Xiaoman Wo, Yuan Yuan, Yong Xu, Yang Chen, Yao Wang, Shuoxuan Zhao, Lexun Lin, Xiaoyan Zhong, Yan Wang, Zhaohua Zhong and Wenran Zhao. TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71[J]. Virologica Sinica, 2021, 36(1): 95-103. doi: 10.1007/s12250-020-00262-x
Citation: Xiaoman Wo, Yuan Yuan, Yong Xu, Yang Chen, Yao Wang, Shuoxuan Zhao, Lexun Lin, Xiaoyan Zhong, Yan Wang, Zhaohua Zhong, Wenran Zhao. TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71 .VIROLOGICA SINICA, 2021, 36(1) : 95-103.  http://dx.doi.org/10.1007/s12250-020-00262-x

肠道病毒A71的3C蛋白酶对TAR DNA结合蛋白43的剪切

  • 通讯作者: 钟照华, zhongzh@hrbmu.edu.cn, ORCID: http://orcid.org/0000-0002-0778-5053
    ; 赵文然, zhaowr@hrbmu.edu.cn, ORCID: http://orcid.org/0000-0003-2495-5138
  • 收稿日期: 2020-03-21
    录用日期: 2020-06-01
    出版日期: 2020-07-21
  • 肠道病毒A71(Enterovirus A71,EV-A71)是手足口病(hand, foot, and mouth disease,HFMD)的主要病原体。HFMD常伴严重的神经系统病变,但其产生机制不明确。TAR DNA结合蛋白43(TAR DNA-binding protein 43,TDP-43)在细胞中的异常位定及聚集是肌萎缩性嵴髓侧索硬化症(amyotrophic lateral sclerosis,ALS)的突出病理表现。然而,EV-A71 感染是否对TDP-43产生影响目前仍未知。本研究发现,EV-A71感染导致 TDP-43的剪切;而导致TDP-43剪切体生成的原因是EV-A71的复制,而不是病毒感染所致的细胞凋亡引起的caspases的活化。EV-A71对TDP-43的剪切发生在TDP-43的331Q 与 332S之间,是由病毒的3C蛋白酶引起的;突变的TDP-43 (Q331A)则不被剪切;如将3C蛋白酶突变使其失去蛋白酶活性,TDP-43也不被剪切。本研究还发现,EV-A71的2A蛋白酶可诱导 TDP-43从细胞核转移到细胞质。综合以上结果,本研究表明,EV-A71感染可导致TDP-43的剪切和异常定位,提示TDP-43可能与EV-A71的发病机制有关。

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71

  • Corresponding author: Zhaohua Zhong, zhongzh@hrbmu.edu.cn Wenran Zhao, zhaowr@hrbmu.edu.cn
  • ORCID: http://orcid.org/0000-0002-0778-5053; http://orcid.org/0000-0003-2495-5138
  • Received Date: 21 March 2020
    Accepted Date: 01 June 2020
    Published Date: 21 July 2020
  • Enterovirus A71 (EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease (HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3C between the residues 331Q and 332S, while mutated TDP-43 (Q331A) was not cleaved. In addition, mutated 3C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2A rather than 3C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71infection.


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    1. Berning BA, Walker AK (2019) The pathobiology of TDP-43 C-terminal fragments in ALS and FTLD. Front Neurosci 13:335
        doi: 10.3389/fnins.2019.00335

    2. Buratti E, Brindisi A, Giombi M, Tisminetzky S, Ayala YM, Baralle FE (2005) TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing. J Biol Chem 280:37572–37584
        doi: 10.1074/jbc.M505557200

    3. Cai Q, Yameen M, Liu W, Gao Z, Li Y, Peng X, Cai Y, Wu C, Zheng Q, Li J, Lin T (2013) Conformational plasticity of the 2A proteinase from enterovirus 71. J Virol 87:7348–7356
        doi: 10.1128/JVI.03541-12

    4. Caine EA, Moncla LH, Ronderos MD, Friedrich TC, Osorio JE (2016) A single mutation in the VP1 of enterovirus 71 is responsible for increased virulence and neurotropism in adult interferon-deficient mice. J Virol 90:8592–8604
        doi: 10.1128/JVI.01370-16

