Characterization of cellular entry factors for enterovirus-A71

Abstract

Several enteroviruses (EVs), including EV-A71 and EV-D68, are capable of causing hand-foot-and-mouth disease and life-threatening encephalitis. Finding human host factors that are essential for the establishment of severe enterovirus infection could facilitate the generation of cell and animal models for studying the pathogenesis of enterovirus and formulation of treatment strategies. In this work, a genome-wide RNAi library screening was performed and identified 118 host factors which knockdown could abolish productive EV-A71 replication. Detail characterization of one of these factors, human tryptophanyl-tRNA synthetase (hWARS), revealed its unexpected cell entry role for enterovirus. Using various in vitro assays, the expression of hWARS was shown to be essential for EV-A71 to mediate virus entry, internalization and infectivity. The requirement of hWARS for EV-A71 to establish productive infection was further confirmed by virus attachment, in vitro pull-down, antibody/antigen blocking, and CRISPR/Cas9 assays. The importance of hWARS in enterovirus infection was extended to other enteroviruses suggesting that hWARS supported a broad range of enterovirus infection. Also, the expression of hWARS could found to be regulated by the level of interferon-gamma (IFN-γ). Hence, induced expression of hWARS by IFN-γ stimulation in semi-permissive (human neuronal NT2) cells or overexpression of hWARS by cDNA transfection in non-permissive (mouse fibroblast L929) cells resulted in sensitization of these cells to EV-A71. Therefore, similar overexpression strategy was used to generate a hWARS transgenic mouse model for studying the EV-A71 infection. It was shown that mice expressing hWARS could support EV-A71 infection with pathological changes similar to patients with severe EV-A71 infections. Further development of this mouse model would be valuable for future studies of enterovirus pathogenesis, development of vaccine and testing of anti-enterovirus drug. Overall, the findings in this work were in line with the detection of a high level of IFNγ was observed in patients with severe EV-A71 infection which could subsequently sensitize semi-permissive or non-permissive cells to infection. The new knowledge generated from my work could enhance the understanding of EV-A71-induced pathogenicity and advance therapeutic options for treating EV-A71-associated severe complications. (Word count: 332)