CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

Abstract

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.

Fig. 1. Generation of a porcine genome-wide lentiviral sgRNA library.

a Pipeline for sgRNA library design and construction. Protein-coding genes from Ensemble, lncRNAs from the domestic-animal lncRNA database (ALDB), and miRNAs from the miRBase database. Primers indicates by blue arrows were used for amplification of the sgRNA targeting sequences from the synthesized oligo array, which were cloned into the lentiviral plasmid. b The number of designed sgRNA targets. c, d Sequencing result of sgRNAs targeting sequences in two CRISPR pooled sgRNA libraries. Plasmid pools (c) and sorted mutant cell populations (d) containing the whole CRISPR pooled sgRNA library were characterized using next-generation sequencing (NGS). The curve indicates the distribution of sgRNAs. e The number of sgRNAs per gene in the genome-wide CRISPR pooled sgRNA library from the designed, plasmid, or mutant cell pools. sgRNA, small guide RNA; PCR, Polymerase chain reaction; hU6, human U6 promoter; Ubi, Ubiquitin promoter; CMV, Human cytomegalovirus promoter; EGFP, Enhanced green fluorescent protein; TTTT, U6 terminator sequence; LTR, long terminal repeat; hspCas9, human codon-optimized Streptococcus pyogenes Cas9; Puro, puromycin; LncRNA, Long non-coding RNA; Designed, sgRNA designed by CRISPR-offinder software; Plasmid, the sequencing result of sgRNA library from plasmid pools; Cell pool, the sequencing result of sgRNA library from sorted mutant cell populations. Source data are provided as Supplementary Data files.

Fig. 2. JEV-resistance screen in PK-15 cells using the porcine genome-scale CRISPR/Cas9 knockout cell collection.

a Workflow and screening strategy for the CRISPR/Cas9 screen. b, c Scatter plots comparing sgRNA targeting sequences frequencies and extent of enrichment vs. the noninoculated control mutant cell pool for the third or fourth (c) rounds of JEV screens after challenge. Counts_JEV3rd and Counts_JEV4th represent the average values of the read counts from paired-end sequencing, respectively. d, e Venn diagrams showing the overlapping enrichment of specific sgRNAs targeting sequences in the third or fourth rounds of JEV screens after challenge. For d, among the top 0.1% of averaged reads for the sgRNAs; for e, among the top 0.5%. f KEGG pathway enrichment analyses for the top 0.5% ranked sgRNA targets from the third and fourth JEV challenge rounds. JEV, Japanese encephalitis virus; MOI, multiplicity of infection; sgRNA, small guide RNA; JEV3rd, the third challenge round of JEV screening; JEV4th, the fourth challenge round of JEV screening; FACS, Fluorescence-activated cell sorting; JEV-RP9, JEV, genotype 3, strain RP9. Source data are provided as Supplementary Data files.

Fig. 3. Significant enrichment of specific sgRNAs targeting 10 genes involved in HSPGs synthesis and metabolic pathways.

a Venn diagram showing the overlapping enrichment of specific sgRNA targeted genes identified in the JEV screen of the cell knockout collection, HSPGs biosynthesis and metabolism, and GAG metabolism pathways. b Enrichment of specific sgRNAs targeting 10 genes involved in HSPGs synthesis and metabolic pathways. Only the top 0.5% of sgRNA was counted. c Schematic diagram, adapted from Tanaka et al. , showing the various classes of chemical components comprising HSPGs with known enzymes for their biosynthesis. Red indicates targeted genes identified in this study. d Schematic diagram, adapted from Blondel et al. , showing the known components and localization information for the sulfurylation modifications known to occur for some HSPGs. Red indicates targeted genes identified in this study. JEV, Japanese encephalitis virus; HS, Heparan sulfate; GAG, Glycosaminoglycan; HSPGs, heparan sulfate proteoglycans; PAPS, 3’-Phosphoadenosine-5’-phosphosulfate; JEV3rd, the third challenge round of JEV screening; JEV4th, the fourth challenge round of JEV screening. Source data are provided as a Supplementary Data file.

Fig. 4. Knockout of genes coding for the HSPGs pathway proteins SLC35B2, HS6ST1, B3GAT3, and GLCE significantly inhibits JEV replication in PK-15 cells.

a Alignment of the nucleic acid sequences of clonal KO cells of SLC35B2, HS6ST1, B3GAT3, and GLCE with WT cells. sgRNA targeting sites are highlighted in red. The red characters “-” indicate the deleted bases in the KO cells. PAM sites are indicated in blue letters. b Virus plaque assays for determination of viral concentration in clonal SLC35B2, HS6ST1, B3GAT3, and GLCE KO cell lines following infection with JEV at an MOI of 0.03 or 0.1. c Absolute quantitative real-time PCR for determination of JEV copy number in clonal SLC35B2, HS6ST1, B3GAT3, and GLCE KO cell lines following infection with JEV at an MOI of 0.03 or 0.1. d Detection of HSPGs sulfurylation level in clonal SLC35B2 and HS6ST1 KO cell lines by immunofluorescence. Scale bar, 200 μm. e, f Rescue assays for ectopic expression of HS6ST1 in HS6ST1-deficient cells. HS6ST1-KO-rescue: Transfection of pcDNA3.1-HS6ST1 vector in HS6ST1-deficient cells; HS6ST1-KO-NTC: Transfection of pcDNA3.1 empty vector in HS6ST1-deficient cells. g, h Rescue assays for ectopic expression of GLCE in GLCE-deficient cells. GLCE-KO-rescue: Transfection of pcDNA3.1-GLCE vector in GLCE-deficient cells; GLCE-KO-NTC: Transfection of pcDNA3.1 empty vector in GLCE-deficient cells. Detection of NS3 by immunofluorescence microscopy, the original data came from Supplementary Fig. 10a (e) and Supplementary Fig. 10c (g), respectively. RT-qPCR assay (f, h) for determination of relative mRNA level of JEV C gene in rescue assays of HS6ST1 and GLCE, respectively. PAM, protospacer adjacent motif; JEV, Japanese encephalitis virus; MOI, multiplicities of infection; hpi, hours post-infection; KO, knockout; WT, wild-type; DAPI, 4’, 6-diamidino-2-phenylindole. Data are represented as means ± S.D.; n = 3 (b, c, e, f, g, h). *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant. P values were determined by two-sided Student’s t-test. Source data are provided as a Source Data file.

