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Published on: October 7, 2011
Muscovy duck reovirus σNS protein triggers autophagy enhancing virus replication
Yijian Wu1,2, Longping Cui1, Erpeng Zhu1,2
1College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, People's Republic of China.
Background:
Muscovy 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.
Methods:
The 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.
Results:
The 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.
Conclusions:
The MDRV σNS nonstructural protein is responsible for MDRV-induced autophagy and benefits virus replication.
Insights
Muscovy duck reovirus (MDRV) induces autophagy through its σNS protein, which benefits viral replication. This study identifies the specific viral protein responsible for this process in infected cells.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Muscovy duck reovirus (MDRV) causes significant mortality in ducklings and shares properties with avian reovirus (ARV).
- Unlike ARV's p17 protein, the mechanism of autophagy induction by MDRV, particularly lacking p17, was unknown.
- Understanding MDRV's autophagy induction is crucial given its impact on duck health and potential for viral replication.
Purpose of the Study:
- To investigate whether Muscovy duck reovirus (MDRV) induces autophagy in host cells.
- To identify the specific viral protein(s) responsible for MDRV-mediated autophagy induction.
- To elucidate the role of autophagy in MDRV replication.
Main Methods:
- Transmission electron microscopy to visualize autophagosome-like structures in MDRV-infected cells.
- Western blot analysis to assess LC3-II levels and mTOR phosphorylation.
- Gene cloning and transfection to determine the role of MDRV p10.8 and σNS proteins in autophagy.
- Quantitative RT-PCR and TCID50 assays to measure viral yields under autophagy modulation.
Main Results:
- Autophagosome-like structures and increased LC3-II levels with decreased phosphorylated mTOR were observed in MDRV-infected cells.
- Autophagy induction was confirmed by modulation with 3-methyladenine, rapamycin, and chloroquine.
- The MDRV σNS protein was identified as a key viral factor inducing increased LC3-II levels.
Conclusions:
- The Muscovy duck reovirus (MDRV) σNS nonstructural protein is responsible for inducing autophagy.
- MDRV-induced autophagy enhances viral replication, as evidenced by increased virus yields.
- The findings reveal a novel mechanism by which MDRV manipulates host cell processes for its benefit.
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