ROS-induced HSP70 promotes cytoplasmic translocation of high-mobility group box 1b and stimulates antiviral autophagy in grass carp kidney cells

Abstract

Autophagy plays many physiological and pathophysiological roles. However, the roles and the regulatory mechanisms of autophagy in response to viral infections are poorly defined in teleost fish, such as grass carp (Ctenopharyngodon idella), which is one of the most important aquaculture species in China. In this study, we found that both grass carp reovirus (GCRV) infection and hydrogen peroxide (H 2 O 2 ) treatment induced the accumulation of reactive oxygen species (ROS) in C. idella kidney cells and stimulate autophagy. Suppressing ROS accumulation with N-acetyl-L-cysteine significantly inhibited GCRVinduced autophagy activation and enhanced GCRV replication. Although ROS-induced autophagy, in turn, restricted GCRV replication, further investigation revealed that the multifunctional cellular protein high-mobility group box 1b (HMGB1b) serves as a heat shock protein 70 (HSP70)-dependent, pro-autophagic protein in grass carp. Upon H 2 O 2 treatment, cytoplasmic HSP70 translocated to the nucleus, where it interacted with HMGB1b and promoted cytoplasmic translocation of HMGB1b. Overexpression and siRNA-mediated knockdown assays indicated that HSP70 and HMGB1b synergistically enhance ROS-induced autophagic activation in the cytoplasm. Moreover, HSP70 reinforced an association of HMGB1b with the C. idella ortholog of Beclin 1 (a mammalian ortholog of the autophagy-associated yeast protein ATG6) by directly interacting with C. idella Beclin 1. In summary, this study highlights the antiviral function of ROS-induced autophagy in response to GCRV infection and reveals the positive role of HSP70 in HMGB1b-mediated autophagy initiation in teleost fish.Autophagy is a fundamental mechanism by which cells degrade dysfunctional organelles, misfolded proteins, and other macromolecules and recycle nutrients from unnecessary cellular components (1-3). Under normal conditions, auto-2 The abbreviations used are: ROS, reactive oxygen species; HSP, heat shock . 16 h after transfection, the cells were treated with 0.15 mM H 2 O 2 , 100 nM rapamycin, or an equal volume of PBS (control) for 24 h. Then the cells were fixed with 10% paraformaldehyde and stained with Hoechst 33342. Finally, the samples were visualized by confocal microscopy. The GFP-LC3 puncta indicate autophagosomes. B, H 2 O 2 promotes fusion between GFP-LC3-labeled autophagosomes and RFP-LAMP2-labeled lysosomes. CIK cells were cotransfected with GFP-LC3 and RFP-LAMP2. Then the cells were treated with rapamycin or H 2 O 2 and prepared for confocal microscopy as described above. The colocalization between GFP-LC3 and RFP-LAMP2 represents autolysosomes. C, quantifying the percentage of cells with autophagosomes and autolysosomes. Autophagosomes were quantified by the number of cells with at least five GFP-LC3-postive puncta per cell, accounting for the GFP-LC3-positive cells. Autolysosomes were calculated as the quantity of cells with GFP-LC3 and RFP-LAMP2 co-localization, accounting for all GFP-LC3 and RFP-LAMP2cotransfected cells. Mean Ϯ S.D.; **, p Ͻ 0.01 compared with...

Interferon Type I Regulates Inflammasome Activation and High Mobility Group Box 1 Translocation in Hepatocytes During Ehrlichia -Induced Acute Liver Injury

“…Extracellular hepatic HMGB1 could also bind to the receptor for advanced glycation end products (RAGE) on uninfected cells (macrophages or HCs), which then triggers caspase-1 activation and pyroptosis as suggested by other studies. (29)(30)(31) Our data are consistent with other studies showing that genetic deletion of Hmgb1 or inhibition with HMGB1-neutralizing antibodies protects mice from lethal endotoxemia and sepsis. (30,31) Notably, our data show that cytosolic translocation of HMGB1 correlates with autophagy induction.…”

“…However, as suggested by others, translocation of HMGB1 to the cytosol following stressful stimuli may promote autophagy by direct interaction with beclin-1 (a key protein that initiates autophagosome formation), which results in separation of beclin-1 from Bcl2 apoptosis regulator (Bcl2). (29,30) This HMGB1-beclin1 complex is controlled at the transcriptional, posttranscriptional, and posttranslational level and is positively regulated by unc-51-like autophagy activating kinase 1 (Ulk1) and mitogen-activated protein kinase (MAPK). (29)(30)(31)(32) Our unpublished data suggest that MAPK plays a role in the induction of autophagy in HCs following IOE infection, as evidenced by phosphorylation and activation of the p38 MAPKs (data not shown).…”

Thoroughly Remold the Localization and Signaling Pathway of TLR22

“…Moreover, CiTLR22a and CiTLR22b in lysosomes can efficiently sense dsRNA. Previous studies reported that GCRV enters cells by caveolae/raft-mediated endocytosis and induces autophagy (30,51). During autophagy, autophagosomes encapsulate GCRV and send them to lysosomes for degradation; thus, viral nucleic acids are fully exposed for binding CiTLR22a and CiTLR22b.…”

“…With the same method, CiTLR22a-myc, CiTLR22b-myc, MyD88-HA, TRIF-HA, and TIRAP-HA were ligated into pCMV-eGFP-CMV-SV40 to obtain overexpression vectors. Other localization fusion vectors (LAMP2-RFP, RAB5-RFP, RAB7-RFP, MyD88-RFP, TRIF-RFP, and TIRAP-RFP) and luciferase reporter plasmids (pIRF3pro-Luc, pIRF7pro-Luc, pIFN1pro-Luc, pIFN3pro-Luc, pIFNγ2pro-Luc, pNF-κB1pro-Luc, and pNF-κB2pro-Luc) were constructed in our previous studies (9,29,30). All the vectors were transfected into CIK cells by FuGENE R 6 Transfection Reagent (Promega) according to the manufacturer's instructions.…”

Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response

“…Overexpression plasmids RIG-I-HA and RIG-I-Flag were saved in our lab [21]. pDsRed1-C1-RAB5, pDsRed1-C1-RAB7, and pDsRed1-C1-LAMP2 for subcellular localization studies were also conserved in our lab [29,30]. For dual-luciferase reporter assays, the valid promoters (RIG-I, IPS-1, STING, TBK1, IRF3, IRF7, IFN1, IFN3, IFNγ2, and NF-kB1, respectively) were cloned into pGL3-basic luciferase reporter vector (Promega), which had been previously constructed in our lab [21,27].…”