The transporters SLC35A1 and SLC30A1 play opposite roles in cell survival upon VSV virus infection

Figures

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  Figure 1. An SLC-focused genetic screen identifies known and novel host factors for IAV and VSV. (a) Schematic overview of the screen workflow. (b) Volcano plot of the genetic screen results in A549 cells infected with IAV-WSN/1933 at 96 h.p.i. (c) Quantification of flow cytometry-based viral replication assay for selected hits. A549 cells carrying sgRNAs targeting SLC35A1 or Renilla luciferase control as well as cells expressing SLC35A1 cDNA were infected with IAV-WSN/1933 at MOI 0.01 and 24 h.p.i. the number of the infected cells was assessed after staining with anti-Influenza Nuclear protein antibody coupled to AF488. (d) Volcano plot of the genetic screens results in A549 cells infected with VSV-GFP at 48 h.p.i. (e,f) Multicolor competition assay (MCA) of SCL30A1 (e) or SLC35A1 (f) knockout cells and Renilla controls. Cells were mixed at 1:1 ratio, infected with VSV-rWT at MOI 5 and cultured until the indicated time points. The percentage of mCherry + (Renilla control) and eGFP+ (gene of interest) cells in the live population (FSC/SSC) were assessed using flow cytometry. Statistical significance was calculated using one-way ANOVA (c) and two-way ANOVA (e,f) with Dunnett’s test. Unless…

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  Figure 2. SLC30A1 inhibits apoptotic caspase activation via regulation of intracellular Zn2+ levels. (a) Intracellular Zn2+ levels determined using Zinpyr1 fluorescent dye. (b,c) Activation of pro-apoptotic caspases in VSV infected A549 (b) and HAP1 (c) SLC30A1 knockout cells. Cropped images are shown for conciseness. Full-length blots are presented in Supplementary Fig. 5. (d) Percentage of apoptotic cells in A549 cells expressing sgRNAs targeting SLC30A1 or Renilla luciferase after infection with VSV (MOI 2) for 24 hours, as measured with AnnexinV-FITC. (e) Quantification of AnnexinV- and PI-stained A549 wild type cells pre-treated with indicated concentrations of ZnCl2 and infected with VSV-rWT (MOI 2) for 24 hours. Statistical significance was assessed using one-way ANOVA (a,d) and two-way ANOVA (e) with Dunnett’s test. Unless otherwise indicated, adjusted P-values in relation to the sgRen control or Hap1wt are shown. Data are represented as mean ± SD of one representative experiment out of at least two independent replicates. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns: not significant.

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  Figure 3. SLC30A1 loss affects VSV infection through modulation of Zn levels. (a–c) VSV-GFP virus replication in SLC30A1 knockout HAP1 cells (a,c) and HAP1 cells overexpressing wild type SLC30A1 and SLC30A1(D254A_H43A) cDNAs (b) and A549 cells (c). Cells were infected with MOI 1 (a), MOI 0.001 (b) and MOI 0.002 (c). The number of GFP+ cells was quantified using flow cytometry at 14 h.p.i. (b,c) or indicated time points (a). (d) Immunoblot analysis of type I IFN/STAT1 signalling pathway activation in the VSV-infected (MOI 2) HAP1 cells carrying sgRNAs against SLC30A1 and Renilla luciferase control. Cropped images are shown for conciseness. Full-length blots are presented in Supplementary Fig. 5. (e) Quantitative RT-PCR of total viral RNA in the HAP1 wild type cells and a SLC30A1 single cell knockout clone at different points after VSV infection (MOI 2). (f) VSV-G protein levels in the HAP1 wild type cells and SLC30A1 single cell knockout clone at different points after VSV infection (MOI 2). Cropped images are shown for conciseness. Full-length blots are presented in Supplementary Fig. 5. (g) Quantification (flow cytometry) of virus titers produced by HAP1wt and two SLC30A1 single cell…

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  Figure 4. SLC35A1 loss promotes cell death and apoptosis upon VSV infection. (a) Percentage of early apoptotic cells (AnnexinV+) and cells that lost membrane integrity due to a late apoptotic/necrotic state (AnnexinV+/PI+) in HAP1 cell lines expressing sgRNAs against SLC35A1 and Renilla luciferase upon infection with VSV at MOI 2. (b) Immunoblot analysis of Golgi stress signalling pathway activation in the VSV-infected (MOI 2) and Brefeldin A-stimulated (3μg/ml) A549 cells carrying sgRNAs against SLC35A1 and Renilla luciferase. Cropped images are shown for conciseness. Full-length blots are presented in Supplementary Fig. 5. (c,d) VSV-GFP virus replication in SLC35A1 knockout HAP1 cells (c) and HAP1 cells overexpressing SLC35A1 cDNA (d) infected with MOI ranging from 0.001 to 0.01 for 14 hours. (e) Quantification (flow cytometry) of virus titers produced by HAP1wt and two SLC35A1 single cell knockout clones infected with MOI of 2 of VSV-GFP for 8 hours. Statistical significance was assessed using one-way ANOVA with Tukey’s (a,d) and Dunnett’s (c,e) tests. Unless otherwise indicated, adjusted P-values in relation to sgRen control or HAP1wt are shown. Data are represented as mean ± SD of one…