Arrayed CRISPR screen with image-based assay reliably uncovers host genes required for coxsackievirus infection

Figures

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  Figure 1.. Plasmid-based sgRNA constructs as a tool for a genetic perturbation screen in human cells. (A,B) HeLa cells were transfected with sgRNA-expressing plasmids (Control, CXADR, and PI4KB) and infected with CVB3 at an MOI of 5. At 8 h post-infection, cells were fixed and stained with anti-3C antibody (green) and DAPI (blue). (A) Quantification of infection. Mean ± SEM for quadruplicate experiments. (**) P < 0.01. (B) Representative images of infection are shown. (C) Mutation frequencies determined after transient transfection of 35 sgRNAs and Cas9 plasmid.

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  Figure 2.. Candidate identification. (A) Schematic presentation of the arrayed CRISPR screen. (B) Quantification of CVB3 infection in HeLa cells transfected with sgRNA-expressing plasmids targeting 10 genes from primary screens. Mean ± SEM for quadruplicate experiments. (**) P < 0.01, (n.s.) nonsignificant. (C) Similar quantification of virus infection in B using newly designed sgRNA. (D) The percentage of CVB3-infected cells (normalized to DMSO control) treated with three known inhibitors of the candidates. Mean ± SD for triplicate experiments. (E) HeLa cells were transfected with ACBD3 and CSDE1 targeting sgRNAs and infected with CVB3. Surviving cells were stained and are shown. Surviving colonies were expanded and the target region was sequenced to confirm the mutations. Red characters indicate the PAM and blue characters indicate insertions. hg19 is the wild-type sequence of each gene. As our target sites have the same sequence in both references (i.e., hg19 and GRCh38), using GRCh38 would not significantly affect our conclusions.

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  Figure 3.. CSDE1 is required for CVB3 infection. (A) Viability of wild-type (HeLa) and CSDE1 knockout (KO) cells after CVB3 infection. (B) Viral replication test of wild-type (HeLa) and CSDE1 KO cells using the CVB3 replicon. Transfection with cDNA encoding the eGFP-CSDE1 protein into CSDE1 KO cells rescued CVB3 replication. (C) Transfection with cDNA encoding the eGFP-CSDE1 protein into CSDE1 KO cells rescued CVB3 infection. After 8 h of CVB3 infection in wild-type (HeLa) and CSDE1 KO cells transfected with the plasmid encoding the eGFP-CSDE1 protein, cells were harvested and lysed for western blot analysis using anti-CSDE1 antibody, anti-VP1 antibody, and anti-beta actin antibody. (D) CVB3 IRES-dependent translation assay of wild-type (HeLa) and CSDE1 KO cells. Dual luciferase reporter plasmids containing the IRES sequence from five different viruses were transfected into cells and the activity of firefly luciferase and Renilla luciferase was measured.

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  Figure 4.. ACBD3 is required for CVB3 replication. (A) Viability of wild-type (HeLa) and ACBD3 knockout (KO) cells after CVB3 infection. (B) Viral replication assay of wild-type (HeLa) and ACBD3 KO cells using the CVB3 replicon. Transfection with cDNA encoding FLAG-tagged ACBD3 protein into ACBD3 KO cells rescued CVB3 replication, as shown by a viral replication assay using the CVB3 replicon. (C) Wild-type HeLa cells and ACBD3 KO cells transfected with control and plasmid encoding FLAG-tagged ACBD3 protein were infected with CVB3 at an MOI of 5. At 8 h post-infection, cells were fixed and stained with anti-3C antibody (green) and DAPI (blue). (D) Interaction between ACBD3 and the 3A protein of PV1 and CVB3 was confirmed using a mammalian two-hybrid system. (E) A fixed number of HeLa cells and KO cells were plated into six-well plates and incubated. On each day, live cells were counted and normalized to the initial number of cells.

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  Figure 5.. HeLa cells were transiently transfected with siRNAs and sgRNAs targeting ACBD3 or CSDE1. (A) Cells were infected with CVB3, fixed, and stained. The percentage of CVB3-infected cells (normalized to sgRNA or siRNA control). Mean ± SEM for triplicate experiments. (**) P < 0.001, (n.s.) nonsignificant. (B,C) Cells were transfected with control siRNA, ACBD3 siRNA, and CSDE1 siRNA. The expression level of each protein was measured by western blot using the anti-ACBD3 antibody (B) and anti-CSDE1 antibody (C).