Editors' ChoiceViral Infection

Replicating Virus in the Autophagosome

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Science Signaling  18 Dec 2012:
Vol. 5, Issue 255, pp. ec325
DOI: 10.1126/scisignal.2003888

Viruses commonly use a cell’s machinery for their own propagation and subvert antiviral defenses, such as autophagy, a cellular degradation process that can degrade intracellular viral particles. Crawford et al. found that rotavirus, which causes severe gastroenteritis and vomiting predominantly in children, depended on the autophagy machinery for its replication, because chemically inhibiting autophagosome formation or deleting critical autophagy genes drastically reduced the intracellular viral load. The rotavirus viroporin NSP4 (nonstructural protein 4), a protein that creates aqueous transmembrane pores, induced calcium release from the endoplasmic reticulum (ER) into the cytoplasm, initiating the early stages of autophagy through the calcium/calmodulin-dependent kinase kinase β (CaMKK-β) pathway. Initiation of autophagy was abrogated in cells treated with a calcium chelator or a CAMKK-β inhibitor. In cells infected with an NSP4 mutant that could not induce a cytosolic calcium increase, the increase in cytosolic calcium was restored when mutant-infected cells were cotreated with the ER calcium pump inhibitor thapsigargin, indicating that rotavirus-induced autophagy was mediated specifically through the viroporin-induced release of calcium that activated CaMKK-β rather than through an ER-mediated unfolded protein response induced by the accumulation of NSP4 in the cell. In a time-course analysis, the processed form of LC3, a component of the autophagosome, was lipidated soon after the NSP4-induced calcium release, followed by formation of autophagosomal puncta containing LC3 and NSP4 that surrounded viroplasms, sites of viral replication. However, autophagy was prevented from progressing to the later stage of lysosomal fusion, in which the virus would be degraded. Using a tandem-tagged red fluorescent protein and green fluorescent protein (RFP-GFP) LC3 probe in which the low pH within lysosomes quenches only the GFP fluorescence, the authors showed that GFP signal was only lost in NSP4-mutant virus-infected cells, whereas cells infected with wild-type rotavirus retained both signals. Furthermore, NSP4 and the rotavirus outer capsid protein VP7 were detected surrounding autophagosome-engulfed viroplasms, indicating completion of particle assembly into new, mature viruses. By inducing autophagosomal formation but inhibiting lysosomal fusion, rotavirus uses the autophagosome as a transient envelope for its own replication and assembly. Their findings demonstrate how the rotavirus requires, but ultimately escapes, the antiviral function of autophagy and suggests that this infectious mechanism may be targeted in treating rotavirus infection and possibly other viral infections in which the virus induces autophagy and increases cytosolic calcium.

S. E. Crawford, J. M. Hyser, B. Utama, M. K. Estes, Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication. Proc. Natl. Acad. Sci. U.S.A.109, E3405–E3413 (2012). [Abstract] [Full Text]

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