Fig 2: UBA3 is the substrate of PFDN and activates ISG production.a Volcano plot of mass spectrometry data from WT and PFDN3 KO cells. Proteins downregulated in both PFDN3 KO and PFDN4 KO cells were shown in blue dots. Statistically significant proteins were identified using a two-sided t test with a permutation-based FDR set at 0.05. b Volcano plot of mass spectrometry data from WT, and PFDN4 KO cells. Proteins downregulated in both PFDN3 KO and PFDN4 KO cells were shown in blue dots. Statistically significant proteins were identified using a two-sided t test with a permutation-based FDR set at 0.05. c HEK293 cells were treated with increasing concentrations of MLN4924 (0, 0.1 μM, 1 μM, and 10 μM) for 24 h and stimulated with or without IFN-β (500 U/ml) for another 24 h. MX1 mRNA was measured by RT-qPCR. Data represents the average of three experiments; error bars indicate SEM (one-way ANOVA with Dunnett’s multiple comparisons test). d WT, PFDN3 KO, and PFDN4 KO HEK293 cell lysates were harvested and examined by western blot with the indicated antibodies. e PFDN4 KO HEK293 cells were transfected with PFDN1-6-Myc plasmids for 24 h. Cell lysates were subject to immunoprecipitation using α-Myc antibody and probed for α-UBA3 antibody. PFDN3 is the top band, and PFDN1-4 and PFDN6 are similar in size, as shown on the bottom. f A His-pulldown assay of PFDN3 and UBA3. His-CDPK1 or His-PFDN3 were incubated with Ni2+ beads and then incubated with GST-UBA3. UBA3 protein in the eluates was detected by western blotting using a mouse anti-UBA3 antibody. g BLI sensorgrams were obtained using a biosensor loaded with His-tagged CDPK1 or PFDN3 (50 nM) (load) and incubated with 50 µM UBA3 protein (association). The dissociation is on the right. The binding of PFDN3 to UBA3 is shown in red. For d–f, experiments were repeated at least three times.

Fig 3: UBA3 activates ISG production.a Western blot detection of UBA3 and GAPDH in WT and UBA3 KO (sgRNA1, sgRNA2) HEK293 cells. Experiments were repeated two times. b WT, UBA3 KO (sgRNA1, sgRNA2), PFDN4 KO, PFDN4 and UBA3 double KO (sgRNA1, sgRNA2) HEK293 cells were stimulated with IFN-β (500 U/ml) for 24 h. MX1 mRNA level was measured by RT-qPCR. Data represents the average of three experiments; error bars indicate SEM (one-way ANOVA with Dunnett’s multiple comparisons test). c WT, UBA3 KO (sgRNA1, sgRNA2), PFDN4 KO, and PFDN4 and UBA3 double KO (sgRNA1, sgRNA2) HEK293 cells were stimulated with IFN-β (500 U/ml) for 24 h. Cell lysates were analyzed by western blot. d WT, UBA3 KO HEK293 cells were infected with or without RV (RRV strain, MOI of 5) for 24 h. MX1 mRNA level was measured by RT-qPCR. Data represents the average of three experiments; error bars indicate SEM (two-way ANOVA with Tukey’s multiple comparisons test). e WT and UBA3 KO HEK293 cells were treated with or without Ruxo (10 μM) and IFN-β (500U/ml) for 24 h, and then infected with UK (MOI = 5) for 72 h. Virus titers were determined by an FFU assay. Data represents the average of three experiments; error bars indicate SEM (one-way ANOVA with Tukey’s multiple comparisons test). f WT and UBA3 KO HEK293 cells were induced with IFN-β (500 U/ml) for 24 h. IRF9 mRNA level was measured by RT-qPCR. Data represents the average of three experiments; error bars indicate SEM (one-way ANOVA with Tukey’s multiple comparisons test). g WT and UBA3 KO HEK293 cells were treated with DMSO or MLN4924 (1 μM) for 24 h, then induced with IFN-β (500 U/ml) for 24 h. Cell lysates were analyzed by western blot.

Fig 4: Amino acids 671D and 677Y within VP3 are crucial for hijacking PFDN and inhibiting the interaction between PFDN and UBA3.a PFDN4 KO HEK293 cells were transfected with PFDN1-6-Myc plasmids for 24 h and infected with or without RV (RRV strain, MOI of 5) for another 24 h. Cell lysates were subject to immunoprecipitation using α-Myc antibody and probed for α-UBA3 antibody. PFDN3 is the top band, and PFDN1-4 and PFDN6 are similar in size, as shown on the bottom. b Schematic diagram of WT and mutant VP3 proteins with defined domains illustrated in colors. c HEK293 cells were transfected with GFP-tagged VP3 mutants for 48 h. Cell lysates were subject to immunoprecipitation using α-GFP antibody and probed for α-PFDN3 antibody. * represents a non-specific band. d A predicted structure of VP3 and PFDN3 complex by AlphaFold-3 (PFDN3: green, VP3: cyan). The orange area was the region 670-689 that interacts with PFDN3. Surface electrostatic charge includes red, positive charge; blue, negative charge; and white, neutral charge. e HEK293 cells were transfected with indicated plasmids for 48 h. Cell lysates were subject to immunoprecipitation using α-GFP beads and probed for α-PFDN3 antibody. f The PFDN3 protein was incubated with 670-689 peptide. BLI sensorgrams obtained using biosensor loaded with His-tagged VP3 (10 nM) (load) and incubated with whether 50 µM PFDN3 protein or PFDN3-670-689 peptide (association). The dissociation is shown on the right. g Amino acid alignment of VP3 (670-689) between different RV strains. Group A rotavirus (RVA: RRV, simian strain; Wa, human strain; SA11, simian strain; OSU, porcine strain; ETD, murine strain), Group C rotavirus (RVC: Bristol, human strain), Group D rotavirus (RVD: 05v0049, chicken strain). h HEK293 cells were transfected with GFP vector, GFP-VP3, or GFP-VP3-D671A/Y677A plasmids for 48 h. Cell lysates were subject to immunoprecipitation using α-GFP beads and probed for α-PFDN3 antibody. i HT-29 cells were infected with SA11 or SA11-GTase-Flag (MOI = 0.1) for 24 h. Cell lysates were subject to immunoprecipitation using α-Flag antibody and probed with α-PFDN3 antibody. For a, c, e, and h–i were repeated at least two times.