• The Roles of Autophagic SNARE proteins SNAP29 and SNAP47 in Autophagy and Enterovirus D68 Replication

      Corona, Abigail; Jackson, William T.; 0000-0003-2271-4541 (2019)
      Enterovirus-D68 (EV-D68) is a positive-sense, single-stranded RNA virus of the Picornaviridae family that causes respiratory disease in children and has been implicated in recent outbreaks of acute flaccid myelitis, a severe paralysis syndrome. We have demonstrated that EV-D68 induces autophagy upon infection and modifies the autophagic process to benefit its own replication. Autophagy is a regulated process of cytosolic degradation in eukaryotic cells which maintains cellular homeostasis by degrading damaged organelles, protein aggregates, microbes and other xenobiotics in the cytoplasm. The autophagic process is characterized by the formation of double-membraned autophagosomes around cytosolic cargo, which then undergo a series of fusion steps with endosomes and lysosomes to degrade the vesicle’s contents. The autophagy pathway is targeted by many pathogens, either to protect themselves from degradation or to utilize components to benefit replication. EV-D68 uses virally-encoded proteases to cleave an autophagosome fusion SNARE protein, SNAP29, blocking delivery of autophagosome contents, including nascent viruses, to the lysosome. Our data show that relocalization occurs for SNAP47 during autophagy induction, and is required for normal virus replication. SNAP47 plays a major role in acidification of autophagosomes into amphisomes, with binding partner VAMP7, which we hypothesize promotes maturation of virions into infectious particles. Using both viral- and non-viral forms of autophagy induction, these data suggest that the cellular network of SNARE proteins is being redirected during infection to promote EV-D68 replication and egress from the cell.
    • Viral and Cellular Determinants of Picornavirus-mediated Autophagy Induction

      Corona Velazquez, Angel; Jackson, William T.; 0000-0002-1157-6908 (2019)
      Macro-autophagy is a basal cellular process that involves the degradation and turnover of cytosolic components, including elimination of damaged organelles and cytosolic cargo. In response to cell stressors, such as but not limited to, starvation and infection from xenobiotics, autophagy is upregulated. The process is controlled by the upstream autophagy signaling ULK complex, composed of the kinases ULK1 or ULK2, and the scaffold proteins ATG13, RB1CC1, and ATG101. This complex serves as a nexus for signaling pathways from nutrient sensitive kinase complexes such as MTORC1 or AMPK. Poliovirus (PV) has been shown to induce autophagy in infected cells, but the mechanism of initiation has not been completely elucidated. Furthermore, the host cellular factors that are involved in this virus-induced autophagy are unknown. We recently have shown that PV does not require the ULK1/2 complex for replication or autophagic signal induction during infection, demonstrating a novel ULK1/2-independent autophagic signaling pathway. We show that knockdown of RB1CC1, a vital scaffold protein for the ULK1/2 complexes, has no effect on PV replication and does not impede the ability of the virus to induce autophagic signals. Furthermore, PV mediates the elimination of this complex during infection in a mechanism that is not dependent on the proteasome. PV causes the cleavage of an autophagic cargo receptor SQSTM1, which was previously described in CV-B3, and therefore impairs our ability to measure bona fide autophagy during infection. We have also found that several members of the Enterovirus genus: Enterovirus D68 (EV-D68), Coxsackievirus B3 (CV-B3), and Rhinovirus A1 (RV-A1) also do not require the ULK complex for replication or with their respective effects on autophagy. We have evidence that suggests that the BECN1 complex, downstream of the ULK complex, is dispensable for PV. Exogenous expression of viral proteins 2BC and 3A from PV and CV-B3 increase the presence of LC3+ puncta but show no acidification of autophagosomes, suggesting the presence of a secondary acidification signal. We discuss the implications of these findings in regards to the ability of picornaviruses to reformat the induction process for their own benefit.