A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis
Fig 2
IGDCC4 is important for the early stage of H5N1 virus replication.
(A) Schematic illustration of the proteins encoded by IGDCC4. The information on the proteins encoded by the IGDCC4 gene was acquired from the UniProtKB website (accession number: Q8TDY8) (www.uniprot.org). The amino acids 62 to 980 in isoform 2 were identical to amino acids 332 to 1250 in isoform 1. (B) IGDCC4 expression level in A549 cells and IGDCC4-KO cells. Both the membrane proteins and the total proteins of the A549 cells and IGDCC4-KO cells were assessed by Western blotting to determine the IGDCC4 protein expression level. (C) The viability of IGDCC4-KO cells was measured by using the CellTiter-Glo assay and compared with that of the control A549 cells. (D) Replication of H5N1 virus in IGDCC4-KO and control A549 cells. IGDCC4-KO and A549 cells were infected with H5N1 virus at an MOI of 0.01; supernatants were collected at the indicated timepoints for virus titration in chicken embryos. (E) The viability of A549 cells transfected with the indicated siRNA was measured by using the CellTiter-Glo assay. (F) Knockdown of IGDCC4 by siRNA in A549 cells. The mRNA level of IGDCC4 in A549 cells transfected with siRNA targeting IGDCC4 was measured by qRT-PCR at 36 h post-transfection and was compared with that in A549 cells transfected with scrambled siRNA. (G) IGDCC4 expression level in A549 cells transfected with different siRNA. (H) Replication of H5N1 virus in IGDCC4 knockdown cells and A549 cells. A549 cells transfected with siRNA targeting IGDCC4 or scrambled siRNA were infected with H5N1 virus at an MOI of 0.01; supernatants were collected at the indicated timepoints for virus titration in MDCK cells by using plaque assays. (I). The effect of IGDCC4 on viral transcription and viral genome replication of H5N1 virus. IGDCC4-KO and A549 cells were infected with H5N1 virus at an MOI of 3. The mRNA and vRNA levels of the NP gene were detected by using qRT-PCR and were normalized with the value of A549 cells at the indicated timepoints. The data shown in panels B–I are the means ± SDs of three independent experiments or replicates. The two-tailed unpaired t-test was used for the statistical analysis. **, p < 0.01, ***, p < 0.001.