    5. Chang PC, Chen SC, Chen KT (2016) The current status of the disease caused by enterovirus 71 infections: epidemiology, pathogenesis, molecular epidemiology, and vaccine development. Int J Environ Res Public Health 13:890
        doi: 10.3390/ijerph13090890

    6. Chen YF, Hu L, Xu F, Liu CJ, Li J (2019) A case report of a teenager with severe hand, foot, and mouth disease with brainstem encephalitis caused by enterovirus 71. BMC Pediatr 19:59
        doi: 10.1186/s12887-019-1428-4

    7. Colombrita C, Onesto E, Megiorni F, Pizzuti A, Baralle FE, Buratti E, Silani V, Ratti A (2012) TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger rnas and differently regulate their post-transcriptional fate in motoneuron-like cells. J Biol Chem 287:15635–15647
        doi: 10.1074/jbc.M111.333450

    8. Donde A, Sun M, Jeong YH, Wen X, Ling J, Lin S, Braunstein K, Nie S, Wang S, Chen L, Wong PC (2020) Upregulation of ATG7 attenuates motor neuron dysfunction associated with depletion of TARDBP/TDP-43. Autophagy 16:672–682
        doi: 10.1080/15548627.2019.1635379

    9. Duan H, Zhu M, Xiong Q, Wang Y, Xu C, Sun J, Wang C, Zhang H, Xu P, Peng Y (2017) Regulation of enterovirus 2A protease-associated viral IRES activities by the cell's ERK signaling cascade: implicating ERK as an efficiently antiviral target. Antiviral Res 143:13–21
        doi: 10.1016/j.antiviral.2017.03.018

    10. Ertel KJ, Brunner JE, Semler BL (2010) Mechanistic consequences of hnRNP C binding to both RNA termini of poliovirus negative-strand RNA intermediates. J Virol 84:4229–4242
        doi: 10.1128/JVI.02198-09

    11. Feng Q, Langereis MA, Lork M, Nguyen M, Hato SV, Lanke K, Emdad L, Bhoopathi P, Fisher PB, Lloyd RE, van Kuppeveld FJ (2014) Enterovirus 2Apro targets MDA5 and MAVS in infected cells. J Virol 88:3369–3378
        doi: 10.1128/JVI.02712-13

    12. Feng M, Guo S, Fan S, Zeng X, Zhang Y, Liao Y, Wang J, Zhao T, Wang L, Che Y, Wang J, Ma N, Liu L, Yue L, Li Q (2016) The preferential infection of astrocytes by enterovirus 71 plays a key role in the viral neurogenic pathogenesis. Front Cell Infect Microbiol 6:192

    13. Fiesel FC, Weber SS, Supper J, Zell A, Kahle PJ (2012) TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR. Nucleic Acids Res 40:2668–2682
        doi: 10.1093/nar/gkr1082

    14. Freibaum BD, Chitta RK, High AA, Taylor JP (2010) Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery. J Proteome Res 9:1104–1120
        doi: 10.1021/pr901076y

    15. Fung G, Shi J, Deng H, Hou J, Wang C, Hong A, Zhang J, Jia W, Luo H (2015) Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis. Cell Death Differ 22:2087–2097
        doi: 10.1038/cdd.2015.58

    16. Gao J, Wang L, Yan T, Perry G, Wang X (2019) TDP-43 proteinopathy and mitochondrial abnormalities in neurodegeneration. Mol Cell Neurosci 100:103396
        doi: 10.1016/j.mcn.2019.103396

    17. Gerhauser I, Hansmann F, Ciurkiewicz M, Loscher W, Beineke A (2019) Facets of theiler's murine encephalomyelitis virus-induced diseases: an update. Int J Mol Sci 20:448
        doi: 10.3390/ijms20020448

    18. Guo Z, Zhong X, Lin L, Wu S, Wang T, Chen Y, Zhai X, Wang Y, Wu H, Tong L, Han Y, Pan B, Peng Y, Si X, Zhang F, Zhao W, Zhong Z (2014) A 3C(pro)-dependent bioluminescence imaging assay for in vivo evaluation of anti-enterovirus 71 agents. Antiviral Res 101:82–92
        doi: 10.1016/j.antiviral.2013.11.002

    19. Hart MP, Gitler AD (2012) ALS-associated ataxin 2 polyq expansions enhance stress-induced caspase 3 activation and increase TDP-43 pathological modifications. J Neurosci 32:9133–9142
        doi: 10.1523/JNEUROSCI.0996-12.2012

