Abstract
Picornaviruses are a leading cause of human and veterinary infections that result in various diseases, including polio and the common cold. As archetypical non-enveloped viruses, their biology has been extensively studied1. Although a range of different cell-surface receptors are bound by different picornaviruses2,3,4,5,6,7, it is unclear whether common host factors are needed for them to reach the cytoplasm. Using genome-wide haploid genetic screens, here we identify the lipid-modifying enzyme PLA2G16 (refs 8, 9, 10, 11) as a picornavirus host factor that is required for a previously unknown event in the viral life cycle. We find that PLA2G16 functions early during infection, enabling virion-mediated genome delivery into the cytoplasm, but not in any virion-assigned step, such as cell binding, endosomal trafficking or pore formation. To resolve this paradox, we screened for suppressors of the ΔPLA2G16 phenotype and identified a mechanism previously implicated in the clearance of intracellular bacteria12. The sensor of this mechanism, galectin-8 (encoded by LGALS8), detects permeated endosomes and marks them for autophagic degradation, whereas PLA2G16 facilitates viral genome translocation and prevents clearance. This study uncovers two competing processes triggered by virus entry: activation of a pore-activated clearance pathway and recruitment of a phospholipase to enable genome release.
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Acknowledgements
The authors thank T. Sixma, L. Wessels, S. Nijman, G. Superti-Furga, W. Fischl, G. Casari and members of the Brummelkamp laboratory for discussions and reading of the manuscript; R. Bin Ali for assistance with generating knockout mice; and K. Kirkegaard and H. Ploegh for providing reagents. This work was supported by SNSF Fellowship PA00P3_145411 to E.V.C., and funding from the Cancer Genomics Center (CGC.nl), Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)–VIDI grant 91711316, European Research Council (ERC) Starting Grant (ERC-2012-StG 309634) to T.R.B. Work in the lab of F.J.M.V.K. was supported by NWO-VICI grant 91812628.
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Contributions
J.S., F.J.M.V.K., A.P., J.E.C. and T.R.B. were responsible for the overall design of the study, J.S. and J.E.C. carried out the host factor screens, V.A.B. performed the bioinformatics analysis, L.G.V.D.H. assisted in mouse experiments and the generation and characterization of knockout cell lines, M.B. performed the suppressor screen, J.N. contributed to microscopy analysis, H.J. performed electron microscopy studies, E.V.C. analysed in vitro experiments, J.B. and H.J.T. helped with preparation of virus stocks, luciferase assays and preparation of the EV-A71 screen. J.S. and T.R.B. wrote the manuscript, all authors commented on the manuscript.
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T.R.B. is a co-founder and advisory board member of Haplogen. J.E.C. and T.R.B. are inventors on a patent describing PLA2G16 as potential antiviral target.
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Reviewer Information Nature thanks J. Bergelson, S. Lemon and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Identification and validation of PLA2G16 as a picornavirus host factor.
a, A haploid genetic screen to identify critical host factors for CV-B3 infection. b, Disruptive (sense orientation, blue) and undisruptive (antisense orientation, yellow) gene-trap insertions mapped to the genomic locus containing phospholipases PLA2G16, RARRES3 and HRASLS2 in different picornavirus screens. An unselected HAP1 dataset is shown for comparison. Disruptive gene-trap integrations are enriched in the PLA2G16 locus. c, Wild-type HAP1 cells, PLA2G16 gene-trapped cells, and PLA2G16 gene-trapped cells expressing cDNA of either wild-type PLA2G16 or PLA2G16-C113A were infected with an increasing amount of PV1 and CV-B1. Surviving cells were stained using crystal violet (n = 3). d, Western blot analysis for the presence of PLA2G16 in different cell lines. CDK4 and Rictor were used as a loading control. For gel source data, see Supplementary Fig. 1. e, Cell lines deficient for PLA2G16 and the respective cells expressing Flag-tagged mouse Pla2g16 were infected with PV1 and stained with a dsRNA antibody to determine infectivity. Cells transduced with a control lentiCRISPR lacking a gene-specific gRNA served as control (values denote mean ± s.d., n = 3). f, Wild-type HeLa cells, ΔPLA2G16 cells, and ΔPLA2G16 cells expressing PLA2G16 cDNA, were infected with different picornaviruses to determine dependency on PLA2G16. Cells were stained 8 h after infection with a dsRNA antibody, and infection efficiencies were quantified (mean ± s.d., n = 4, chi-square test, ****P < 0.0001).
Extended Data Figure 2 The generation and characterization of Pla2g16-deficient mice.
a, Overview of Pla2g16-targeted locus. Insertion of a trapping cassette, containing a splice acceptor and polyadenylation sequence, perturbs the Pla2g16 gene. b, Western blot analysis for the presence of PLA2G16 in different wild-type and ΔPla2g16 mouse tissues. α-Tubulin was used as a loading control. For gel source data, see Supplementary Fig. 1. c, Wild-type and homozygous ∆Pla2g16 mice were exposed to a high-fat diet for a period of 50 weeks. d, Weight of wild-type (n = 10) and homozygous ∆Pla2g16 (n = 11) mice on a high-fat diet (error bars represent s.d.). e, Wild-type and ∆Pla2g16 mouse-tail fibroblasts were infected with CV-A10 and stained with a dsRNA antibody to determine infectivity (mean ± s.d., n = 3, chi-square test, ****P < 0.0001).
