Identification of Cables1 as a critical host factor that promotes ALV-J replication via genome-wide CRISPR/Cas9 gene knockout screening

Figures

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  Figure 1. Genome-wide genetic screening identifies host factors required for ALV-J infection.A, identification of the infection efficiency of ALV-J reporter virus RCAS(J)GFP in ChGeCKO cells at MOI of 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.016, 0.018 and 0.2. After 48, 60, and 72 h following infection, the infection efficiency was analyzed using a flow cytometer. B, venn diagrams indicate the overlapping enrichment genes from the three rounds of screening of host factors associated with ALV-J replication using the ChGeCKO library. In the initial, second, and third rounds of screening, there were 222, 218, and 207 enriched genes, respectively. Genes listed in the middle were identified in three screens. C, gene ontology analysis of the identical 42 candidate host genes from three ALV-J challenge rounds. D, KEGG pathway enrichment analyses of the identical 42 candidate host genes from three ALV-J challenge rounds. E, sequence analysis of the Cables1, CDK1 and DHFR knockout DF-1 cells lines. F-H, DHFR, CDK1, and Cables1 are essential genes for ALV-J infection. To detect the susceptibility of host factors to ALV-J, DHFR, CDK1, and Cables1 knockout cell lines were inoculated with…

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  Figure 2. Cables1 is a host factor required for ALV-J replication.A-B, knockout of Cables1 inhibits ALV-J HPRS103 replication. The WT DF-1 cells and Cables1 knockout cell lines were infected with ALV-J HPRS103 strain at MOI of 0.01. The cells were harvested from 2 days to 6 days. The viral p27 expression level was determined based on the value of the ratio of the sample to the positive control (the standard p27 protein sample) (S/P) for p27 antigen detection (A), and viral titers were quantified by TCID50 assay (B). C-D, overexpression of Cables1 promoted replication of ALV-J HPRS103 strain. C, Western blotting assay to detect the Cables1-Flag protein. The Cables1-Flag overexpression cell lines were constructed by lentiviral expression systems, and the expression of Cables1-Flag was detected by Western blotting using mouse anti-Flag mAb. The WT DF-1 cells and Cables1 overexpression cell lines were infected with ALV-J HPRS103 strain at an MOI of 0.01. The viral titers were quantified by TCID50 assay D, a representative experiment out of three repeats is shown, and data are shown as means ± SD for triplicates from the representative experiment. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns,…

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  Figure 3. Cables1 interacted with ALV-J p15.A-C, interaction between Cables1 and viral proteins was detected by Co-IP in HEK293T cells. A, HEK293T cells were co-transfected with pFlag-Cables1 and pHA-p12, pHA-p15, pHA-p19, pHA-p27, pHA-IN, pHA-RT, pHA-gp37, pHA-gp85, respectively. After 36 h, the cells were harvested and lysed. The lysates were incubated with anti-Flag mAb and detected with the indicated antibodies. B and C, HEK293T cells were co-transfected with pFlag-Cables1 and pHA-p15 or pHA-IN for 36 h. The lysates were incubated with anti-Flag mAb or mouse-derived IgG. D, confocal assays were used to assess the co-localization between Cables1 and p15. DF-1 cells were co-transfected with pFlag-Cables1 and pHA-p15. After 24 h following transfection, the cells were harvested and incubated with the corresponding antibodies. The interaction between Cables1 and p15 was analyzed via confocal assay. Scale bar: 5 μm. A representative experiment out of three repeats is shown.

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  Figure 4. Cables1 enhanced ALV-J p15 ubiquitination via Lys26.A, cables1 promoted ALV-J p15 ubiquitination. The pHA-Ub and pFlag-p15 plasmids were co-transfected with or without pMyc-Cables1 into HEK293T cells for 36 h. Empty vector pCAGGS (eukaryotic expression-empty vector) was transfected into HEK293T cells for 36 h as negative control. The lysates were incubated with anti-Flag mAb and detected using the corresponding antibody. B, cables1 enhanced the K63-linked polyubiquitination of ALV-J p15. pFlag-p15 and pHA-Ub-K63 (the ubiquitin with all lysine substituted by arginine but for the lysine 63 site) plasmids were co-transfected with pMyc-Cables1 or empty vector pCAGGS into HEK293T cells for 36 h. Then, the lysates were incubated with indicated mouse anti-Flag mAb and detected using IP. C and D, schematic representation of the potential ubiquitination site of ALV-J p15. And the p15 residual sequence from residues 1 to 124 of ALV-J p15. Lysine residues are displayed in boldface. E, Lys26 is the key site for the increase in ALV-J p15 ubiquitination mediated by Cables1. The ALV-J p15 plasmids including the WT and different mutants and pHA-Ub were co-transfected with pMyc-Cables1 or empty…

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  Figure 5. The replication ability of recombinant viruses rHPRS103-K26R, rHPRS103-K73R, and rHPRS103 under in vivo and in vitro conditions.A, the schematic diagram shows the construction of the recombinant viruses rHPRS103-K26R and rHPRS103-K73R. Lys26 and Lys73 of rHPRS103 p15 were mutated to Arg for constructing the recombinant virus rHPRS103-K26R and rHPRS103-K73R, respectively, based on the rHPRS103 backbone. B, ELISA assay. The rescued viruses rHPRS103-K26R, rHPRS103-K73R, and rHPRS103 were detected with mouse anti-p27 mAb, rHPRS103 as a reference. C, Western blotting. DF-1 cells were infected with recombinant viruses rHPRS103-K26R, rHPRS103-K73R, and rHPRS103 and then analyzed with an anti-p27 monoclonal antibody at 72 h following infection. D-F, TCID50 assay. The rescued viruses rHPRS103-K26R, rHPRS103-K73R, and rHPRS103 were titrated using the Reed-Muench method (D). The replication activities of rHPRS103-K26R, rHPRS103-K73R, and rHPRS103 were detected using the TCID50 assay in vitro. Cables1 knockout cells (E) and WT DF-1 cells (F) were infected with rHPRS103-K26R, rHPRS103-K73R, and rHPRS103 at MOI of 0.01. Subsequently, the samples were harvested at two, three, four, five and six…