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Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease.

ABSTRACT: Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in suckling piglets, leading to severe economic losses worldwide. There is an urgent need to find new therapeutic methods to prevent and control PEDV. Not only is there a shortage of commercial anti-PEDV drugs, but available commercial vaccines fail to protect against highly virulent PEDV variants. We screened an FDA-approved library of 911 natural products and found that tomatidine, a steroidal alkaloid extracted from the skin and leaves of tomatoes, demonstrates significant inhibition of PEDV replication in Vero and IPEC-J2 cells in vitro. Molecular docking and molecular dynamics analysis predicted interactions between tomatidine and the active pocket of PEDV 3CL protease, which were confirmed by fluorescence spectroscopy and isothermal titration calorimetry (ITC). The inhibiting effect of tomatidine on 3CL protease was determined using cleavage visualization and FRET assay. Tomatidine-mediated blocking of 3CL protease activity in PEDV-infected cells was examined by western blot detection of the viral polyprotein in PEDV-infected cells. It indicates that tomatidine inhibits PEDV replication mainly by targeting 3CL protease. In addition, tomatidine also has antiviral activity against transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), encephalo myocarditis virus (EMCV) and seneca virus A (SVA) in vitro. These results may be helpful in developing a new prophylactic and therapeutic strategy against PEDV and other swine disease infections.

SUBMITTER: Wang P

PROVIDER: S-EPMC7656508 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Interferon-induced BST2 (bone marrow stromal cell antigen 2) inhibits viral replication by tethering enveloped virions to the cell surface to restrict viral release and by inducing the NFKB-dependent antiviral immune response. However, the mechanism by which BST2 uses the selective autophagy pathway to inhibit viral replication is poorly understood. In this study, we showed that BST2 expression was significantly increased during porcine epidemic diarrhea virus (PEDV) infection of Vero cells by IRF1 targeting its promoter. We also showed that BST2 suppressed PEDV replication by binding and degrading the PEDV-encoded nucleocapsid (N) protein. The downregulation of N protein was blocked by macroautophagy/autophagy inhibitors but not a proteasome inhibitor, implying that the N protein was degraded via the selective autophagy pathway. Both the BST2 and N protein interacted with the E3 ubiquitin ligase MARCHF8/MARCH8 and the cargo receptor CALCOCO2/NDP52, and the ubiquitination of N protein was necessary for the degradation of N mediated by the BST2-MARCHF8 axis. The knockdown of MARCHF8 or ATG5 with small interfering RNAs blocked the selective autophagy pathway, rescued the protein abundance of PEDV N in 293T cells, and prevented the inhibition of PEDV replication by BST2 in Vero cells. Together, our data demonstrate the novel mechanism of BST2-mediated virus restriction, in which BST2 recruits MARCHF8 to catalyze the ubiquitination of the PEDV N protein. The ubiquitinated N protein is then recognized by CALCOCO2/NDP52, which delivers it to autolysosome for degradation through the selective autophagy pathway. Abbreviations: 3MA: 3-methyladenine; ATG: autophagy-related; Baf A1: bafilomycin A1; BST2: bone marrow stromal cell antigen 2; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CC: coiled-coil; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; CT: cytoplasmic tail; DAPI: 4',6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; IRF1: interferon regulatory factor 1; ISG: IFN-stimulated gene; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PED: porcine epidemic diarrhea; PEDV: porcine epidemic diarrhea virus; RT: room temperature; siRNA: small interfering RNA; STAT: signal transducer and activator of transcription; TCID50: 50% tissue culture infectious doses; TM: transmembrane.

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Tomatidine inhibits porcine epidemic diarrhea virus replication by targeting 3CL protease.

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