Fig 1.
Genome-wide CRISPR/Cas9 screening of A549 cells for host factors required for highly pathogenic H5N1 influenza virus infection.
(A) Schematic of the generation of the A549-GeCKO libraries by using lentivirus sgRNA libraries and A549 cells expressing Cas9 (A549-Cas9). A549-Cas9 cells were transduced with lentivirus containing sgRNA libraries A and B, respectively, and were maintained in puromycin for 14 days to generate the A549-GeCKO library, which was then deep sequenced. (B) Coverage of amplified library plasmids and GeCKO library cells compared with sgRNA lists from Addgene detected by high-throughput sequencing. (C) Schematic of screening host factors associated with H5N1 virus infection. (D) The heat map plots showing sgRNA abundance of the selected host factors in different screens. (E) H5N1 virus replication titers in different gene knockout cells. The cells were infected with H5N1 virus at an MOI of 0.01; the supernatants were collected at 48 h post-infection for viral titration in chicken embryos. The data shown are the means ± SDs of three biological repeats. The two-tailed unpaired t-test was used for the statistical analysis. **, p < 0.01, ***, p < 0.001.
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.
Fig 3.
Colocalization of IGDCC4 and viral NP protein in A549 cells and IGDCC4-KO cells infected with H5N1 virus at different timepoints.
(A) Cells were infected with H5N1 virus at an MOI of 5 and incubated at the indicated temperature and for the indicated time as described in the text. They were then fixed and stained with a rabbit anti-NP antibody and a mouse anti-IGDCC4 antibody, followed by incubation with Alexa Fluor 633 goat anti-rabbit IgG(H+L) (red) and 488 donkey anti-mouse IgG(H+L) (green). The nuclei were stained with DAPI (blue). (B) Quantitative analysis of NP localization in virus-infected cells. The ratio of cells showing colocalization of virus NP and nucleus was calculated from 100 virus-infected cells.
Fig 4.
IGDCC4 is required for influenza virus internalization.
(A) Sialic acid expression in different cells. IGDCC4-KO and A549 control cells treated with or without neuraminidase (NA) were stained with Alexa Fluor 647 conjugated with wheat germ agglutinin, and after three washes with PBS, the cells were suspended to detect WGA binding by use of flow cytometry. (B) NP protein of viruses attached to the surface of IGDCC4-KO and A549 cells. IGDCC4-KO and A549 cells were incubated with H5N1 virus at an MOI of 5 at 4°C for 1 h and then washed with cold PBS (pH = 7.2) or cold acidic PBS (pH = 1.5), which can elute virus particles on the cell surface that have not been internalized. The cells were collected and the NP protein of the viruses attached to the cells was evaluated by Western blotting. (C) NP protein of viruses internalized into IGDCC4-KO and A549 cells. A549 cells treated with or without dynasore and IGDCC4-KO cells were incubated with H5N1 virus at an MOI of 5 at 4°C for 1 h and at 37°C for 1 h. After being washed with cold PBS (pH = 7.2) or acidic PBS (pH = 1.5), the cells were collected and the NP protein level of the total attached and internalized viruses (normal PBS washed) or the internalized viruses (acidic PBS washed) was determined by using Western blotting. (D) Overexpression of IGDCC4 restores the internalization of influenza virus into IGDCC4-KO cells. A549 cells and IGDCC4-KO cells transfected with the indicated plasmids were infected with H5N1 virus at an MOI of 5. After being incubated at 4°C for 1 h and 37°C for 1 h, the cells were washed three times with ice-cold acidic PBS. The washed cells were then collected to detect the NP protein level by Western blotting, and (E) the RNA level by qRT-PCR. The band intensities of the Western blots from three assays were quantified by using ImageJ software, and the relative gray values of NP to GAPDH are presented. The data shown represent or are from three independent experiments or replicates (means ± SDs). The two-tailed unpaired t-test was used for the statistical analysis. ***, p < 0.001.
Fig 5.
IGDCC4 interacts with the HA protein of influenza virus.
(A) Interaction of IGDCC4 with the HA, NA, M1, and M2 proteins of H5N1 virus. HEK293T cells were transfected individually or in combination with plasmids for the expression of IGDCC4 fused with Myc tag and viral proteins fused with Flag tag. (B) IGDCC4 interaction with the HA protein of H5N1 virus was confirmed by immunoprecipitation with the anti-Myc antibody. (C) Schematic presentation of IGDCC4. (D) The extracellular domain of IGDCC4 interacts with the HA of H5N1 virus. (E) The endocytic domain of IGDCC4 does not interact with the HA of H5N1 virus. (F) A549 cells were co-transfected with a plasmid expressing IGDCC4 and a plasmid expressing HA, NA, or M2 of H5N1 virus, and the interaction of IGDCC4 with these proteins was confirmed by using a colocalization assay. (G) The HA of H5N1 virus interacted with IGDCC4 expressed in HEK293T cells treated with neuraminidase. The data shown are representative of three independent experiments.
Fig 6.
Effect of IGDCC4 on H1N1 influenza virus and H9N2 influenza virus.
(A) Replication titers of H1N1 virus and H9N2 virus in A549 cells and IGDCC4-KO cells. A549 cells and IGDCC4-KO cells were infected with the indicated virus at an MOI of 0.01 and the supernatants were harvested at the indicated time for virus titration in MDCK cells. The data shown are from three biological replicates (means ± SDs) (B) Interaction of IGDCC4 with the HA protein of H1N1 virus and H9N2 virus. HEK293T cells were transfected individually or in combination with plasmids for the expression of IGDCC4 fused with Myc tag and viral HA protein fused with Flag tag. Cell lysates were immunoprecipitated with a mouse anti-Flag monoclonal antibody. The data shown are representative of three independent experiments. The two-tailed unpaired t-test was used for the statistical analysis. ***, p < 0.001.
Fig 7.
IGDCC4 does not affect the internalization of vesicular stomatitis virus-green fluorescent protein (VSV-GFP) and transferrin.
The A549 cells and IGDCC4-KO cells infected with VSV-GFP at an MOI of 10 were incubated and washed differently before being collected for detecting viral attachment (A) or (B) internalization by using qRT-PCR, the values of IGDCC4-KO cells were normalized with that of A549 cells. Data shown are from three replicates (means ± SDs). (C) and (D) Internalization of transferrin in IGDCC4-KO and A549 cells were evaluated by incubating cells with 50 μg/ml fluorescently labeled transferrin at 37°C for 30 min before receiving an acid wash to quench extracellular transferrin and were subsequently processed for confocal microscopy. Automatic image analysis quantified a total of 100 cells in three independent experiments and normalized to the A549 cells. The data shown in A, B, and D are from three independent experiments or biological replicates (means ± SDs).
Fig 8.
Replication and lethality of H5N1 virus in wild-type mice and IGDCC4 knockout mice.
(A) Viral titers in organs of different mice. Wild-type mice and IGDCC4 knockout mice were euthanized on Day 3 post-infection with 5 MLD50 of H5N1 virus, and their organs were collected for virus titration in chicken embryos. The data are presented as means ± SDs for organ samples of three mice. (B) Body weight change of wild-type mice and IGDCC4 knockout mice after H5N1 virus infection. The values are means ± SDs from surviving mice at the indicated timepoints. (C) Lethality of H5N1 virus to wild-type mice and IGDCC4 knockout mice. The two-tailed unpaired t-test was used for the statistical analysis.
Table 1.
Sequence information of primers used in this studya.