Fig. 5. The EMC3 is required for JEV replication.

a Alignment of the nucleic acid sequences of two clonal KO cells of EMC3 with WT cells. sgRNA targeting sites are highlighted in red. The red characters “-” indicate the deleted bases, and black “^” characters indicate the inserted bases in KO cells. PAM site is indicated in blue letters. b Western blot analysis of EMC3. c Virus plaque assays. d Absolute quantitative real-time PCR. e Immunofluorescence for detection of NS3 expressed in EMC3 KO cell lines following infection with JEV. Scale bar, 200 μm. f Western blot was performed to examine the expression of the NS3 in EMC3 KO cell lines following infection with JEV. g Real-Time Cell Analyzer assay was performed to record the cell survival curves. h, i, j Rescue assays for ectopic expression of EMC3 in KO cells. RT-qPCR assay for determination of relative mRNA level of EMC3 (h); Detection of NS3 by Immunofluorescence, the original data came from Supplementary Fig. 16a (i). RT-qPCR assay for determination of relative mRNA level of JEV C gene (j). EMC3-KO-1-rescue: Transfection of pcDNA3.1-EMC3 vector in KO cells; EMC3-KO-1-NTC: Transfection of pcDNA3.1 empty vector in KO cells. k Evaluation of the effects of EMC3 knockout on virus particle assembly by negative-staining electron microscopy. Numerous scattered virus-like particles were present in the diluted cisternae of the ER (Red arrow). The pink arrow indicates KO cells displayed dramatic changes in ER morphology after JEV infection. Scale bar, 1 µm or 100 nm. PAM, protospacer adjacent motif; JEV, Japanese encephalitis virus; Mock, non-infected cells was included as negative control; MOI, multiplicities of infection; hpi, hours post-infection; KO, knockout; WT, wild-type; DAPI, 4’, 6-diamidino-2-phenylindole; kDa, kilodalton. The experiments were repeated three times with similar results and representative results shown (k). Data are represented as means ± S.D.; n = 3 (c, d, h, i, j). *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant. P values were determined by two-sided Student’s t-test.

Fig. 6. The CALR protein of the endoplasmic reticulum lumen is required for JEV replication.

a Alignment of the nucleic acid sequences of KO cells of CALR with WT cells. sgRNA targeting sites are highlighted in red. The black “^” characters indicate the inserted bases in KO cells. PAM site is indicated in blue letters. b Western blot analysis of CALR in KO and WT cells. c Virus plaque assays. d Absolute quantitative real-time PCR. e Immunofluorescence for detection of NS3 expressed in CALR KO cell line following infection with JEV. Scale bar, 200 μm. f Real-Time Cell Analyzer assay was performed to record the cell survival curves. g, h, i Rescue assays for ectopic expression of CALR in KO cells. RT-qPCR assay for determination of relative mRNA level of CALR (g); Detection of NS3 by Immunofluorescence, the original data came from Supplementary Fig. 16b (h). RT-qPCR assay for determination of relative mRNA level of JEV C gene for restoration of CALR expression in KO cells following infection with JEV (i). CALR-KO-rescue: Transfection of pcDNA3.1-CALR vector in KO cells; CALR-KO-NTC: Transfection of pcDNA3.1 empty vector in KO cells. j Evaluation of the effects of CALR knockout on virus particle assembly by negative-staining electron microscopy. Numerous scattered virus-like particles were present in the mitochondrial (Red arrow). The pink arrow indicates KO cells displaying dramatic changes in mitochondrial morphology after JEV infection. Scale bar, 1 µm or 100 nm. PAM, protospacer adjacent motif; JEV, Japanese encephalitis virus; Mock, non-infected cells was included as negative control; MOI, multiplicities of infection; hpi, hours post-infection; DAPI, 4’, 6-diamidino-2-phenylindole; KO, knockout; WT, wild-type; kDa, kilodalton. The experiments were repeated three times with similar results and representative results shown (j). Data are represented as means ± S.D.; n = 3 (c, d, g, h, i). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant. P values were determined by two-sided Student’s t-test. Source data are provided as a Source Data file.

Fig. 7. A proposed model for JEV entry and replication in porcine cells.

The JEV-infection cycle starts with binding to co-factors HSPGs, and/or unknown cellular receptors, followed by viral entry to enable replication. The EMC complex protein (EMC3 and EMC6) and CALR calcium-binding protein of the ER lumen are involved in JEV replication of host cells. Subsequently, the JEV RNA genome is replicated, viral particles are matured and packaged, and are released from cells. JEV, Japanese encephalitis virus; HSPG, heparan sulfate proteoglycan; PAPS, 3′-Phosphoadenosine-5′-phosphosulfate; PAP, 3′-phosphoadenosine-5′-phosphate; ATP, Adenosine triphosphate; ADP, Adenosine diphosphate; APS, Adenosine 5′ phosphosulfate; PPI, pyrophosphate; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated protein degradation; EMC, endoplasmic reticulum membrane protein complex.