Duplicate US1 Genes of Duck Enteritis Virus Encode a Non-essential Immediate Early Protein Localized to the Nucleus

Figure 1. Prokaryotic expression of DEV ICP22 and identification of the ICP22 protein. (A) M, Protein molecular mass marker; Lane 1, untreated samples; lane 2, recombinant bacterial supernatant after IPTG induction; lane 3, the purified ICP22 protein. (B) Identification of the ICP22 protein by the prepared anti-ICP22 antibody in the eukaryotic expression plasmid EGFP-ICP22 transfection group (left panel) or DEV infection group (right panel).

Figure 2. Transcriptional analysis of the DEV US1 gene in infected cells with 10 MOI DEV; the average relative content of the DEV US1 gene transcripts was calculated at the indicated time points using GraphPad Prism 8.0 software. Each time point was examined in triplicate with the standard error.

Figure 3. Dynamic expression of ICP22 in DEV-infected cells. Expression of ICP22 in DEFs with 10 MOI DEV was examined by Western blot assay (top panel). The quantitative result of WB is shown in the bottom panel. Mock-infected DEFs were used as a control.

Figure 4. Identification of the US1 gene type in DEV-infected cells. M, DL2000 Marker; ACV group, DEV-infected cells treated with 300 μg/mL nucleic acid synthesis inhibitor ACV; CHX group, DEV-infected cells treated with 50 μg/mL the protein synthesis inhibitor CHX; NC group, DEV-infected cells without drugs; Mock group, mock-infected DEFs were used as a control.

Figure 5. Construction of DEV CHv-BAC-ΔUS1 and DEV CHv-BAC-2ΔUS1. (A) Schematic diagram of constructing the duplicated US1 deletion mutant using the Red recombinant system. (B) DEV CHv-BAC-ΔUS1 and DEV CHv-BAC-2ΔUS1 were rescued in DEF cells.

Figure 6. Identification of BAC-ΔUS1 and BAC-2ΔUS1. (A) Identification of BAC-ΔUS1 and BAC-2ΔUS1 by enzyme digestion. M, 1 kb ladder marker; lane 4, negative control. (B) Identification of DEV-ΔUS1 and DEV-2ΔUS1 by PCR. (C) Identification of BAC-ΔUS1 and BAC-2ΔUS1 by IFA. (D) Identification of BAC-ΔUS1 and BAC-2ΔUS1 by Western blot analysis.

Figure 7. Viral titres and viral copies of one-step and multi-step replication kinetics. (A) Viral titer in the cytoplasm, supernatant and total of one-step growth assays (top panel) and multi-step replication kinetics (bottom panel). (B) Viral copies in the cytoplasm and total of one-step growth assays (top panel) and multi-step replication kinetics (bottom panel). Each time point was measured in triplicate, with the standard error indicated. A representative experiment with at least three repeats was performed. Ns represents no significance, *p < 0.1, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 8. Intracellular localization of ICP22 in DEFs. (A) Intracellular localization of ICP22 in infected DEFs at different time points with 10 MOI; mock was used as a control. (B) Intracellular localization of ICP22 at different time points in EGFP-ICP22-transfected DEFs. pEGFP-N1 was used as a control.

Figure 9. Screening for key amino acids in NLS after transfection. (A) Schematic diagram of the ICP22 plasmids. (B) Localization of ICP22 in different plasmids transfected DEFs. mock was used as a control. (C) Localization of ICP22 in different mutation plasmids transfected DEFs.

Figure 10. Intracellular localization of ICP22 and viral titres of one-step and multi-step replication kinetics. (A) Intracellular localization of ICP22 in infected DEFs at different time points with 10 MOI BAC-ΔUS1, BAC-ΔUS1 R309A, and BAC-ΔUS1 308-312AA. (B) Viral titer in the cytoplasm, supernatant, and total of one-step growth assays (top panel) and multi-step replication kinetics (bottom panel). Each time point was measured in triplicate, with the standard error indicated. A representative experiment with at least three repeats was performed. Ns represents no significance, *p < 0.1, **p < 0.01, ***p < 0.001 and ****p < 0.0001.