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Endophilin-A2-mediated endocytic pathway is critical for enterovirus 71 entry into caco-2 cells.

ABSTRACT: Enterovirus 71 (EV71) is typically transmitted by the oral-faecal route and initiates infection upon crossing the intestinal mucosa. Our limited understanding of the mechanisms by which it crosses the intestinal mucosa has hampered the development of effective therapeutic options. Here, using an RNA interference screen combined with chemical inhibitors or the overexpression of dominant negative proteins, we found that EV71 entry into Caco-2 cells, a polarized human intestinal epithelial cell line, does not involve clathrin- and caveolae-dependent endocytic pathways or macropinocytosis but requires GTP-binding protein dynamin 2 and cytoskeleton remodelling. The use of siRNAs targeting endophilin family members revealed that endophlin-A2 is essential for the uptake of EV71 particles by Caco-2 cells. Subcellular analysis revealed that internalized EV71 virions largely colocalized with endophilin-A2 at cytomembrane ruffles and in the perinuclear area. Combined with viral entry kinetics, these data suggest that EV71 enters Caco-2 cells mainly via an endophilin-A2-mediated endocytic (EME) pathway. Finally, we showed that internalized EV71 virions were transported to endosomal sorting complex required for transport (ESCRT)-related multivesicular bodies (MVBs). These data provide attractive therapeutic targets to block EV71 infection.

SUBMITTER: Chen SL

PROVIDER: S-EPMC6542187 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Endophilin-A2-mediated endocytic pathway is critical for enterovirus 71 entry into caco-2 cells.

Chen Sheng-Lin SL Liu Yan-Gang YG Zhou Yong-Tao YT Zhao Ping P Ren Hao H Xiao Man M Zhu Yong-Zhe YZ Qi Zhong-Tian ZT

Emerging microbes & infections 20190101 1

Enterovirus 71 (EV71) is typically transmitted by the oral-faecal route and initiates infection upon crossing the intestinal mucosa. Our limited understanding of the mechanisms by which it crosses the intestinal mucosa has hampered the development of effective therapeutic options. Here, using an RNA interference screen combined with chemical inhibitors or the overexpression of dominant negative proteins, we found that EV71 entry into Caco-2 cells, a polarized human intestinal epithelial cell lin ...[more]

PMID: 31132962

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Project description:A close relative of poliovirus, enterovirus 71 (EV71) is regarded as an important neurotropic virus of serious public health concern. EV71 causes Hand, Foot and Mouth Disease and has been associated with neurological complications in young children. Our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. Here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during EV71 infection of motor-neuron NSC-34 cells, which are found at the neuromuscular junctions where EV71 is believed to enter the central nervous system. Meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in NSC-34 cells. Among the candidate proteins, we focused our attention on prohibitin (PHB), a protein that is involved in multiple cellular functions and the target of anti-cancer drug Rocaglamide (Roc-A). We demonstrated that cell surface-expressed PHB is involved in EV71 entry into neuronal cells specifically, while membrane-bound mitochondrial PHB associates with the virus replication complex and facilitates viral replication. Furthermore, Roc-A treatment of EV71-infected neuronal cells reduced significantly virus yields. However, the inhibitory effect of Roc-A on PHB in NSC-34 cells was not through blocking the CRAF/MEK/ERK pathway as previously reported. Instead, Roc-A treated NSC-34 cells had lower mitochondria-associated PHB and lower ATP levels that correlated with impaired mitochondria integrity. In vivo, EV71-infected mice treated with Roc-A survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. Together, this study uncovers PHB as the first host factor that is specifically involved in EV71 neuropathogenesis and a potential drug target to limit neurological complications.

Project description:BackgroundTactile and mechanical pain are crucial to our interaction with the environment, yet the underpinning molecular mechanism is still elusive. Endophilin A2 (EndoA2) is an evolutionarily conserved protein that is documented in the endocytosis pathway. However, the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear.MethodsMale and female C57BL/6 mice (8-12 weeks) and male cynomolgus monkeys (7-10 years old) were used in our experiments. Nerve injury-, inflammatory-, and chemotherapy-induced pathological pain models were established for this study. Behavioral tests of touch, mechanical pain, heat pain, and cold pain were performed in mice and nonhuman primates. Western blotting, immunostaining, co-immunoprecipitation, proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms.ResultsThe results showed that EndoA2 was primarily distributed in neurofilament-200-positive (NF200+) medium-to-large diameter dorsal root ganglion (DRG) neurons of mice and humans. Loss of EndoA2 in mouse NF200+ DRG neurons selectively impaired the tactile and mechanical allodynia. Furthermore, EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons. Moreover, as an adaptor protein, EndoA2 also bound to kinesin family member 5B (KIF5B), which was involved in the EndoA2-mediated membrane trafficking process of Piezo2. Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents, and re-expression of EndoA2 rescued the MA currents. In addition, interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates.ConclusionsOur data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals. EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons. Our findings identify a potential new target for the treatment of mechanical pain.

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Endophilin-A2-mediated endocytic pathway is critical for enterovirus 71 entry into caco-2 cells.

Publications

Endophilin-A2-mediated endocytic pathway is critical for enterovirus 71 entry into caco-2 cells.

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