    20. Holmes CW, Koo SS, Osman H, Wilson S, Xerry J, Gallimore CI, Allen DJ, Tang JW (2016) Predominance of enterovirus B and echovirus 30 as cause of viral meningitis in a UK population. J Clin Virol 81:90–93
        doi: 10.1016/j.jcv.2016.06.007

    21. Huang CC, Liu CC, Chang YC, Chen CY, Wang ST, Yeh TF (1999) Neurologic complications in children with enterovirus 71 infection. N Engl J Med 341:936–942
        doi: 10.1056/NEJM199909233411302

    22. Huang Q, Wang Y, Si C, Zhao D, Wang Y, Duan Y (2017) Interleukin-35 modulates the imbalance between regulatory T cells and T helper 17 cells in enterovirus 71-induced hand, foot, and mouth disease. J Interferon Cytokine Res 37:522–530
        doi: 10.1089/jir.2017.0080

    23. Hung HC, Chen TC, Fang MY, Yen KJ, Shih SR, Hsu JT, Tseng CP (2010) Inhibition of enterovirus 71 replication and the viral 3d polymerase by aurintricarboxylic acid. J Antimicrob Chemother 65:676–683
        doi: 10.1093/jac/dkp502

    24. Ju Y, Tan Z, Huang H, Chen M, Tan Y, Zhang C, Wang J, Wang H, Chen M (2020) Clinical and epidemiological characteristics of coxsackievirus a6- and enterovirus 71-associated clinical stage 2 and 3 severe hand, foot, and mouth disease in Guangxi, Southern China, 2017. J Infect 80:121–142

    25. Lee KY (2016) Enterovirus 71 infection and neurological complications. Korean J Pediatr 59:395–401
        doi: 10.3345/kjp.2016.59.10.395

    26. Lei X, Liu X, Ma Y, Sun Z, Yang Y, Jin Q, He B, Wang J (2010) The 3C protein of enterovirus 71 inhibits retinoid acid-inducible gene I-mediated interferon regulatory factor 3 activation and type I interferon responses. J Virol 84:8051–8061
        doi: 10.1128/JVI.02491-09

    27. Lei X, Sun Z, Liu X, Jin Q, He B, Wang J (2011) Cleavage of the adaptor protein TRIF by enterovirus 71 3C inhibits antiviral responses mediated by toll-like receptor 3. J Virol 85:8811–8818
        doi: 10.1128/JVI.00447-11

    28. Lei X, Zhang Z, Xiao X, Qi J, He B, Wang J (2017) Enterovirus 71 inhibits pyroptosis through cleavage of gasdermin D. J Virol 91:e01069

    29. Levengood JD, Tolbert M, Li ML, Tolbert BS (2013) High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71. RNA Biol 10:1136–1145
        doi: 10.4161/rna.25107

    30. Li B, Yue Y, Zhang Y, Yuan Z, Li P, Song N, Lin W, Liu Y, Gu L, Meng H (2017) A novel enterovirus 71 (ev71) virulence determinant: the 69th residue of 3C protease modulates pathogenicity. Front Cell Infect Microbiol 7:26

    31. Li J, Gao F, Hao SB, Cheng D, Zhang WQ, Lin B, Zhao L, Yu XJ, Wang ZY, Wen HL (2017) Contribution of 3CD region to the virulence of enterovirus 71. Biomed Environ Sci 30:767–771

    32. Masaki K, Sonobe Y, Ghadge G, Pytel P, Roos RP (2019) TDP-43 proteinopathy in theiler's murine encephalomyelitis virus infection. PLoS Pathog 15:e1007574
        doi: 10.1371/journal.ppat.1007574

    33. McCluskey LF, Elman LB, Martinez-Lage M, Van Deerlin V, Yuan W, Clay D, Siderowf A, Trojanowski JQ (2009) Amyotrophic lateral sclerosis-plus syndrome with TAR DNA-binding protein-43 pathology. Arch Neurol 66:121–124

    34. Park N, Schweers NJ, Gustin KE (2015) Selective removal of FG repeat domains from the nuclear pore complex by enterovirus 2A(pro). J Virol 89:11069–11079
        doi: 10.1128/JVI.00956-15

