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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):5163-5177. 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

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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 3

hWARS interacts directly with EV-A71.

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hWARS interacts directly with EV-A71.
(A) cDNA-transfected L929 cells we...
(A) cDNA-transfected L929 cells were inoculated with high-titer EV-A71 (TCID50/ml = 190,000). Forty-eight hours after inoculation, the cell lysates were harvested for Western blot analyses using specific antibodies. Mock-transfected L929 and EV-A71–infected RD cells were included as positive and negative controls, respectively. (B) Immunoprecipitation (IP) of hWARS from RD or hWARS-transfected L929 cells followed by pulldown of EV-A71. Endogenous hWARS from RD cells (lane 2) and overexpressed hWARS from hWARS-transfected L929 cells (lane 4) were first separately immunoprecipitated by anti-hWARS antibodies. The complexes were then inoculated with EV-A71 overnight at 4oC. After washing away unbound viruses, the complexes were dissociated by anti–EV-A71 antibodies for Western blot (WB) analyses. Asterisks indicate nonspecific bands. (C) Pulldown of EV-A71 clinical isolates by recombinant hWARS protein. Three different clinical EV-A71 isolates were inoculated with recombinant hWARS protein immunocomplexes and detected using Western blot analyses as described in B. (D) Anti-hWARS antibody blockage of EV-A71 infection. Surface hWARS on RD cells was blocked with anti-hWARS antibodies for 1 hour before EV-A71 infection. Virus production in conditioned supernatants and virus protein expression in infected cell lysates are shown. (E) Saturation of EV-A71 virions by recombinant hWARS protein (ranging from 18.8 to 75.2 nM). EV-A71 was preincubated with recombinant hWARS protein before challenging the RD cells. Virus production and viral protein expression were measured as described in D. (F) Western blot analyses of EV-A71, hWARS, and hSCARB2 proteins in RD CRISPR/Cas9 (CRISPR) cell clones. Endogenous γ-tubulin was used as a loading control. The r value represents the Pearson’s correlation coefficient. Data in A–G are representative of 3 independent experiments, and data in D and E represent the mean ± SEM.
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