Abstract
Despite autophagy’s pivotal role in the replication of viruses such as duck Tembusu virus (DTMUV), which has caused massive economic losses to the poultry industry in the world, the specific relationships between DTMUV and cellular autophagy remain largely unknown. In response, we investigated the interactions between autophagy and DTMUV, the effects of the structural and non-structural proteins of DTMUV on autophagy, and the autophagy-related signaling pathways induced by DTMUV. Among the results, DTMUV increased the autophagy flux in duck embryo fibroblasts (DEF) and BHK-21 cells, while autophagy facilitated viral replication. After we pharmacologically induced autophagy with rapamycin (RAPA), the replication of DTMUV increased by 15.23-fold compared with the control group of DEF cells. To identify which DTMUV protein primarily induced autophagy, all three structural proteins and seven non-structural proteins of DTMUV were transfected into cells, and the results showed that non-structural protein 3 (NS3) induced significant autophagy in DEF cells. By means of Western blot, immunofluorescence, and transmission electron microscopy, we confirmed that NS3 protein could significantly induce autophagy and autophagy flux. Furthermore, we showed that NS3 induced autophagy in DEF cells through extracellular signal-regulated kinase 2 (ERK2) and phosphatidylinositol-3-kinase (PI3K)/AKT and the mammalian target of rapamycin (mTOR) signaling pathways using specific inhibitors and RNA interference assays. Finally, autophagy induced by NS3 promoted DTMUV replication. These results provide novel insight into the relationship between DTMUV and autophagy, broadening the current understanding of the molecular pathogenesis of DTMUV.
Highlights
In April 2010, duck Tembusu virus (DTMUV) was identified as the causative agent of egg drop syndrome, a condition among egg-laying and breeder ducks in China characterized by depression, a substantial decrease in egg laying and growth, and signs of neurological degradation [1]
Studies have shown that the NS4A of a variety of flaviviruses plays a vital role in inducing autophagy, such as dengue virus (DENV) [30], West Nile virus (WNV) [31], and Zika virus (ZIKV) [32]
Further expression of individual DTMUV proteins indicated that E, nonstructural protein 3 (NS3), NS4A, and NS4B proteins could significantly increase the expression of light chain 3 (LC3)-II, and we further confirmed that DTMUV NS3 protein played a key role in inducing autophagy of duck embryo fibroblasts (DEF) cells by immunofluorescence, Western blot, and transmission electron microscopy (TEM) experiments, and
Summary
In April 2010, duck Tembusu virus (DTMUV) was identified as the causative agent of egg drop syndrome, a condition among egg-laying and breeder ducks in China characterized by depression, a substantial decrease in egg laying and growth, and signs of neurological degradation [1]. Since expanding host profiles of DTMUV infection have revealed that it can infect ducks but chickens and geese as well [2]. When positive DTMUV antibodies were detected in the sera of workers at duck farms [4], the virus became known for causing tremendous economic losses in the poultry industry and as a potential zoonotic pathogen that could threaten public health [5]. DTMUV possesses a genome approximately 11 kb long that encodes three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [7]. NS3 plays a non-enzymatic role in autophagy, fatty acid biosynthesis, and actin polymerization, all of which are mediated by its recruitment of host proteins from various cellular pathways [12]
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