Extended Data Figure 3 Characterization of PLA2G16 function during viral infection.
a, Detection of Cy5-labelled PV1 (MOI of ~20, red) virus bound to the cell surface of wild-type and ∆PLA2G16 HeLa cells. Cells were co-stained with wheat-germ agglutinate (WGA, green) to label cell membranes and counterstained with Hoechst33342 (blue) for DNA. ∆PVR cells served as negative control. Insets represent magnifications of dashed boxes. b, Internalization of Cy5-PV1 (MOI of ~20, red) over time. c, Incubation of Cy5-PV1 (red) in the presence of pleconaril (1 μM) on live GFP–PLA2G16 (green)-expressing HeLa cells, 15 minutes after infection. Individual channels for different regions are shown. d, Electron micrograph of PV1 replication in the indicated genotypes. Cells were infected for 6 h with PV1 (MOI of ~5). Abundant modification of intracellular membranes was observed in wild-type cells or cells reconstituted with PLA2G16 cDNA. e, Quantification of PV1-DsRed infected and transfected cells (mean ± s.d., n = 3, chi-square test, ****P < 0.0001). f, Yield of viral progeny after transfection of PV1-DsRed. TCID50 was determined on wild-type HeLa cells after 1 cycle of replication (mean ± s.d. of three biological replicates).
Extended Data Figure 4 Loss of ATG7 or LGALS8 restores virus susceptibility in ΔPLA2G16 cells.
a, Absolute numbers of disruptive mutations in PVR and ATG14 genes in DsRed-negative (low) and -positive (high) channels. Mutation index is determined by the relative number of integrations in the high channel divided by the relative number of integrations in the low channel. b, ∆PLA2G16 HeLa cells expressing GFP–LGALS8 were exposed to PV1, or CV-B1 (MOI of ~20), leading to the formation of GFP–LGALS8-positive foci. Cells were counterstained with Hoechst33342 (blue) for DNA. Insets represent zoom in of dashed boxes. c, Western blot analysis for the expression of PLA2G16 and ATG7 in indicated genotypes. Rictor was used as a loading control. For gel source data, see Supplementary Fig. 1. d, e, LGALS8 deficiency (∆LGALS8) or ATG7 deficiency (ΔATG7) restores virus infection in ΔPLA2G16 HeLa cells. f, ΔATG7 restores virus infection in ΔPLA2G16 HAP1 cells. The indicated genotypes were infected with an increasing amount of CV-B3–GFP and PV1 (stained with a dsRNA antibody to detect infected cells, n = 4).
Extended Data Figure 5 Viral susceptibility of HeLa cells lacking ATG7 and LGALS8.
a, b, Experiments presented in Fig. 3c, d (a) and Extended Data Fig. 4d, e (b) were quantified to determine susceptibility to PV1, and CV-B3–GFP in wild-type, ∆LGALS8, ∆ATG7, ∆PLA2G16, ∆PLA2G16/∆ LGALS8, and ∆PLA2G16/∆ATG7 HeLa cells. Cells were infected, stained with antibodies against dsRNA and counted to determine percentage of infection (mean ± s.d., n = 4, chi-square test, ****P < 0.0001). c, Yield of viral progeny of CV-B3–GFP after one round of replication on indicated genotypes. TCID50 was determined on wild type HeLa cells (mean ± s.d., n = 4).
Extended Data Figure 6 The N-terminal CRD of LGALS8 is required for recognition of picornavirus induced membrane damage.
Indicated genotypes were reconstituted with the N-terminal CRD (R69H), or the C-terminal CRD (R232H) GFP–LGALS8 mutants, with wild-type GFP–LGALS8 serving as a control or with the corresponding empty vector. a, b, Cells were assessed for infectivity with CV-B3 (numbers denote mean ± s.d., n = 4) (a), and for the formation of GFP-positive galectin-8 (GFP–LGALS8) foci during PV1 infection (b). Insets represent magnifications of dashed boxes.
Extended Data Figure 7 PLA2G16 and LGALS8 respond to membrane damage.
a, GFP–LGALS8 (green) forms foci when cells are exposed to hypotonic shock in both wild-type and ∆PLA2G16 HeLa cells. Insets represent magnifications of dashed boxes. Untreated cells are shown as a negative control. b, Hypotonic shock induces occasional co-localization of GFP–PLA2G16 and mCherry-LGALS8. Live cells were imaged 10 min after hypotonic shock and individual channels for selected regions are shown. Insets represent magnifications of dashed boxes. c, GFP–PLA2G16 forms foci after hypotonic shock in both wild type as well as ΔLGALS8 cells. Untreated GFP–PLA2G16-expressing cells serve as a negative control. Cells were counterstained with Hoechst33342 (blue) for DNA.
