Abstract
Autophagy is an intrinsic cellular process that can degrade cytoplasmic components. It has been reported that several pathogens hijack this process to facilitate their replication. Coxsackievirus B3 (CVB3), a member of the family Picornaviridae, induces autophagy upon infection. However, the details of CVB3-induced autophagy remain a subject of debate. This study applied a combination of multiple assays for the measurement of autophagy and demonstrated that CVB3 induces a complete autophagic flux. Experiments with infected HEK293A cells revealed that autophagosomes were induced upon CVB3 infection. Most of these autophagosomes were mCherry positive in mCherry-GFP-LC3 cells. Conversely, mCherry-positive autophagosomes were rescued to green positive when treated with the acidification inhibitors chloroquine (CQ) and bafilomycin A1 (BAF), suggesting that autophagosomes fused with late endosomes or lysosomes. The co-localization of LC3-positive puncta with lysosome-associated membrane protein 1 (LAMP1) or LysoTracker confirmed that the autophagosomes fused primarily with lysosomes. Interestingly, the disruption of autophagosome formation by 3-methyladenine (3-MA) or ATG5 siRNA treatment during viral infection significantly decreased CVB3 replication. However, inhibitors of lysosomal acidification, fusion, or degradation did not affect viral replication. Therefore, autolysosomes may not be critical for viral replication in vitro.
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Acknowledgments
The authors would like to thank Prof. William Jackson at Medical College of Wisconsin for providing the mCherry-GFP-LC3 plasmid. The authors also thank Prof. Zhaohua Zhong at Harbin Medical University for providing the CVB3-eGFP plasmid. The anti-CVB3-VP1 polyclonal antibody was kindly provided by Dr. Fei Deng at the Wuhan Institute of Virology, Chinese Academy of Science.
This work was supported by grants from Major State Basic Research Development Program of China (2013CB531502, 2013CB530501), the National Natural Science Foundation of China (31270977, 81101257, 31470848), the Scientific Research Foundation for Returned Overseas Chinese Scholars from State Education Ministry to C.D., and Jiangsu Provincial Innovative Research Team. The authors declare that there are no conflicting financial interests.
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X. Shi and Z. Chen contributed equally to the work.
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Supplementary data 1 HEK293A mCherry-GFP-LC3 stable cells starved with or without 50 nM CQ treatment. At 8 h post-starvation, the cells were observed under confocal microscopy. The bar represents 10 μm. (JPEG 2103 kb)
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Shi, X., Chen, Z., Tang, S. et al. Coxsackievirus B3 infection induces autophagic flux, and autophagosomes are critical for efficient viral replication. Arch Virol 161, 2197–2205 (2016). https://doi.org/10.1007/s00705-016-2896-6
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DOI: https://doi.org/10.1007/s00705-016-2896-6