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A viroporin-like 2B protein of duck hepatitis A virus 1 that induces incomplete autophagy in DEF cells.

ABSTRACT: Duck hepatitis A virus 1 (DHAV-1) can cause high morbidity and fatal acute infectious hepatitis in ducklings, which seriously endangers animal husbandry. Viroporin is a small molecular weight hydrophobic transmembrane protein encoded by the virus, that has been suggested to induce autophagy in host cells by increasing the membrane permeability through disturbing the ion balance. In this study, we aimed to investigate whether the DHAV-1 2B protein can induce autophagy in DEF cells with a viroporin-like function. Bioinformatics analysis has indicated that the 2B protein is characterized by a viroporin domain, which is consistent with the type IA viroporin transmembrane protein. We experimentally confirmed that the 2B protein disturbed the Ca2+ balance of infected cells by elevating the intracellular Ca2+ concentration. Eukaryotic expression of the 2B protein upregulates the expression of microtubule-associated protein 1 light chain 3 II (LC3-II) and the number of autophagosomes in the cell. Interestingly, the Western Blot (WB) results showed that 2B protein expression induced less protein degradation of the autophagic substrate sequestosome 1 (SQSTM1/p62) than the positive control, while microscopy observations showed that the autophagosomes did not colocalize with the lysosomes. In summary, 2B protein expression induced autophagy in host cells, but the autophagic flow was incomplete. The results of this experiment are expected to provide reference scientific data for elucidating the infective and pathogenic mechanism of DHAV-1.

SUBMITTER: Liu Z

PROVIDER: S-EPMC8368021 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1?) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1?. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca(2+) level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca(2+) did not reduce virus-induced IL-1? secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca(2+) flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.

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A viroporin-like 2B protein of duck hepatitis A virus 1 that induces incomplete autophagy in DEF cells.

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