Article Figures & Data
Figures
- FIG 1
Avibirnavirus polymerase protein VP1 undergoes ubiquitination during infection. (A) Mass molecular shift modified bands of VP1 during IBDV infection. Lysates from 293T cells, DF-1 cells, and BF cells infected with IBDV were analyzed by Western blotting with mouse anti-VP1 MAb. Mock-infected cells and BF cells were used as negative controls. (B) Purified IBDV virions were probed by Western blotting with anti-VP1 MAb and anti-Ub polyclonal antibody. (C) Dynamic profiles of VP1 ubiquitination during infection. DF-1 cells infected with IBDV at 5 MOI were cultured at the indicated time points before immunoprecipitation with anti-VP1 antibody plus protein A/G. Immunoblotting was performed with anti-Ub pAb, anti-VP1 MAb, and anti-VP3 MAb. (D) VP1 was modified by ubiquitin during transfection. Flag-VP1 and HA-Ub were cotransfected into 293T cells for 48 h. Cell lysates were used for in vivo ubiquitination assays and subjected to Western blotting with the indicated antibodies. Flag-VP1 or HA-Ub transfected alone was used as the control.
- FIG 2
K63-linked ubiquitination of VP1 during IBDV replication. (A) Schematic diagram of ubiquitin K48 and K63 mutants. (B) VP1 was modified by K63-linked ubiquitin during transfection. 293T cells were cotransfected with Flag-VP1 and either HA-K63 or HA-K48 for 48 h. The cell lysates were subjected to in vivo ubiquitination assays and Western blotting with indicated antibodies. (C) VP1 was modified by K48R mutant-linked ubiquitin during transfection. 293T cells were cotransfected with Flag-VP1 and HA-K63R or HA-K48R for 48 h. The lysates were subjected to in vivo ubiquitination assay and Western blotting with indicated antibodies. (D and E) VP1 was modified by K63-linked ubiquitin (D), rather than by K48-linked ubiquitin (E), during virus infection. DF-1 cells infected with IBDV at 5 MOI for 12 h were used in an in vivo ubiquitination assay with anti-VP1 antibody and for Western blotting with K63/K48-specific antibodies. All input proteins were detected by Western blotting with the indicated antibodies; β-actin expression was used as a loading control.
- FIG 3
Lys 751 of VP1 is the target site for ubiquitination. (A) The C terminus of VP1 was modified by ubiquitin. 293T cells were cotransfected with HA-Ub and the indicated Flag-tag VP1 subdomains for 48 h. The lysates were subjected to immunoprecipitation and Western blotting with the indicated antibodies. (B) C-terminal residual sequence from residues 658 to 878 of VP1 of IBDV. Lysine residues are displayed in boldface. (C) Screening the ubiquitin-modified K residues of VP1. 293T cells were cotransfected with HA-Ub and individual specific K-to-R mutant constructs of VP1 for 48 h, respectively. Transfected cells were lysed and analyzed by in vivo ubiquitination assay and Western blotting with the indicated antibodies. (D) K751R mutant of VP1 impaired its ubiquitination. 293T cells were cotransfected with HA-Ub and WT VP1, K751R mutant VP1, and K787R mutant VP1 for 48 h, respectively. Cell lysates were subjected to in vivo ubiquitination assay and Western blotting with the indicated antibodies. (E) The K751R mutant of VP1 abolished K63-linked ubiquitination. 293T cells were cotransfected with HA-K63 and WT VP1 or K751R mutant VP1 for 48 h. Lysates were subjected to in vivo ubiquitination assay and Western blotting with the indicated antibodies.