    35. Riku Y, Watanabe H, Yoshida M, Tatsumi S, Mimuro M, Iwasaki Y, Katsuno M, Iguchi Y, Masuda M, Senda J, Ishigaki S, Udagawa T, Sobue G (2014) Lower motor neuron involvement in TAR DNA-binding protein of 43 kDa-related frontotemporal lobar degeneration and amyotrophic lateral sclerosis. JAMA Neurol 71:172–179
        doi: 10.1001/jamaneurol.2013.5489

    36. Riku Y, Watanabe H, Yoshida M, Mimuro M, Iwasaki Y, Masuda M, Ishigaki S, Katsuno M, Sobue G (2016) Marked involvement of the striatal efferent system in TAR DNA-binding protein 43 kDa-related frontotemporal lobar degeneration and amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 75:801–811
        doi: 10.1093/jnen/nlw053

    37. Rohn TT (2009) Cytoplasmic inclusions of TDP-43 in neurodegenerative diseases: a potential role for caspases. Histol Histopathol 24:1081–1086

    38. Romano M, Buratti E, Romano G, Klima R, Del Bel Belluz L, Stuani C, Baralle F, Feiguin F (2014) Evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins functionally interact with human and drosophila TAR DNA-binding protein 43 (TDP-43). J Biol Chem 289:7121–7130
        doi: 10.1074/jbc.M114.548859

    39. Shimonaka S, Nonaka T, Suzuki G, Hisanaga S, Hasegawa M (2016) Templated aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) by seeding with TDP-43 peptide fibrils. J Biol Chem 291:8896–8907
        doi: 10.1074/jbc.M115.713552

    40. Sun D, Chen S, Cheng A, Wang M (2016) Roles of the picornaviral 3C proteinase in the viral life cycle and host cells. Viruses 8:82
        doi: 10.3390/v8030082

    41. Takahashi S, Liao Q, Van Boeckel TP, Xing W, Sun J, Hsiao VY, Metcalf CJ, Chang Z, Liu F, Zhang J, Wu JT, Cowling BJ, Leung GM, Farrar JJ, van Doorn HR, Grenfell BT, Yu H (2016) Hand, foot, and mouth disease in china: modeling epidemic dynamics of enterovirus serotypes and implications for vaccination. PLoS Med 13:e1001958
        doi: 10.1371/journal.pmed.1001958

    42. Teoh HL, Mohammad SS, Britton PN, Kandula T, Lorentzos MS, Booy R, Jones CA, Rawlinson W, Ramachandran V, Rodriguez ML, Andrews PI, Dale RC, Farrar MA, Sampaio H (2016) Clinical characteristics and functional motor outcomes of enterovirus 71 neurological disease in children. JAMA Neurol 73:300–307
        doi: 10.1001/jamaneurol.2015.4388

    43. Tolbert M, Morgan CE, Pollum M, Crespo-Hernandez CE, Li ML, Brewer G, Tolbert BS (2017) HnRNP A1 alters the structure of a conserved enterovirus IRES domain to stimulate viral translation. J Mol Biol 429:2841–2858
        doi: 10.1016/j.jmb.2017.06.007

    44. Tudor EL, Galtrey CM, Perkinton MS, Lau KF, De Vos KJ, Mitchell JC, Ackerley S, Hortobagyi T, Vamos E, Leigh PN, Klasen C, McLoughlin DM, Shaw CE, Miller CC (2010) Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology. Neuroscience 167:774–785
        doi: 10.1016/j.neuroscience.2010.02.035

    45. Ule J (2008) Ribonucleoprotein complexes in neurologic diseases. Curr Opin Neurobiol 18:516–523
        doi: 10.1016/j.conb.2008.09.018

    46. Wang B, Xi X, Lei X, Zhang X, Cui S, Wang J, Jin Q, Zhao Z (2013) Enterovirus 71 protease 2Apro targets MAVS to inhibit anti-viral type I interferon responses. PLoS Pathog 9:e1003231
        doi: 10.1371/journal.ppat.1003231

    47. Wang C, Sun M, Yuan X, Ji L, Jin Y, Cardona CJ, Xing Z (2017) Enterovirus 71 suppresses interferon responses by blocking janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling through inducing karyopherin-alpha1 degradation. J Biol Chem 292:10262–10274
        doi: 10.1074/jbc.M116.745729

    48. Wang T, Wang B, Huang H, Zhang C, Zhu Y, Pei B, Cheng C, Sun L, Wang J, