Foot-and-Mouth Disease Virus Induces Autophagosomes during Cell Entry via a Class III Phosphatidylinositol 3-Kinase-Independent Pathway

Abstract

Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, ϳ50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication. Macroautophagy is a highly conserved degradation pathway that recycles cytoplasmic organelles and proteins in response to starvation (48). During macroautophagy (here referred to as autophagy), cup-shaped isolation membranes expand to form double-membrane autophagosomes that engulf cytosolic cargo and then fuse with late endosomes or lysosomes for degradation (49). Autophagosome formation is regulated by the mTORC1 (mammalian target of rapamycin complex 1) kinase, which is a negative regulator of autophagy. Autophagy can therefore be activated in the absence of starvation by mTORC1 inhibitors, such as rapamycin (46) or torin (43). mTORC1 regulates a multimeric complex (the ULK-1 complex) made up of ULK-1 (unc-51-like kinase), FIP200 (focal adhesion kinase family interacting protein of 200 kDa), Atg13, and Atg101 (4, 8-11, 13, 17, 26). Other key autophagy components, such as the phosphatidylinositol 3-kinase (PI3-kinase) complex (Atg6/beclin-Atg14-Vps15-Vps34) and the Atg12 (Atg12-Atg5-Atg16) and LC3 conjugation systems operate downstream of ULK-1 (13, 47). Increased PI3-kinase activity leads to recruitment of the Atg12-Atg5-Atg16 complex to isolation membranes and lipidation of LC3 to generate LC3II. Membrane association of LC3II facilitates expansion of the isolatio...

Reactive Oxygen Species-Mediated c-Jun NH 2 -Terminal Kinase Activation Contributes to Hepatitis B Virus X Protein-Induced Autophagy via Regulation of the Beclin-1/Bcl-2 Interaction

“…The class III PI3K(VPS34)/beclin-1 complex is well known for its role in autophagy induction (16,17), although there are also reports of class III PI3K-independent autophagosome formation (57,58). By downregulating the level of VPS34 or beclin-1 expression by the siRNA technique, we demonstrated that the VPS34/beclin-1 complex is required for HBx-induced autophagosome formation.…”

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles

“…Several mammalian autophagy receptors have been implicated in the targeting of intracellular bacterial and viral pathogens in a process called "xenophagy" (8)(9)(10). For instance, the cargo receptor p62 (SQSTM1) was shown to bind directly to and mediate autophagic clearance of different viral capsid proteins (11)(12)(13).…”

Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidylinositol-4-kinases

“…It has been demonstrated that FMDV induces the formation of autophagosomes from the endocytic pathway to facilitate cell entry, but this does not appear to be involved in viral replication. Autophagosome formation is induced in a PI3K-independent manner (Berryman et al, 2012), whereas inhibition of both PI3K and PI4KIIIa significantly inhibits HCV replication in cells (Berger et al, 2009;Gosert et al, 2003). There is evidence from the literature that reorganization of cellular membranes during FMDV infection is different to that seen during infection with other picornaviruses.…”