Genome-wide CRISPR screen for Zika virus resistance in human neural cells

Abstract

Zika virus (ZIKV) is a neurotropic and neurovirulent arbovirus that has severe detrimental impact on the developing human fetal brain. To date, little is known about the factors required for ZIKV infection of human neural cells. We identified ZIKV host genes in human pluripotent stem cell (hPSC)-derived neural progenitors (NPs) using a genome-wide CRISPR-Cas9 knockout screen. Mutations of host factors involved in heparan sulfation, endocytosis, endoplasmic reticulum processing, Golgi function, and interferon activity conferred resistance to infection with the Uganda strain of ZIKV and a more recent North American isolate. Host genes essential for ZIKV replication identified in human NPs also provided a low level of protection against ZIKV in isogenic human astrocytes. Our findings provide insights into host-dependent mechanisms for ZIKV infection in the highly vulnerable human NP cells and identify molecular targets for potential therapeutic intervention.

Keywords: CRISPR screen; Zika virus; fetal CNS infection; human pluripotent stem cells; neural progenitors.

Fig. 1.

Genome-wide screen for ZIKV^U resistance in human iPSC-derived NPs. (A) Schematic diagram of CRISPR screen with a genome-wide library in iPS-wt5 NPs. (B and C) Surviving NPs displayed minimal level of ZIKV protein (B) and greatly diminished viral RNA (vRNA) release into the medium (C). Control NPs were infected with multiplicity of infection (MOI) 1 ZIKV^U and collected at 48 h (B) or 72 h (C) postinfection. (Scale bars in B: 100 μm.) (D) Surviving NPs rechallenged with MOI 1 ZIKV^U and ZIKV^PR displayed low ZIKV infectivity at 7 d postinfection. Control NPs infected with ZIKV^U and ZIKV^PR were collected at 3 d and 7 d postinfection, respectively. (Scale bars in D: 100 μm.) (E) Six main pathways were identified as protective against ZIKV^U infection, upon gene knockouts. The size of the circles represents the magnitude of the increase in sgRNA representation after ZIKV^U exposure. *P < 0.05.

Fig. 2.

Secondary CRISPR knockout screen in human NPs against ZIKV^U. (A) Schematic diagram of CRISPR screen with a focused library in iPS-wt5 NPs. (B) Focused library contained gene hits with confirmed expression in human NPs on the baseline and 24 h after multiplicity of infection (MOI) 1 ZIKV^U infection. (C) Focused screen in iPS-wt5 NPs identified most of the hits previously identified in the genome-wide screen. The size of the colored circles represents the increase in sgRNA representation after ZIKV^U exposure. FPKM, fragments per kilobase of exon model per million reads mapped. *P < 0.05.

Fig. 3.

Validation of ZIKV host factors with individual sgRNAs in human NPs. (A and B) Individually targeted iPS-wt5 NPs infected with multiplicity of infection (MOI) 0.5 ZIKV^U (A) and ZIKV^PR (B) showed reduced viral RNA released into the supernatant at 72 h postinfection, compared with ZIKV-infected control NPs. All experimental sgRNA results were significant (P < 0.001) compared with control sgRNA, except sgRNA-2 against STAT3 infected with ZIKV^PR. (C) Immunostaining for ZIKV envelope (green) and DAPI (blue) showed reduced infection by MOI 0.5 ZIKV^PR in targeted human NPs, 96 h postinfection. (Scale bar: 1,000 μm.) Three technical replicates. Results are mean ± SEM.

Fig. 4.

Validation of host pathways using pharmacological compounds and in gene-edited brain organoids. (A and B) Depletion of cellular heparan sulfate with sodium chlorate reduced viral RNA load (A) and envelope protein staining (B) upon ZIKV^U and ZIKV^PR exposure. iPS-wt5 NPs were treated with multiplicity of infection (MOI) 0.1 ZIKV^U or MOI 1 ZIKV^PR and collected at 48 h or 72 h postinfection, respectively. (Scale bars in B: 100 μm.) (C and D) Blockade of acidification with bafilomycin A1 (BafA1) decreased ZIKV^U viral envelope protein staining. Low concentration (1 nM) bafilomycin A1 was sufficient to reduce ZIKV^U infection as quantified by viral RNA load (D). NPs were infected with MOI 0.1 ZIKV^U and collected at 24 h (C) or 48 h (D) postinfection. (Scale bars in C: 100 μm.) (E and F) NPs Pretreated with IFN-γ showed reduced infection by ZIKV^U and ZIKV^PR, as observed by viral RNA load (E) and ZIKV envelope protein staining (F). NPs were infected with MOI 1 ZIKV^U and ZIKV^PR and collected at 48 h (E) or 72 h (F) postinfection. (Scale bars in E: 100 μm.) (G–I) ISG15 mutant WIBR3-derived brain organoids lacked ISG15 protein (G) and showed reduced ZIKV^PR infection, as shown by ZIKV protein (H) and viral RNA load (I). (Scale bars in H: 100 μm.) Two technical replicates. Results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5.

Investigation of the role of ZIKV host factors in human astrocytes. (A and B) Human astrocytes derived from iPS-wt5 were susceptible to ZIKV^U (A) and ZIKV^PR (B) infection. Astrocytes were infected with multiplicity of infection (MOI) 0.1 ZIKV^U or MOI 0.6 ZIKV^PR, and collected at 48 h. (Scale bars: 1,000 μm.) (C and D) Astrocytes targeted with individual sgRNAs show varying degrees of infection by ZIKV^U (C) and ZIKV^PR (D), as measured by viral RNA release into the medium. Astrocytes were infected with MOI 1 ZIKV^U or ZIKV^PR and collected at 72 h. Experimental sgRNA results that showed significant reduction compared with control sgRNA were noted. Two technical replicates. Results are mean ± SEM. *P < 0.05, ***P < 0.001, **P < 0.01, ****P < 0.0001.