Enterovirus D-68 (EV-D68) causes severe respiratory distress in children and may be associated with acute flaccid myelitis and Guillain-Barr syndrome. Outbreaks of EV-D68 have increased in frequency and size over the last decade. Despite being discovered over 50 years ago, little is known about the specifics of the intracellular life cycle of this picornavirus. Here we present data showing that EV-D68 induces autophagosome formation to promote its own replication. Autophagosomes are double-membraned vesicles found in cells and all the hallmark of autophagy, a pathway of degradation which is used by cells to degrade and recycle damaged proteins and organelles. Autophagy is also a response to stresses such as starvation or infection. EV-D68 infection, like infection with other picornaviruses, induces autophagy in host cells. For other viruses, the autophagy pathway is involved in maturation of the virus particle into its infectious form and release of virus in enveloped packets. We have found that EV-D68 dysregulates autophagy in several novel ways to promote its own replication. EV-D68 induces cleavage of the SQSTM1/p62 autophagic cargo receptor, which likely results in inhibition of cargo loading into autophagosomes. The SNARE protein SNAP29, part of a complex that mediates fusion between acidic autophagosomes and lysosomes, is cleaved by the viral 3C protease during infection. We hypothesize that SNAP29 cleavage promotes redirection of virus-containing autophagosome- derived vesicles, preventing them from fusing with lysosomes and promoting fusion with the plasma membrane and release of virus. We have investigated another SNARE in the same family, SNAP47, which associates with the endosomal SNARE VAMP7, and found that it is required for normal autophagy and EV-D68 replication. Since endosomes are required for acidifying autophagosomes, and we have shown that acidification promotes capsid maturation of the related virus poliovirus, we predict SNAP47 may promote virion maturation into infectious virus. These data indicate that the virus disrupts autophagic degradation by at least two mechanisms: blocking cargo loading into autophagosomes, and inhibiting autophagosome delivery to lysosomes. To understand the mechanisms of EV-D68 inhibiting and redirecting the autophagy pathway, in the first Aim we will define the roles of SNAP29 at multiple points of the virus life cycle.
In Aim II, we investigate the specific role of SNAP47 in autophagy and EV-D68 replication. In the final Aim, we will analyze mechanisms by which these redirected membranes fuse with the plasma membrane and are released from cells, including SNARE proteins STX17 and SNAP23. We hypothesize that dysregulation of autophagic degradation is an important part of virus dissemination from cell to cell within a host, and we anticipate that understanding this pathway will point the way to a class of therapeutics which could limit continued spread of infections within the lung, or importantly, spread of the virus to other organs including the central nervous system.
Enterovirus D-68 is responsible for an increasing number of recent outbreaks of respiratory disease in children over recent years and is suspected to be associated with a flaccid paralysis similar to that caused by the closely related pathogen poliovirus. This project analyzes the viral factors involved in converting the cell into a virus factory, and how their interactions with host proteins redirect traffic in the cell to promote virus growth and release from cells. Understanding these virus-host interactions may lead to identification of potential drug targets to prevent the virus from triggering and redirecting cell trafficking pathways, thereby providing new therapeutics to treat virus infections.
