Protein kinase Cdc7 supports viral replication by phosphorylating Avibirnavirus VP3 protein.

IMPORTANCE: The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.

TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1.

Tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin ligase, plays a critical role in the host antiviral response. However, the mechanism and antiviral spectrum of TRIM21 in influenza A virus (IAV) remain unclear. Here, we report that TRIM21 inhibits the replication of various IAV subtypes by targeting matrix protein 1 (M1) from H3/H5/H9, but not H1 and H7 M1. Mechanistically, TRIM21 binds to the residue R95 of M1 and facilitates K48 ubiquitination of M1 K242 for proteasome-dependent degradation, leading to the inhibition of H3, H5, and H9 IAV replication. Interestingly, the recombinant viruses with M1 R95K or K242R mutations were resistance to TRIM21 and exhibited more robust replication and severe pathogenicity. Moreover, the amino acid sequence M1 proteins, mainly from avian influenza such as H5N1, H7N9, H9N2, ranging from 1918 to 2022, reveals a gradual dominant accumulation of the TRIM21-driven R95K mutation when the virus jumps into mammals. Thus, TRIM21 in mammals' functions as a host restriction factor and drives a host adaptive mutation of influenza A virus.

[Identification of host proteins interacting with African swine fever virus inner envelope protein p17].

African swine fever virus (ASFV) infection leads to a mortality rate of up to 100%, causing devastating disasters to the pig industry. Understanding the ASFV infection and replication is therefore of great importance. ASFV has more than 150 open reading frames, among which the inner coat protein p17 encoded by the D117L gene is involved in the formation of the icosahedral structure of the virus. However, little is known about the mechanism how p17 regulates host cell function. In this study, the potential host proteins interacting with ASFV p17 were screened by immunoprecipitation technique combined with protein profiling analysis. The interactions of p17 with mitochondrial membrane protein TOMM70 and heat shock protein HSPA8 were confirmed by co-immunoprecipitation technique and laser confocal experiments. This study provides important information for further exploring the function of p17 during ASFV infection.

TRAF6 autophagic degradation by avibirnavirus VP3 inhibits antiviral innate immunity via blocking NFKB/NF-κB activation.

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-ß (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-ß: 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.

DeSUMOylation of Apoptosis Inhibitor 5 by Avibirnavirus VP3 Supports Virus Replication.

SUMOylation is a reversible posttranslational modification involved in the regulation of diverse biological processes. Growing evidence suggests that virus infection can interfere with the SUMOylation system. In the present study, we discovered that apoptosis inhibitor 5 (API5) is a SUMOylated protein. Amino acid substitution further identified that Lys404 of API5 was the critical residue for SUMO3 conjugation. Moreover, we found that Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. In addition, our results further revealed that viral protein VP3 inhibited the SUMOylation of API5 by targeting API5 and promoting UBC9 proteasome-dependent degradation through binding to the ubiquitin E3 ligase TRAF3. Furthermore, we revealed that wild-type but not K404R mutant API5 inhibited IBDV replication by enhancing MDA5-dependent IFN-ß production. Taken together, our data demonstrate that API5 is a UBC9-dependent SUMOylated protein and deSUMOylation of API5 by viral protein VP3 aids in viral replication. IMPORTANCE Apoptosis inhibitor 5 (API5) is a nuclear protein initially identified for its antiapoptotic function. However, so far, posttranslational modification of API5 is unclear. In this study, we first identified that API5 K404 can be conjugated by SUMO3, and Avibirnavirus infectious bursal disease virus (IBDV) infection significantly decreased SUMOylation of API5. Mechanically, viral protein VP3 directly interacts with API5 and inhibits SUMOylation of API5. Additionally, the cellular E3 ligase TNF receptor-associated factor 3 (TRAF3) is employed by VP3 to facilitate UBC9 proteasome-dependent degradation, leading to the reduction of API5 SUMOylation. Moreover, our data reveal that SUMOylation of API5 K404 promotes MDA5-dependent beta interferon (IFN-ß) induction, and its deSUMOylation contributes to IBDV replication. This work highlights a critical role of conversion between SUMOylation and deSUMOylation of API5 in regulating viral replication.

Inhibition of Antiviral Innate Immunity by Avibirnavirus VP3 via Blocking TBK1-TRAF3 Complex Formation and IRF3 Activation.

