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

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.V iruses are obligate intracellular parasites that, due to their limited coding capacity, have evolved strategies that usurp cellular processes to facilitate their own propagation. Macroautophagy (hereafter referred to as autophagy) is a cellular catabolic process used to maintain homeostasis by delivering cytoplasmic material to lysosomes for degradation via an intracellular membrane trafficking pathway (1). Autophagy also has intracellular antimicrobial properties and plays a role in the initiation of innate and adaptive immune responses to viral and bacterial infections. Numerous pathogens, including a number of DNA and RNA viruses, have been shown to evade or subvert autophagy (2); however, for most of these viruses, the mechanisms used to initiate autophagy and subvert the normal autophagy process have not been elucidated.The formation of autophagy membranes is complex and not completely understood, but the autophagy (Atg) proteins comprise the core molecular machinery involved in this dynamic membrane rearrangement (3). Autophagy, which is repressed by the mammalian target of rapamycin (mTOR), can be activated by nutrient deprivation; growth factor depletion; or cellular stress, such as hypoxia, energy depletion, endoplasmic reticulum (ER) stress, high temperature, or high cell density conditions (4). Following nutrient deprivation, mTOR is inhibited and a complex composed of Atg13/ULK1/FIP200/Atg101 forms to initiate nucleation of an isolation membrane, or phagophore (5). The phagophore elongates and subsequently encloses cytoplasmic components, forming a double-membrane vacuole, the autophagosome. The elongation phase requires two ubiquitin-like conjugation ...

Activation of the Endoplasmic Reticulum Calcium Sensor STIM1 and Store-Operated Calcium Entry by Rotavirus Requires NSP4 Viroporin Activity

“…Depletion of ER Ca 2ϩ needed to sustain chronic STIM1 activation seems incompatible with the need for ER Ca 2ϩ to assemble VP7 onto RV particles during morphogenesis. Our recent data showing that NSP4 viroporin activity induces autophagy to traffic NSP4 and VP7 to viroplasms (28) suggests that final virus morphogenesis takes place at discrete ER microdomains, which are likely isolated from the ER-PM junctions that are the sites of Ca 2ϩ entry and resequestration into the ER. This predicts that RV may generate a Ca 2ϩ microdomain within membranes that surround viroplasms, but further investigation is needed to determine how reduced ER Ca 2ϩ , induction of SOCE, and RV assembly are integrated during infection.…”

“…Previous studies showed the formation of NSP4 punctate structures is dependent on elevation of [Ca 2ϩ ]cyto (8,28,34). Therefore, we hypothesize activation of STIM1 should correlate with NSP4 puncta formation because STIM1 activation would activate Ca 2ϩ entry, which would then promote NSP4 puncta formation due to elevated [Ca 2ϩ ]cyto.…”

“…YFP-STIM1 cells were seeded into ⌬T Dishes (Bioptics, Butler, PA) and transfected with plasmids expressing RFP-tagged wild-type NSP4 (NSP4-WT) or mutant NSP4 (NSP4-Mut) RFP-labeled proteins. We quantitated the number of cells containing diffuse or punctate YFP-STIM1 similar to a previously described NSP4/LC3 puncta formation assay (28). At ϳ20 h posttransfection, random fields from two experiments were imaged by deconvolution microscopy, and the images were collected.…”

“…Determination of infectious rotavirus yield by fluorescent focus assay (FFA) was performed as previously described (28). Results are reported as the number of infectious virus per cell.…”

Topology and biological function of enterovirus non-structural protein 2B as a member of the viroporin family

“…Autophagic vesicle membranes are used to transfer viral proteins from the ER to the corresponding replication and assembly sites [48]. The maturation of autophagosomes is inhibited during enterovirus infection, and intracellular vesicles become abundant during enterovirus 2B protein process.…”