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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. [ABSTRACT FROM AUTHOR]

Accumulation of toxic protein assemblies and damaged mitochondria are key features of neurodegenerative diseases, which arise in large part from clearance defects in the Macroautophagy/autophagy-lysosome system. The autophagy cargo receptor SQSTM1/p62 plays a major role in the clearance of ubiquitinated cargo through Ser403 phosphorylation by multiple kinases. However, no phosphatase is known to physiologically dephosphorylate SQSTM1 on this activating residue. RNAi-mediated knockdown and overexpression experiments using genetically encoded fluorescent reporters and defined mutant constructs in cell lines, primary neurons, and brains show that SSH1, the canonical CFL (cofilin) phosphatase, mediates the dephosphorylation of phospho-Ser403-SQSTM1, thereby impairing SQSTM1 flux and phospho-MAPT/tau clearance. The inhibitory action of SSH1 on SQSTM1 is fully dependent on SQSTM1 Ser403 phosphorylation status and is separable from SSH1-mediated CFL activation. These findings reveal a unique action of SSH1 on SQSTM1 independent of CFL and implicate an inhibitory role of SSH1 in SQSTM1-mediated clearance of autophagic cargo, including phospho-MAPT/tau. Abbreviations: AAV: adeno-associated virus; Aβ42O: amyloid β1-42 oligomers; AD: Alzheimer disease; CA3: cornu Ammonis 3; CSNK2/CK2: casein kinase 2; FCCP: 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile; FTLD: frontotemporal lobar degeneration; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; SQSTM1/p62: sequestosome-1; PLA: proximity ligation assay; RFP: red fluorescent protein; RIPA: radioimmunoprecipitation assay; shRNA: short hairpin RNA; siRNA: small interfering RNA; Ser403: Serine403; SSH1: slingshot protein phosphatase 1; TBK1: TANK-binding kinase 1; ULK: unc-51 like kinase 1 [ABSTRACT FROM AUTHOR]

PDPK1 (3-phosphoinositide dependent protein kinase 1) is a phosphorylation-regulated kinase that plays a central role in activating multiple signaling pathways and cellular processes. Here, this study shows that PDPK1 turns on macroautophagy/autophagy as a SUMOylation-regulated kinase. In vivo data demonstrate that the SUMO modification of PDPK1 is a physiological feature in the brain and that it can be induced by viral infections. The SUMOylated PDPK1 regulates its own phosphorylation and subsequent activation of the AKT1 (AKT serine/threonine kinase 1)-MTOR (mechanistic target of rapamycin kinase) pathway. However, SUMOylation of PDPK1 is inhibited by binding to PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3). The nonSUMOylated PDPK1 then tethers LC3 to the endoplasmic reticulum to initiate autophagy, and it acts as a key component in forming the autophagic vacuole. Collectively, this study reveals the intricate molecular regulation of PDPK1 by post-translational modification in controlling autophagosome biogenesis, and it highlights the role of PDPK1 as a sensor of cellular stress and regulator of autophagosome biogenesis. Abbreviations: AKT1: AKT serine/threonine kinase 1; ATG14: autophagy related 14; Co-IP: co-immunoprecipitation; ER: endoplasmic reticulum; hpi: hours post-infection; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; pAb: polyclonal antibody; PDPK1: 3-phosphoinositide dependent protein kinase 1; PI3K: phosphoinositide 3-kinase; PIK3C3: phosphatidylinositol 3-kinase catalytic, subunit type 3; RPS6KB1: ribosomal protein S6 kinase B1; SGK: serum/glucocorticoid regulated kinase; SQSTM1: sequestosome 1; SUMO: small ubiquitin like modifier; UBE2I/UBC9: ubiquitin conjugating enzyme E2 I; UVRAG: UV radiation resistance associated [ABSTRACT FROM AUTHOR]

Iron metabolism is involved in numerous physiological processes such as erythropoiesis, oxidative metabolism. However, the in vivo physiological functions of the iron metabolism-related gene Hfe in immune response during viral infection remain poorly understood. Here, we identified 5 iron metabolism-associated genes specifically affected during RNA virus infection by a high-throughput assay and further found that HFE was a key negative regulator of RIG-I-like receptors (RLR)-mediated type I interferons (IFNs) signaling. RNA virus infection inhibited the binding of HFE to MAVS (mitochondrial antiviral signaling protein) and blocked MAVS degradation via selective autophagy. HFE mediated MAVS autophagic degradation by binding to SQSTM1/p62. Depletion of Hfe abrogated the autophagic degradation of MAVS, leading to the stronger antiviral immune response. These findings established a novel regulatory role of selective autophagy in innate antiviral immune response by the iron metabolism-related gene Hfe. These data further provided insights into the crosstalk among iron metabolism, autophagy, and innate immune response. Abbreviations: ATG: autophagy-related; BAL: bronchoalveolar lavage fluid; BMDMs: bone marrow-derived macrophages; CGAS: cyclic GMP-AMP synthase; CQ: chloroquine; Dpi: days post-infection; ELISA: enzyme-linked immunosorbent assay; GFP: green fluorescent protein; HAMP: hepcidin antimicrobial peptide; Hpi: hours post-infection; HJV: hemojuvelin BMP co-receptor; IFNs: interferons; IL6: interleukin 6; IRF3: interferon regulatory factor 3; ISRE: interferon-stimulated response element; Lipo: clodronate liposomes; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MEFs: mouse embryonic fibroblasts; SLC40A1/FPN1: solute carrier family 40 (iron-regulated transporter), member 1; flatiron; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1/STING: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1; TFRC/TfR1: transferrin receptor; TNF/TNFα: tumor necrosis factor; WT: wild type. [ABSTRACT FROM AUTHOR]

Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection. [ABSTRACT FROM PUBLISHER]