Muscovy duck reovirus σNS protein triggers autophagy enhancing virus replication

Abstract

BackgroundMuscovy duck reovirus (MDRV) causes high morbidity and mortality in Muscovy ducklings at 10 days old and can persist in an infected flock until the ducklings of 6 weeks old. It shares common physicochemical properties with avian reovirus (ARV) and differs in coding assignment and pathogenicity. The ARV p17 protein has been shown to trigger autophagy via activation multiple signaling pathways, which benefits virus replication. Since MDRV lacks the p17 protein, whether and how MDRV induces autophagy remains unknown. The aim of this study was to explore whether MDRV induces autophagy and which viral proteins are involved in MDRV-induced autophagy.MethodsThe autophagosome-like structures in MDRV-infected cells was observed under transmission electron microscopy. MDRV-induced autophagy was examined by analyzing the LC3-II level and phosphorylated form of mammalian target of rapamycin (mTOR) by Western blot assays. The effects of 3-methyladenine, rapamycin, chloroquine on viral yields were measured with quantitative(q) real-time reverse transcription (RT)-polymerase chain reaction (PCR) and 50% tissue culture infective dose (TCID50) assays, respectively. Additionally, to determine which viral protein is responsible for MDRV-induced autophagy, both p10.8- and σNS-encoding genes of MDRV were cloned into the pCI-neo-flag vector and transfected into DF-1 cells for detection of LC3-II.ResultsThe typical double-membrane vesicles containing cytoplasmic inclusions were visible in MDRV-infected immortalized chicken embryo fibroblast (DF-1) cells under transmission electron microscopy. Both primary Muscovy duck embryo fibroblasts (MDEF) and DF-1 cells infected with MDRV exhibited a significant increased levels of LC3-II accompanied with downregulation of phosphorylated form of mTOR, further confirming that MDRV is capable of inducing autophagy. Autophagy could be suppressed by 3-methylademine and induced by rapamycin and chloroquine. Furthermore, we found that σNS induces an increased levels of LC3-II, suggesting that the MDRV σNS protein is one of viral proteins involved in induction of autophagy. Both qRT-PCR and TCID50 assays showed that virus yield was increased in rapamycin treated DF-1 cells following MDRV infection. Conversely, when infected cells were pretreated with chloroquine, virus yield was decreased.ConclusionsThe MDRV σNS nonstructural protein is responsible for MDRV-induced autophagy and benefits virus replication.

Endoplasmic Reticulum Stress Mediated MDRV p10.8 Protein-Induced Cell Cycle Arrest and Apoptosis Through the PERK/eIF2α Pathway

“…MDRV could cause injury of the small intestinal mucosal immune barrier and mucosal immune function in sick Muscovy ducklings ( Wu et al, 2017b ). MDRV σNS protein could trigger autophagy to enhance its replication ( Wu et al, 2017a ). The current study revealed that MDRV could induce ER stress and that p10.8 protein was the critical factor; these results provided new evidence for elucidating the molecular pathogenic mechanism of MDRV.…”

“…The DF1 cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS. Cells, at 70% confluence were transfected with plasmids (pCI-neo-flg, pCI-neo-flg-p10.8) using Lipofectamine 2000 reagent (Promega, Madison, WI, United States), as previously described ( Wu et al, 2017a ).…”

“…DF1 cell line is the important model cell of avian virus research ( Wang et al, 2015 ; Wu et al, 2017a ). DF-1 cells were transfected with plasmids (pCI-neo-flag-p10.8, pCI-neo-flag) or infected with MDRV (MOI = 2) for 24 h, respectively, then were collected and lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), supplemented with a complete protease inhibitor cocktail (Roche, Switzerland)].…”

“…The protein A-Sepharose mixture was washed five times with NETN (900 mmol/L NaCl). Finally, the samples were analyzed by Western blot ( Wu et al, 2017a ).…”

Bombyx mori Nuclear Polyhedrosis Virus (BmNPV) Induces Host Cell Autophagy to Benefit Infection

“…There has been a great deal of research into the relationship between viral infection and host autophagy. Autophagy commonly serves as a defense mechanism against viral infection [ 12 , 13 ]; however, many viruses have evolved to escape, or even to exploit, this mechanism to favor their survival or replication in different ways [ 6 , 14 , 15 , 16 , 17 ]. Thus, the role of autophagy in host-virus interactions is diverse for different viruses.…”