These authors contributed equally to this work.Keywords: AKT-MTOR pathway, autophagy, avibirnavirus, HSP90AA1, viral protein VP2Abbreviations: ANOVA, analysis of variance; ATG5, autophagy-related 5; BCA, bicinchoninic acid; BECN1, Beclin 1, autophagyrelated; cDNA, complementary DNA; CoIP, coimmunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; dsRNA, double-stranded RNA; EBSS, Earle's balanced salt solution; EIF2AK2, eukaryotic translation initiation factor 2-alpha kinase 2; EIF2S1, eukaryotic translation initiation factor 2, subunit 1 alpha; eGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; Gg, Gallus gallus (chicken); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GOPC, golgi-associated PDZ and coiled-coil motif containing; GST, glutathione S-transferase; HE-IBDV, heat-inactivated IBDV; hpi, hours post-infection; Hs, Homo sapiens (human); HSP90AA1, heat shock protein 90 kDa alpha (cytosolic), class A member 1; HSV-1, herpes simplex virus 1; IBDV, infectious bursal disease virus; IgG, immunoglobulin G; LPS, lipopolysaccharide; mAb, monoclonal antibody; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin (serine/threonine kinase); Ni-NTA, nickel-nitrilotriacetic acid; PAMP, pathogen-associated molecular patterns; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-kinase; PRR, pattern recognition receptors; RNAi, RNA interference; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; shRNA, short hairpin RNA; SQSTM1, sequestosome 1; TCID 50 , 50% tissue culture infectious doses; TLR, tolllike receptors; TSC, tuberous sclerosis complex; VP, viral protein; SVP, subviral particle.Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa a [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon...
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 404 I and 406 I 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 I 404 C/T and I 406 C/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 404 I and 406 I 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.S UMOylation is the covalent attachment of SUMO molecules to lysine (K) on the target protein (1). A SUMO molecule has an approximate molecular weight of 12 kDa; however, the covalent attachment of one SUMO moiety adds 20 kDa to the substrate (2). The mature SUMO peptide is activated by E1-activating enzyme (SUMOactivating enzymes 1 and 2 [SAE1/2]) in an ATP-dependent manner. The active SUMO is then transferred to Ubc9, the E2 conjugation enzyme (3). In contrast to the ubiquitination process, E3 SUMO ligase is optional for transferring the SUMO peptide to the substrate (4). As posttranslational modifications, the SUMOylation process could be also reversed by sentrin-specific proteases (SENPs), which act as de-SUMOylation enzymes (5). Generally, the lysine following the motif ⌿KXE/D (where ⌿ represents a large on July 11, 2020 by guest http://jvi.asm.org/ Downloaded from FIG 3 Identification of SIM3 required for VP1 SUMO1 modification. (A) Identification of the candidate SUMOylation sites of VP1. 293T cells were cotransfected with Myc-Ubc9, HA-SUMO1, and Flag-VP1 or its truncation mutants for 36 h. (B to D) Iden...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.