Abstract
Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Here we use a human genome-wide RNAi screen to identify 256 host factors involved in EV71 replication in human rhabdomyosarcoma cells. Enrichment analyses reveal overrepresentation in processes like mitotic cell cycle and transcriptional regulation. We have carried out orthogonal experiments to characterize the roles of selected factors involved in cell cycle regulation and endoplasmatic reticulum-associated degradation. We demonstrate nuclear egress of CDK6 in EV71 infected cells, and identify CDK6 and AURKB as resistance factors. NGLY1, which co-localizes with EV71 replication complexes at the endoplasmatic reticulum, supports EV71 replication. We confirm importance of these factors for EV71 replication in a human neuronal cell line and for coxsackievirus A16 infection. A small molecule inhibitor of NGLY1 reduces EV71 replication. This study provides a comprehensive map of EV71 host factors and reveals potential antiviral targets.
Highlights
Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood
While much of the EV71 host factor discovery work has benefitted from the progress made in more established fields of poliovirus and coxsackievirus B3 (CB3) research[15,19], others have tackled the problem through mapping transcriptomic and proteomic profiles of EV71-infected cells[22,23]
The genome-wide small interfering RNA (siRNA) library consists of pools of four siRNAs per gene arrayed in a series of 384-well plates. siRNA pools against SCARB2 and MMP20 were added to empty wells of each 384-well plate to serve as positive (EV71-inhibitory) controls while a non-targeting (NT) siRNA pool serves as the negative control (Supplementary Fig. 1)
Summary
Enterovirus 71 (EV71) is a neurotropic enterovirus without antivirals or vaccine, and its host-pathogen interactions remain poorly understood. Coyne et al.[25] reported a comparative screen of the druggable genome library (B5500 genes) for poliovirus and CB3 but no genome-wide screen has been reported for EV71 or any other human enterovirus to date In this context, we carried out an immunofluorescence-based phenotypic screen against a genome-wide siRNA library of 21,121 human genes in rhabdomyosarcoma cells (RD) to identify cellular factors that facilitate (host susceptibility factors, HSFs) or suppress (host resistance factors, HRFs) EV71 replication. Our screen identified 256 host factors involved in EV71 replication Among these hits, we showed that the cell cycle regulators aurora kinase B (AURKB) and cyclin-dependent kinase 6 (CDK6) act as HRFs and that EV71 potentially regulates CDK6 by inducing its nuclear egress. Our findings here provide a comprehensive map of host factors involved in EV71 replication in a human cell line
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