LC3s hire membrane breakers to attack viral shelters

The MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) conjugation system is a key functional module in macroautophagy (henceforth autophagy) for efficient formation of autophagosomes and selective degradation of autophagosomal cargoes through the lysosome. Recently, it has been appreciated that LC3 and homologs (LC3s) have roles outside of canonical autophagy. One such example is the presence of LC3s on the membrane of phagosomes containing certain pathogen-associated molecular patterns. In this process, termed LC3-associated phagocytosis (LAP), the LC3s modulate fusion between the phagosome and the lysosome. Adding to this theme, we have uncovered another crucial function of LC3s on the membranes of pathogencontaining vacuoles, where they recruit antimicrobial proteins to the LC3-marked membrane. We dubbed this process targeting by autophagy proteins (TAG). In our new study, TAG was identified to mediate a novel antiviral mechanism against the membranous shelters of viruses, known as viral replication complexes (RCs). All known viruses with positive-sense RNA (CRNA) genomes reorganize cellular membranes to form their RCs. These structures are critical in the viral life cycle, shielding viruses from immune detection during genome replication. Although the formation of these RCs has been intensely studied, there have been few studies on how these structures are counteracted by the host immune system. While studying a potential role of autophagy in CRNA virus replication, we found that IFNG/IFN-gamma inhibits the RC of murine norovirus (MNV) and consequently its replication. Intriguingly, this IFNG-mediated inhibition of the MNV RC requires the ATG12–ATG5ATG16L1 complex but not lysosomal degradation through canonical autophagy. The E3-like ligase complex, ATG12– ATG5-ATG16L1, localizes to the RC of MNV, suggesting that the LC3 homologs might be conjugated to the membrane of the MNV RC. However, it was unknown how the ATG12–ATG5ATG16L1 complex functions in this lysosome-independent, IFNG-mediated inhibition of theMNV RC. A clue came from a eukaryotic parasite, Toxoplasma gondii, which forms a parasitophorus vacuole (PV) from the host plasma membrane during its invasion and survives within this cytoplasmic vacuole. Upon activation of cells with IFNs, IFNinducible GTPases are expressed and targeted to the PV membrane (PVM) of T. gondii. These dynamin-like GTPases oligomerize on the targeted membranes and disrupt the structure through vesiculation. Surprisingly, we and others found that the proper targeting of these GTPases to the PVM is dependent on the ATG12–ATG5-ATG16L1 complex but not canonical autophagy. Furthermore, we demonstrated that the ATG12–ATG5ATG16L1 complex marks the PVM of T. gondii with LC3s and that the LC3s are necessary and sufficient to recruit the IFNinducible GTPases to the LC3-marked membranes. Based on this novel function of the LC3 conjugation system in cell-autonomous immune defense, we speculated that TAG may be responsible for the IFNG-mediated inhibition of the MNV RC. Indeed, in our new study, we found that the antiviral activity of IFNG is analogous to the role of IFNG in controlling T. gondii. The ATG12–ATG5ATG16L1 complex is required to mark the MNV RC with LC3s, allowing proper targeting of IFN-inducible GTPases to the RC membrane. The targeting of RCs by the membranolytic GTPases leads to the subsequent disruption of these membrane structures. The role of LC3s in TAG is fundamentally different from those in canonical autophagy and LAP, of which the endgame is fusion with the lysosome and degradation of vacuolar contents (Fig. 1). In fact, the end result of TAG in the IFN-activated cells is to release the vacuolar contents to the cytoplasm, contrary to canonical autophagy. Such a fundamental difference raises 2, if not more, important questions. First, what brings the ATG12–ATG5-ATG16L1 complex to the membranous shelters of pathogens? In canonical autophagy, WIPI2B (WD repeat domain, phosphoinositide interacting 2B) binds to phosphatidylinositol 3-phosphate (PtdIns3P) at the site of autophagosome formation and recruits the ATG12–ATG5ATG16L1 complex to this membrane via its interaction with ATG16L1. In contrast, PtdIns 3-kinase activity is not required to bring the ATG12–ATG5-ATG16L1 complex to the membrane of pathogen shelters, suggesting a novel mechanism of