The host innate immune system develops various strategies to antagonize virus infection, and the pathogen subverts or evades host innate immunity for self-replication. In the present study, we discovered that Avibirnavirus infectious bursal disease virus (IBDV) VP3 protein significantly inhibits MDA5-induced beta interferon (IFN-ß) expression by blocking IRF3 activation. Binding domain mapping showed that the CC1 domain of VP3 and the residue lysine-155 of tumor necrosis factor receptor-associated factor 3 (TRAF3) are essential for the interaction. Furthermore, we found that the CC1 domain was required for VP3 to downregulate MDA5-mediated IFN-ß production. A ubiquitination assay showed that lysine-155 of TRAF3 was the critical residue for K33-linked polyubiquitination, which contributes to the formation of a TRAF3-TBK1 complex. Subsequently, we revealed that VP3 blocked TRAF3-TBK1 complex formation through reducing K33-linked polyubiquitination of lysine-155 on TRAF3. Taken together, our data reveal that VP3 inhibits MDA5-dependent IRF3-mediated signaling via blocking TRAF3-TBK1 complex formation, which improves our understanding of the interplay between RNA virus infection and the innate host antiviral immune response.IMPORTANCE Type I interferon plays a critical role in the host response against virus infection, including Avibirnavirus. However, many viruses have developed multiple strategies to antagonize the innate host antiviral immune response during coevolution with the host. In this study, we first identified that K33-linked polyubiquitination of lysine-155 of TRAF3 enhances the interaction with TBK1, which positively regulates the host IFN immune response. Meanwhile, we discovered that the interaction of the CC1 domain of the Avibirnavirus VP3 protein and the residue lysine-155 of TRAF3 reduced the K33-linked polyubiquitination of TRAF3 and blocked the formation of the TRAF3-TBK1 complex, which contributed to the downregulation of host IFN signaling, supporting viral replication.

PDPK1 regulates autophagosome biogenesis by binding to PIK3C3.

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.

Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway.

Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication. ABBREVIATIONS: ATG14/Barkor: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; ER: endoplasmic reticulum; hpi: hours post-infection; IBDV: infectious bursal disease virus; IP: co-immunoprecipitation; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PDPK1: 3-phosphoinositid-dependent protein kinase-1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; vBCL2: viral BCL2 apoptosis regulator.

SUMO1 Modification Facilitates Avibirnavirus Replication by Stabilizing Polymerase VP1.

SUMOylation is a posttranslational modification that has crucial roles in diverse cellular biological pathways and in various viral life cycles. In this study, we found that the VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV), regulates virus replication by SUMOylation during infection. Our data demonstrated that the polymerase VP1 is efficiently modified by small ubiquitin-like modifier 1 (SUMO1) in avibirnavirus-infected cell lines. Mutation analysis showed that residues 404I and 406I within SUMO interaction motif 3 of VP1 constitute the critical site for SUMO1 modification. Protein stability assays showed that SUMO1 modification enhanced significantly the stability of polymerase VP1 by inhibiting K48-linked ubiquitination. A reverse genetic approach showed that only IBDV with I404C/T and I406C/F mutations of VP1 could be rescued successfully with decreased replication ability. Our data demonstrated that SUMO1 modification is essential to sustain the stability of polymerase VP1 during IBDV replication and provides a potential target for designing antiviral drugs targeting IBDV.IMPORTANCE SUMOylation is an extensively discussed posttranslational modification in diverse cellular biological pathways. However, there is limited understanding about SUMOylation of viral proteins of IBDV during infection. In the present study, we revealed a SUMO1 modification of VP1 protein, the RNA-dependent RNA polymerase of avibirnavirus infectious bursal disease virus (IBDV). The required site of VP1 SUMOylation comprised residues 404I and 406I of SUMO interaction motif 3, which was essential for maintaining its stability by inhibiting K48-linked ubiquitination. We also showed that IBDV with SUMOylation-deficient VP1 had decreased replication ability. These data demonstrated that the SUMOylation of IBDV VP1 played an important role in maintaining IBDV replication.

BECN1-dependent CASP2 incomplete autophagy induction by binding to rabies virus phosphoprotein.

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.

The C-terminal amyloidogenic peptide contributes to self-assembly of Avibirnavirus viral protease.

Unlike other viral protease, Avibirnavirus infectious bursal disease virus (IBDV)-encoded viral protease VP4 forms unusual intracellular tubule-like structures during viral infection. However, the formation mechanism and potential biological functions of intracellular VP4 tubules remain largely elusive. Here, we show that VP4 can assemble into tubules in diverse IBDV-infected cells. Dynamic analysis show that VP4 initiates the assembly at early stage of IBDV infection, and gradually assembles into larger size of fibrils within the cytoplasm and nucleus. Intracellular assembly of VP4 doesn't involve the host cytoskeleton, other IBDV-encoded viral proteins or vital subcellular organelles. Interestingly, the last C-terminal hydrophobic and amyloidogenic stretch (238)YHLAMA(243) with two "aggregation-prone" alanine residues was found to be essential for its intracellular self-assembly. The assembled VP4 fibrils show significantly low solubility, subsequently, the deposition of highly assembled VP4 structures ultimately deformed the host cytoskeleton and nucleus, which was potentially associated with IBDV lytic infection. Importantly, the assembly of VP4 significantly reduced the cytotoxicity of protease activity in host cells which potentially prevent the premature cell death and facilitate viral replication. This study provides novel insights into the formation mechanism and biological functions of the Avibirnavirus protease-related fibrils.