviral sgRNA library Brunello (MOI, 0.3). Thirty million transduced cells per replicate were selected with 1 μg/ml puromycin to obtain a mutant cell population to cover at least 300× the library. Selective pressure via synthetic long dsRNA (1 μg/ml) was applied to induce cell death (in red). DNA libraries from input cells and cells surviving the dsRNA treatment as three independent biological replicates were sequenced on an Illumina HiSeq 4000 instrument. Comparisons of the relative sgRNA boundance under the input and dsRNA conditions were done using the MAGeCK standard pipeline. (B) Median normalized read count distribution of all sgRNAs for the input (in black) and dsRNA (in red) replicates. (C) Bubble plot of the candidate genes. Significance of robust rank aggregation (RRA) score was calculated for each gene in the dsRNA condition compared with that of input using the MAGeCK software. The number of enriched sgRNAs for each gene is represented by the bubble size. The gene ontology pathways associated with the significant top hits are indicated in orange and green. (D) Viability assay. Cells were transfected (80,000 cells; 1 μg/ml) or nucleofected (200,000 cells; 400 ng) with synthetic long dsRNA, and cell viability was quantified 24 h (nucleofection) or 48 h (transfection) posttreatment using PrestoBlue reagent. The average of at least three independent biological experiments ± SD is shown. One-way ANOVA analysis; *, P < 0.05. (E, F) Cy5-labeled dsRNA (80,000 cells; 1 μg/ml) was transfected into HCT116cas9, B4GALT7#1 and 2, and SLC35B2#1 and #2 cells; and Cy5 fluorescence was quantified using FACS (10,000 events). The relative number of the Cy5-positive (Cy5+) cells (E) and the relative median of Cy5 intensity of fluorescence (F) compared to those of HCT116cas9 cells are shown. The average of three independent biological experiments ± SD is shown. Paired t test analysis; *, P < 0.05. (G) Quantification of extracellular heparan sulfates. FACS analysis of HCT116 control or KO clones stained with the HS-specific antibody 10E4 (in red) compared to unstained samples (in blue) (10,000 events). One representative experiment out of three is shown.
FIG 2
FIG 2
COG4 is a novel host susceptibility factor to long dsRNA-induced cell death. (A) Viability assay. Cells (80,000 cells; 1 μg/ml) were transfected with dsRNA, and then the viability of the cells was quantified 48 h posttransfection using PrestoBlue reagent. Data from at least three independent biological experiments are shown. One-way ANOVA analysis; *, P < 0.05. (B, C) Cy5-labeled dsRNA transfection (80,000 cells; 1 μg/ml) in HEK293T, KO COG4, and rescued cells. Cy5 fluorescence was quantified using the FACSCalibur platform (10,000 events). The percentage of Cy5+ cells (B) and the relative median of Cy5 intensity of fluorescence (C) compared to parental HEK293T cells are shown. Average of three experiments ± SD is shown. Paired t test analysis; *, P < 0.05. (D, E) qPCR quantification of dsRNA and IFN-β. Cells (300,000 cells; 1 μg/ml) were transfected with synthetic long dsRNA. Total RNA was extracted 24 h posttransfection and quantified by RT-qPCR. The histogram represents the expression fold change of synthetic dsRNA (D) and IFN-β mRNA (E) relative to GAPDH mRNA in dsRNA-transfected HEK293T KO COG4 rescued compared to HEK293T KO COG4. The average of three independent biological experiments ± SD is shown. Paired t test analysis; *, P < 0.05. (F, G) poly(I·C) rhodamine transfection and immunofluorescence in HEK293T and HEK293T KO COG4. Cells were transfected with rhodamine-labeled poly(I·C) (in red) and with a Rab5-GFP plasmid (in green). Images were acquired using a spinning disk microscope at different times posttransfection. Representative pictures (E) and the approximative number of rhodamine-positive foci per cells quantified by counting 7 fields per conditions (F) are shown. Two-way ANOVA analysis; *, P < 0.05.
FIG 3
FIG 3
CRISPR-Cas9 screen identifies COG4 as a permissivity factor to SINV. (A) Schematic representation of the CRISPR-Cas9 approach. HCT116 cells stably expressing a human codon-optimized S. pyogenes Cas9 protein were transduced with the lentiviral sgRNA library Brunello (MOI, 0.3). Thirty million transduced cells per replicate were selected with 1 μg/ml puromycin to obtain a mutant cell population to cover at least 300× the library. Selective pressure via SINV infection (MOI, 0.1) was applied to induce cell death (in red). DNA libraries from input cells and cells surviving the dsRNA treatment as three independent biological replicates were sequenced on an Illumina HiSeq 4000 instrument. Comparisons of the relative sgRNA boundance under the input and dsRNA conditions were done using the MAGeCK standard pipeline. (B) Median normalized read count distribution of all sgRNAs for the input (in black) and SINV (in red) replicates. (C) Bubble plot of the candidate genes. Significance of RRA score was calculated for each gene under the dsRNA condition compared with that of input using the MAGeCK software. The number of enriched sgRNAs for each gene is represented by the bubble size. The gene ontology pathways associated with the significant top hits are indicated in orange and green. (D) Viability of cells upon SINV infection. Cells were infected with SINV at MOI of 0.1, and then the viability of the cells was quantified 24, 48, and 72 h postinfection using PrestoBlue reagent. One-way ANOVA analysis; *, P < 0.05. (D) SINV GFP plaque assay. WT, COG4KO, and rescued HEK293T cells were infected with SINV GFP for 24 and 48 h at an MOI of 1, and the supernatant was collected in order to measure viral production. The fold change in titer relative to HEK293T arbitrarily set to 1 is shown. The average of three independent biological experiments ± SD is shown. Paired t test analysis; *, P < 0.05.
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