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PNAS 109 (50): 3405-3413

Copyright © 2012 by the National Academy of Sciences.



Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication

Sue E. Crawford, Joseph M. Hyser, Budi Utama, and Mary K. Estes1

Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030

Contributed by Mary K. Estes, September 25, 2012 (sent for review May 23, 2012)


This study describes a unique mechanism of virus-initiated autophagy and exploitation of autophagy membranes for virus replication. Autophagy is a highly regulated cellular process in which cells destroy and recycle their own components in lysosomes. The mechanism most viruses use to induce autophagy is unknown. We show a rotavirus pore-forming protein activates a calcium-dependent signaling pathway to initiate autophagy. Rotavirus hijacks autophagy membranes to transport viral proteins to sites of virus replication for assembly of infectious particles and interferes with autophagy maturation. Inhibition of the signaling pathway blocks virus production, suggesting a therapeutic target to fight infection.

Abstract: Autophagy is a cellular degradation process involving an intracellular membrane trafficking pathway that recycles cellular components or eliminates intracellular microbes in lysosomes. Many pathogens subvert autophagy to enhance their replication, but the mechanisms these pathogens use to initiate the autophagy process have not been elucidated. This study identifies rotavirus as a pathogen that encodes a viroporin, nonstructural protein 4, which releases endoplasmic reticulum calcium into the cytoplasm, thereby activating a calcium/calmodulin-dependent kinase kinase-β and 5' adenosine monophosphate-activated protein kinase-dependent signaling pathway to initiate autophagy. Rotavirus hijacks this membrane trafficking pathway to transport viral proteins from the endoplasmic reticulum to sites of viral replication to produce infectious virus. This process requires PI3K activity and autophagy-initiation proteins Atg3 and Atg5, and it is abrogated by chelating cytoplasmic calcium or inhibiting calcium/calmodulin-dependent kinase kinase-β. Although the early stages of autophagy are initiated, rotavirus infection also blocks autophagy maturation. These studies identify a unique mechanism of virus-mediated, calcium-activated signaling that initiates autophagy and hijacks this membrane trafficking pathway to transport viral proteins to sites of viral assembly.

Key Words: viroplasm • morphogenesis • LC3 • STO-609

Author contributions: S.E.C., J.M.H., B.U., and M.K.E. designed research; S.E.C., J.M.H., and B.U. performed research; S.E.C., J.M.H., B.U., and M.K.E. analyzed data; and S.E.C., J.M.H., and M.K.E. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at

1To whom correspondence should be addressed. E-mail: mestes{at}

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