Human tryptophanyl-tRNA synthetase is an IFN-γ–inducible entry factor for Enterovirus
Man Lung Yeung, … , Shin-Ru Shih, Kwok-Yung Yuen
Man Lung Yeung, … , Shin-Ru Shih, Kwok-Yung Yuen
Published August 28, 2018
Citation Information: J Clin Invest. 2018;128(11):e99411. https://doi.org/10.1172/JCI99411.
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Research Article Infectious disease Microbiology

Human tryptophanyl-tRNA synthetase is an IFN-γ–inducible entry factor for Enterovirus

Abstract

Enterovirus A71 (EV-A71) receptors that have been identified to date cannot fully explain the pathogenesis of EV-A71, which is an important global cause of hand, foot, and mouth disease and life-threatening encephalitis. We identified an IFN-γ–inducible EV-A71 cellular entry factor, human tryptophanyl-tRNA synthetase (hWARS), using genome-wide RNAi library screening. The importance of hWARS in mediating virus entry and infectivity was confirmed by virus attachment, in vitro pulldown, antibody/antigen blocking, and CRISPR/Cas9-mediated deletion. Hyperexpression and plasma membrane translocation of hWARS were observed in IFN-γ–treated semipermissive (human neuronal NT2) and cDNA-transfected nonpermissive (mouse fibroblast L929) cells, resulting in their sensitization to EV-A71 infection. Our hWARS-transduced mouse infection model showed pathological changes similar to those seen in patients with severe EV-A71 infection. Expression of hWARS is also required for productive infection by other human enteroviruses, including the clinically important coxsackievirus A16 (CV-A16) and EV-D68. This is the first report to our knowledge on the discovery of an entry factor, hWARS, that can be induced by IFN-γ for EV-A71 infection. Given that we detected high levels of IFN-γ in patients with severe EV-A71 infection, our findings extend the knowledge of the pathogenicity of EV-A71 in relation to entry factor expression upon IFN-γ stimulation and the therapeutic options for treating severe EV-A71–associated complications.

Authors

Man Lung Yeung, Lilong Jia, Cyril C. Y. Yip, Jasper F. W. Chan, Jade L. L. Teng, Kwok-Hung Chan, Jian-Piao Cai, Chaoyu Zhang, Anna J. Zhang, Wan-Man Wong, Kin-Hang Kok, Susanna K. P. Lau, Patrick C. Y. Woo, Janice Y. C. Lo, Dong-Yan Jin, Shin-Ru Shih, Kwok-Yung Yuen

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Figure 2

hWARS affects early EV-A71 replication.

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hWARS affects early EV-A71 replication. (A) Amount of EV-A71 RNA in hWAR...
(A) Amount of EV-A71 RNA in hWARS-KD and control RD cells during early infection. (B) Immunostained EV-A71 protein in hWARS-KD cells transfected with control RNA or purified EV-A71 RNA and control RD cells transfected with EV-A71 RNA. Histogram shows the estimated percentages of transfected cells. (C) Confocal microscopy showing endogenous hWARS expression. RD cells were stained with mouse monoclonal anti-hWARS antibodies (green). Also shown are the bright-field image and the overlapping image of hWARS protein. Arrows indicate positively stained hWARS. (D) Flow cytometric assays of EV-A71 virus attachment to hWARS, hSCARB2, and hPSGL1. High-titer EV-A71 was incubated with nonpermissive L929 cells overexpressing hWARS, hSCARB2, and hPSGL1, separately, at 4oC for 2 hours, and then washed to remove unbound viruses before fixation for flow cytometric analyses. EV-A71–inoculated mock-transfected L929 cells were included as a negative control. Max, maximum. (E) Assays of EV-A71 virus attachment to hWARS, hSCARB2, and hPSGL1 by confocal microscopy. Immunostaining for EV-A71 (green), hWARS (red), hSCARB2 (red), and hPSGL1 (red) was performed. Arrowheads indicate the colocalization (yellow) of EV-A71 and surface proteins. Immunostaining for mock-transfected L929 cells was also included as a negative control. Data in A, B, and D represent the mean ± SEM of 3 independent experiments. Images shown in B, D, and E are representatives of 3 independent experiments. Scale bars: 80 μm.