Fig. 6: 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.