Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model.

Ying-Ray Lee,Po-Shun Wang,Jen-Ren Wang,Hsiao-Sheng Liu

doi:10.1186/s12929-014-0080-4

Ying-Ray Lee, Po-Shun Wang + Show 2 more

Open Access

https://doi.org/10.1186/s12929-014-0080-4

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Abstract

BackgroundWe previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.ResultsWe demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH4Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.ConclusionsIn this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.

Highlights

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo
EV71 infection of human neuroblastoma SK-N-SH cells induced amphisome formation and autophagic flux We previously reported that EV71 infection can induce autophagy activity, which further promotes viral replication, in human rhabdomyosarcoma RD and neuronal SKN-SH cells [36]
We further revealed that the EV71 VP1 and 2C proteins colocalized with the MPR protein (Mannose-6-phosphate receptor, a marker of late endosome) (Figure 1B, arrow), which parallel with the increased colocalization of the LC3 and MPR proteins in EV71-infected cells (Figure 1C, arrow)

Summary

Introduction

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. The effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice. CNS infection by EV71 has been reported in animal models, including mice, and cynomolgus and rhesus monkeys [14,15,16,17,18]. In order to develop effective vaccines and antiviral therapies against EV71, it is important to understand the pathogenesis of EV71 infection. Aberrant autophagy may lead to various pathogenic conditions, including diabetes, neuron degeneration, heart disease, and cancers [20,21]

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