Abstract
Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.
Highlights
Enterovirus 71 (EV-A71) is the major etiological agent of hand, foot and mouth disease (HFMD)frequently seen in young children and has been implicated in severe neurological manifestations, Viruses 2017, 9, 169; doi:10.3390/v9070169 www.mdpi.com/journal/virusesViruses 2017, 9, 169 including meningitis, encephalitis, and acute flaccid paralysis [1]
Formation of Autolysosomes during EV-A71 Infection Is Important for Viral Replication
A significantly higher amount of RFP signals compared to GFP was observed in EV-A71-infected RD cells at 8 hpi compared to mock-infected cells, which suggests the formation of autolysosomes during EV-A71 infection (Figure 1D,E). These findings indicate that EV-A71 induces autolysosome formation, which is required for viral replication
Summary
Viruses 2017, 9, 169 including meningitis, encephalitis, and acute flaccid paralysis [1]. EV-A71 belongs to the Picornaviridae family, and consists of a single-stranded RNA of positive polarity. The length of its genome is approximately 7.4 kilobases. This virus encodes a single polyprotein that is proteolytically cleaved to a. Several precursors exist prior to cleavage, including VP0, 2BC, 3AB, and 3CD. The maturation of an immature viral particle into infectious viral particle involves the RNA-mediated cleavage of VP0 into VP2 and VP4 [2]. The genomic replication of enteroviruses occurs at cytosolic membranous vesicles. The vesicles observed during late infection resemble the membranous compartments induced by cellular autophagy [2,3,4]
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