Fig 1.
Design of the pooled genetic screen for the identification of VV pro-viral factors.
A pool of cells with single gene inactivations was obtained by transducing the GeCKO sgRNA lentiviral library. Subsequently, the cell population is subjected to consecutive rounds of infection to enrich for resistant cells. Surviving cells are analyzed by high-throughput sequencing of the sgRNA region in the integrated lentiviral construct, which was amplified by PCR. Finally, results are analyzed using MaGeCK and ScreenBEAM algorithms for hit determination.
Fig 2.
A) Example of results obtained in one screen experiment (1_1) analyzed with MaGeCK. Hits with FDR < 0.05 are labelled with gene name tags. B) Heatmap summarizing the 27 experiments/experiment variations. Experiments are named with the starting cell pool followed by a number for the conditions used for that particular sample (see S1 Table). Genes with FDR < 0.05 in at least 1 experiment are included in the analysis. Enrichment results after selection are shown in the left panel as a heatmap representing the gene rank obtained with MaGeCK. For comparison, the right panel shows the log2-fold change for each gene in non-infected control experiment. Dots to the right of the red dotted line indicate enrichment in non-infected cultures for that particular gene inactivation. For instance, CMIP KOs are depleted from the pool during passages in the absence of infection, whereas CSK KOs are enriched.
Fig 3.
Reactome Pathway Analysis was performed using the top 1% list of genes according to MaGeCK rank. Class I MHC mediated antigen processing & presentation pathway was the most enriched pathway with 12 genes and p-adjust value < 0.05. Genes associated to this pathway by Reactome Pathway Analysis were B2M, UBE4A, CUL3, KLHL22, PSME1, UBE2F, RNF41, PSMC3, TRIM4, PSMA8, TRIP12, ASB17.
Fig 4.
Screen hits related to the ITGB1/AXL activated AKT pathway.
Genes in bold letters were hits in the screen analysis (MaGeCK or ScreenBEAM). Genes in light gray letters encode proteins with related functions that were not screen hits. Activation of AKT by ITGB1 signaling pathway results in actin cytoskeleton reorganization, leading to VV endocytosis. CMIP and AHR are new suggested players in the pathway (see main text).
Fig 5.
Effect of B2M KO on VV replication.
A) Analysis of cell lysates using anti-B2M antibody. B) Immunofluorescence images of HeLa and HeLa-B2M KO cells labelled with anti-B2M antibody. C) Virus production in HeLa and HeLa-B2M KO. Infected cell monolayers (m.o.i. = 0.05) were collected 30 h.p.i. and VV titers were obtained by a standard plaque infectivity assay. Error bars indicate SD of three independent experiments. D) and E): HAP1 and derived cell lines were infected at m.o.i. = 0.05. At different time points, a fraction of supernatant was collected to determine extracellular virus production (D). Error bars indicate SD of three independent experiments. In the same cultures, fluorescence images were obtained at different times (E). p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05, ns > 0.05.
Fig 6.
Effect of B2M inactivation on the early steps of Vaccinia virus infection.
A) MW6 well plaques with HeLa and B2M KO clones 1 and 2 were infected with 200 pfu/well of V-GFP. Plaques were counted at 24 h.p.i B) Images of representative plaques under the microscope by GFP fluorescence. C) and D) Early luciferase expression was taken as a measure of virus entry. HeLa (C) and HAP1 (D) cells were infected with V-e.Luc at m.o.i. = 0.8 and luciferase activity was measured 3 h.p.i. Error bars indicate SD of three independent experiments. E) Infection of B2M deficient cells with extracellular virus (EV) and intracellular virus (IV). F) Time course expression of A26-defficient VV in B2M KO cells. nLuc expression was measured in triplicates in the culture medium of cells infected with the indicated viruses. G) Effect of A26 deletion on virus entry. nLuc activity at 40 min after virus adsorption is shown. p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05, ns > 0.05.
Fig 7.
VV binding is not affected by B2M absence.
A) HeLa and B2M KO cells were infected with V-A4-cherry at m.o.i = 30. After 1 h of adsorption at 4°C, cells were washed with PBS and viral particles bound to the cell were counted. To standardize measurements data is given in relation to cell surface area. B) Example showing the labeling with WGA-488 to visualize the cell surface. In the lower panel, mCherry labeled virus particles are shown together with the cell contour as defined by WGA labeling. p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05, ns > 0.05. Scale bars: 10 μm.
Fig 8.
B2M gene inactivation affects VV internalization.
A) and B) HeLa and B2M KO cells were infected with V -A4-cherry at m.o.i. = 4 during 1 h at 4°C to avoid internalization. Cells were incubated for different times at 37°C and non-permeabilized cells were stained with antibody to VV-A27 (green) to visualize cell surface virus particles. White arrow-heads mark internalized virions (only red in merged image) and yellow-empty arrow-heads indicate non-internalized virions (green or yellow). Cell nucleus was labeled in blue by Hoechst staining. Internalized virions were quantified for each time point showing significant differences between HeLa and B2M KO cell lines. p-values: **** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05, ns > 0.05. Scale bars: 5 μm.
Fig 9.
Cell-cell fusion induced by VV in HeLa B2M KO cells.
Cells expressing either GFP or Scarlet fluorescent proteins were incubated with 100 PFU/cell of purified VV, and then treated for 3 min with buffer at pH 7.4 or 5.0. Two hours later, cells were fixed and photographed. The number of nuclei (n>600) in syncytia and in individual cells were counted from multiple images. The percentage of nuclei in syncytia is represented in lower panel. ns, not significant.
Fig 10.
VV, B2M and ITGB1 partial colocalization.
A) HeLa cells were infected with V-A4-cherry (red) for 1 h at 4°C and unbound virus was removed by washing (m.o.i. = 5). Cells were stained with anti-B2M antibody (green) to analyze colocalization. Colocalization of VV with B2M is indicated with white arrow-heads. B) Non permeabilized HeLa cells were stained with anti-B2M (green) and anti-ITGB1 (red) to analyze colocalization. Scale bars: 5 μm.