Fig 2: 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.

Fig 3: 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.