Extended Data Figure 8 PLA2G16 and LGALS8 respond to membrane damage independently.
a, b, GFP–PLA2G16 (green) (a) and GFP–LGALS8 (green) (b) expressed in the respective knockout cell lines localize on LAMP1-positive lysosomes (red). c, d, mCherry–LGALS8 (red), and GFP–PLA2G16 (green) colocalize after 30 min of treatment with 200 μM l-leucyl-l-leucine methyl ester (LLOme, which is converted to the lysomotropic membranolytic form (Leu-Leu)n-OMe (n ≥ 3) polymers by a lysosomal thiol protease, dipeptidyl peptidase I). e, f, LLOme treatment of ∆LGALS8 cells expressing GFP–PLA2G16 (e) and ∆PLA2G16 cells expressing GFP–LGALS8 (f). g, Localization of GFP–PLA2G16 (green) and Cy5-PV1 (MOI of ~10, red) in live ∆LGALS8 HeLa cells 15 min after infection. h, LLOme treatment of ∆PLA2G16 cells expressing full-length and truncated GFP–PLA2G16. Lysosomes were stained for LAMP1 (red) and cells were counterstained with Hoechst33342 (blue) for DNA. Insets represent magnifications of dashed boxes. Individual channels for different regions are shown.
Extended Data Figure 9 Detection of PV1 genome using single-molecule FISH.
a, Frequency of LGALS8 foci in each infected cell 30 min after infection with PV1 (n = 192 and n = 191 cells, for wild type and ΔPLA2G16, respectively) or CV-B1 (n = 189 and n = 191 cells, for wild type and ΔPLA2G16, respectively) shown as a box plot (measured over n = 5 independent experiments). To estimate the significance of the difference between genotypes, an unpaired Welch-corrected t-test was performed between the mean values in each of the five independent experiments. b, In situ hybridization of PV1 RNA (red) bound to wild-type or ΔPLA2G16 cells. Cells were stained with WGA (green) to label cell membranes and counterstained using Hoechst33342 (blue) for DNA. ∆PVR cells served as negative control. Insets represent magnifications of dashed boxes. c, In situ hybridization of PV1 genomes (MOI of ~100) over time. Indicated genotypes were infected with PV1 and co-stained for VP1 (green) and viral genomes (red). Cells were counterstained with Hoechst33342 (blue) for DNA. d, Protein lysates of PV1 infected cells (MOI of ~10, 2 h after infection) probed for eIF4G to measure 2A cleavage of eIF4G in the indicated genotypes. GHL was added 30 min before infection to inhibit viral genome replication. For gel source data, see Supplementary Fig. 1.
Supplementary information
Supplementary Figure 1
This file contains the uncropped blots with protein standards in kDa. (PDF 562 kb)
Supplementary Table 1 - Identified host-factors for PV1
The number of disruptive integrations retrieved from poliovirus-selected cells is compared per gene to a dataset of unselected cells. A one-sided Fisher exact test is used to gauge the enrichment of mutations in a selected versus the unselected population. (XLSX 737 kb)
Supplementary Table 2 - Identified host-factors for CV-B1
The number of disruptive integrations retrieved from poliovirus-selected cells is compared per gene to a dataset of unselected cells. A one-sided Fisher exact test is used to gauge the enrichment of mutations in a selected versus the unselected population. (XLSX 530 kb)
Supplementary Table 3 - Identified host-factors for CV-B3
The number of disruptive integrations retrieved from poliovirus-selected cells is compared per gene to a dataset of unselected cells. A one-sided Fisher exact test is used to gauge the enrichment of mutations in a selected versus the unselected population. (XLSX 514 kb)
Supplementary Table 4 - Identified host-factors for CV-A7
The number of disruptive integrations retrieved from poliovirus-selected cells is compared per gene to a dataset of unselected cells. A one-sided Fisher exact test is used to gauge the enrichment of mutations in a selected versus the unselected population. (XLSX 703 kb)
Supplementary Table 5 - Identified host-factors for EV-A71
The number of disruptive integrations retrieved from poliovirus-selected cells is compared per gene to a dataset of unselected cells. Genes without integrations in in the control dataset were corrected. A one-sided Fisher exact test is used to gauge the enrichment of mutations in a selected versus the unselected population. (XLSX 836 kb)
Supplementary Table 6 - Suppressors restricting infectivity in δPLA2G16 cells
The disruptive integrations in δPLA2G16 cells cells with high signal for PV1-DsRed are compared to those with a low signal. Genes without mutations in one population were corrected by the addition of 1. A two-sided Fisher exact test is used to determine mutations that render δPLA2G16 cells cells more or less susceptible. (XLSX 1391 kb)
Supplementary Table 7
This table contains the generated knock out cell lines. (PDF 84 kb)
Live cell imaging of HeLa cells
Live cell imaging of HeLa cells expressing GFP-PLA2G16 (green) infected with PV1-Cy5 (red). (MP4 8086 kb)
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Staring, J., von Castelmur, E., Blomen, V. et al. PLA2G16 represents a switch between entry and clearance of Picornaviridae. Nature 541, 412–416 (2017). https://doi.org/10.1038/nature21032
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DOI: https://doi.org/10.1038/nature21032
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