Ubiquitination is an important reversible post-translational modification. Many viruses hijack the host
ubiquitin system to enhance self-replication. In the present study, we found that Avibirnavirus VP3
protein was ubiquitinated during
infection and supported virus replication by ubiquitination. Mass spectrometry and mutation analysis showed that VP3 was ubiquitinated at residues K73, K135, K158, K193, and K219. Virus rescue showed that ubiquitination at sites K73, K193, and K219 on VP3 could enhance the replication abilities of infectious bursal disease virus (IBDV), and that K135 was essential for virus survival. Binding of the zinc finger domain of
TRAF6 (
TNF receptor associated factor 6) to VP3 mediated K11- and K33-linked ubiquitination of VP3, which promoted its nuclear accumulation to facilitate virus replication. Additionally, VP3 could inhibit TRAF6-mediated NFKB/NF-κB (
nuclear factor kappa B) activation and IFNB/IFN-β (
interferon beta) production to evade host innate immunity by inducing
TRAF6 autophagic degradation in an SQSTM1/p62 (sequestosome 1)-dependent manner. Our findings demonstrated a macroautophagic/autophagic mechanism by which Avibirnavirus
protein VP3 blocked NFKB-mediated IFNB production by targeting
TRAF6 during
virus infection, and provided a potential drug target for
virus infection control.Abbreviations: ATG: autophagy related; BafA1:
bafilomycin A1; CALCOCO2/NDP52:
calcium binding and coiled-coil domain 2; Cas9:
CRISPR-associated protein 9; CHX:
cycloheximide; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; GAPDH:
glyceraldehyde-3-phosphate dehydrogenase; GST:
glutathione S-transferase; IBDV: infectious bursal disease virus; IF: indirect immunofluorescence; IFNB/IFN-β:
interferon beta; mAb:
monoclonal antibody; MAP1LC3/LC3:
microtubule associated protein 1 light chain 3; MOI: multiplicity of
infection; MS: mass spectrometry; NFKB/NF-κB:
nuclear factor kappa B; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; pAb: polyclonal antibody;
PRRs:
pattern recognition receptors; RNF125: ring finger
protein 125; RNF135/Riplet: ring finger
protein 135; SQSTM1/p62: sequestosome 1; TAX1BP1: tax1 binding protein1; TCID50: 50% tissue culture infective dose;
TRAF3:
TNF receptor associated factor 3;
TRAF6:
TNF receptor associated factor 6; TRIM25: tripartite motif containing 25; Ub:
ubiquitin; Wort:
wortmannin; WT: wild type.