- FIG 4
The K751 ubiquitination of VP1 is essential for its polymerase activity. (A) Schematic diagram of the minigenome reporter system. The ORF of VP1 gene between the 5′ and 3′ UTRs of IBDV genomic segment B was replaced by the ORF of the firefly luciferase gene. The resulting cassette was subcloned into vector pCDNA3.0 by an antisense orientation between HamR and HDR. (B) The minigenome reporter system could work efficiently. Western blotting, RT-qPCR, and polymerase activity assays were performed using 293T cells expressing pCDNA3.0-A, together with a minigenome reporter system and pRL-TK in the absence of VP1 (Con) or presence of VP1 (WT), D402A, or D416A mutant VP1. (C and D) Wild-type ubiquitin (C) and K63-linked ubiquitins (D) promoted VP1 polymerase activity. Polymerase activity assays were performed using 293T cells expressing segment A and segment B, along with minigenome and pRL-TK, as well as increasing amounts of HA-Ub and HA-K63. Protein expression and luciferase mRNA were analyzed by Western blotting and RT-qPCR. (E) Ubiquitin-deficient VP1 showed a significant reduction in polymerase activity. Polymerase activity and luciferase mRNA levels were assessed in 293T cells expressing IBDV K751R mutant (K751R) and WT segment B (WT), together with minigenome and pRL-TK. (F) The polymerase activity and luciferase mRNA level of the K751R mutant VP1 was not improved with an increasing expression of WT ubiquitin and K63-linked ubiquitin. Polymerase activity assays were performed using 293T cells expressing pCDNA3.0-A, pCDNA3.0-B, or K751R mutant, along with minigenome and pRL-TK, as well as increasing doses of HA-Ub or HA-K63. (G) Stability difference between K751R mutant and WT VP1. CHX assays were performed to estimate the degradation half-life of WT VP1 and K751R mutant VP1. Data are presented as the means ± the SD of three independent experiments. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 5
K751 ubiquitination of VP1 did not disturb VP1 interaction with VP3 and eIF4AII. (A) The K751R mutant VP1 was colocalized with eIF4AII. DF-1 cells transfected with Myc-eIF4AII for 18 h were infected with K751R mutant and WT IBDV for 12 h. Immunofluorescence analysis was performed with anti-VP1 MAb and anti-Myc pAb as the primary antibodies and FITC-labeled goat anti-rabbit and Alexa Fluor 546-conjugated anti-mouse antibodies as the secondary antibodies. (B) K751R mutant VP1 was colocalized with VP3. DF-1 cells were infected with K751R mutant and WT IBDV for 12 h. Immunofluorescence analysis was performed with anti-VP1 pAb and anti-VP3 MAb as the primary antibodies and Alexa Fluor 546-conjugated anti-mouse and FITC-labeled goat anti-rabbit antibodies as the secondary antibodies. DAPI staining revealed nuclei. Scale bars, 10 μm. (C) Ubiquitination of VP1 did not affect the formation of the VP1-VP3 complex. Fresh 293T cells were transfected by the indicated plasmids for 48 h. The lysates were subjected to immunoprecipitation with Flag beads, and Western blotting was performed with the indicated antibodies. (D) Ubiquitination of VP1 was not affected by VP3. 293T cells were transfected with indicated plasmids for 48 h. The lysates were subjected to immunoprecipitation with Flag beads, and Western blotting was performed with the indicated antibodies. (E) Ubiquitination of VP1 did not alter the interaction of VP1 with eIF4AII. The lysates were subjected to immunoprecipitation with Flag beads, and Western blotting was performed with the indicated antibodies. (F) Ubiquitination of VP1 was not affected by eIF4AII. 293T cells were transfected with the indicated plasmids for 48 h. The lysates were subjected to immunoprecipitation with Flag beads, and Western blotting was performed with the indicated antibodies. Cell lysates were immunoprecipitated with anti-Flag resin and immunoblotted with the indicated antibodies. (G and H) The K751R mutant VP1 still interacted with VP3 (G) and eIF4AII (H). Lysates from 293T cells cotransfected with Flag-VP1 or Flag-K751R VP1 and Myc-VP3 or Myc-eIF4AII for 48 h were subjected to immunoprecipitation and Western blotting with the indicated antibodies.
- FIG 6
K751 ubiquitination of VP1 facilitates IBDV replication. (A) IBDV with the K751R mutation of VP1 was successfully rescued by IBDV CT stain rescue system using a T7 promoter system combined with the HDR sequence. CPE and IFAs of IBDV were observed. (a, d, and g) Mock-infected cells; (b, e, and h) WT IBDV; (c, f, and i) rescued K751 mutant IBDV. Scale bar, 100 μm. (B) Western blotting of viral proteins from K751R and WT IBDV. DF-1 cells were infected with K751R IBDV or WT IBDV at 1 MOI and cultured for the indicated times. Cell lysates were subjected to Western blotting with the indicated viral protein antibodies. Data are presented as the means ± the SD of three independent experiments. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. β-Actin was used as a loading control. (C) One-step growth curves of WT and mutant IBDV. DF-1 cells were infected with WT and K751R mutant IBDV at 1 MOI, respectively. The cells were harvested at the indicated time points, and the TCID50 was calculated as described in Materials and Methods. Data are presented as the means ± the SD of three independent experiments. ns, P > 0.05; *, P < 0.05; **, P < 0.01.
