EV71 3C protease induces apoptosis by cleavage of hnRNP A1 to promote apaf-1 translation

RESEARCH ARTICLE EV71 3C protease induces apoptosis by cleavage of hnRNP A1 to promote apaf-1 translation Mei-Ling Li1, Jing-Yi Lin2, Bo-Shiun Chen3, Kuo-Feng Weng3, Shin-Ru Shih3,4, Jesse Davila Calderon5, Blanton S. Tolbert5, Gary Brewer1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Biochemistry and Molecular Biology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, United States of America, 2 Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan, 3 Research Center for Emerging Viral Infections, Chang Gung University, Tao-Yuan, Taiwan, 4 Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Tao-Yuan, Taiwan, 5 Department of Chemistry, Case Western Reserve University, Cleveland, OH, United States of America * Abstract OPEN ACCESS Citation: Li M-L, Lin J-Y, Chen B-S, Weng K-F, Shih S-R, Calderon JD, et al. (2019) EV71 3C protease induces apoptosis by cleavage of hnRNP A1 to promote apaf-1 translation. PLoS ONE 14(9): e0221048. https://doi.org/10.1371/journal. pone.0221048 Editor: Eric Jan, University of British Columbia, CANADA Received: January 25, 2019 Accepted: July 29, 2019 Enterovirus 71 (EV71) induces apoptosis to promote viral particle release. Earlier work showed that EV71 utilizes its 3C protease to induce apoptosis in a caspase-3-dependent pathway, though the mechanism is unknown. However, work from Vagner, Holcik and colleagues showed that host protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds the IRES of cellular apoptotic peptidase activating factor 1 (apaf-1) mRNA to repress its translation. In this work, we show that apaf-1 expression is essential for EV71-induced apoptosis. EV71 infection or ectopic expression of 3C protease cleaves hnRNP A1, which abolishes its binding to the apaf-1 IRES. This allows IRES-dependent synthesis of apaf-1, activation of caspase-3, and apoptosis. Thus, we reveal a novel mechanism that EV71 utilizes for virus release via a 3C protease–hnRNP A1–apaf-1–caspase-3–apoptosis axis. Published: September 9, 2019 Copyright: © 2019 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data are available in Figshare: https://figshare.com/articles/Apaf-1/ 9728039/1 (DOI: 10.6084/m9.figshare.9728039). Funding: This work was funded by National Institutes of Health R01 GM126833 (G.B. & B.S. T.), Taiwan MOST105-2320-B-038-020-MY3 (J.-Y. L.), and Research Center for Emerging Viral Infections, Chang Gung University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Introduction Enterovirus 71 (EV71) is a positive-stranded RNA virus in the genus Enterovirus, family Picornaviridae. It is one of the major pathogens causing hand, foot and mouth disease (HFMD) particularly in the Asia-Pacific region. Some EV71 strains can lead to severe neurological complications ranging from aseptic meningitis with or without pulmonary edema to brain stem encephalitis and poliomyelitis-like acute flaccid paralysis, particularly among children under five years old [1–4]. Outbreaks of EV71 infection have occurred worldwide [5]. To date, there is no FDA-approved vaccine or antiviral agent against EV71. Apoptosis is a highly regulated, programmed cell death to eliminate damaged, aged, or virally infected cells. EV71 infection can induce apoptosis in various cell types through different mechanisms [6]. For example, EV71 infection modulates the expression of miR-146a or miR-370 to induce apoptosis through targeting Son of sevenless homolog 1 (SOS1) and Growth arrest and DNA damage-inducible protein 45β (GADD45β) [7]. EV71 infection PLOS ONE | https://doi.org/10.1371/journal.pone.0221048 September 9, 2019 1 / 16 EV71 3C cleaves hnRNP A1 to induce apoptosis Taiwan and the Ministry of Science and Technology (MOST), Taiwan (MOST 107-3017-F-182-001)(S.R.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. activates calpain via calcium flux to induce a caspase-independent apoptotic pathway [8]. EV71 2B protein (2B) induces cell apoptosis by recruiting the pro-apoptotic protein Bax to mitochondria and inducing Bax conformational activation [9]. Cleavage of eukaryotic initiation factor 4G (eIF4G) by EV71 2A protease also triggers apoptosis [10]. Some years ago, we also reported that EV71 3C protease triggers apoptosis through activation of caspase-3 [11]. However, the underlying molecular mechanisms involved in this event are not well understood. Apoptotic peptidase activating factor 1 (apaf-1) plays an essential role in the apoptosis regulatory network. Upon binding cytochrome c and dATP, apaf-1 assembles into an oligomeric apoptosome which is responsible for autocatalytic activation of pro-caspase-9 (apaf-3). This in turn activates caspase-3 and apoptosis [12]. Our previous studies showed that EV71 infection activates caspase-9 and caspase-3 to induce apoptosis [6, 11]. The 5’ UTR of apaf-1 contains an IRES for translation initiation to ensure continuous expression of apaf-1 despite an overall reduction of protein synthesis under apoptosis conditions [13–15]. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an RNA-binding protein that shuttles between the nucleus and the cytoplasm, is involved in several RNA metabolic processes such as pre-mRNA splicing and RNA trafficking. hnRNP A1 is also an IRES trans-acting factor (ITAF) that binds to the IRES of EV71, human rhinovirus-2 (HRV-2), and apaf-1 mRNA to regulate their translation [16, 17]. Binding of hnRNP A1 to the IRES of EV71 and HRV-2 enhances translation, while binding to the apaf-1 IRES blocks its translation to inhibit apoptosis [16–18]. Essential to the viral life cycle, the 3C protease cleaves the EV71 polyprotein post-translation. In addition to processing viral polyprotein, 3C protease cleaves several host factors and impairs host transcription and translation. For example, 3C proteases of EV71, polio-, and human rhino viruses cleave the RNA-binding protein AUF1 to promote viral translation [19, 20]. Despite the observations described above, connections are lacking between EV71 infection, 3C protease, the ITAF hnRNP A1, IRES-dependent regulation of apaf-1 translation, and activation of apoptosis, which permits virus release. In the present study, we show that 3C protease cleaves hnRNP A1, thereby reducing hnRNP A1 association with the apaf-1 IRES. This allows apaf-1 translation and triggers apoptosis for virus release. Thus, our study reveals that, in addition to interact (...truncated)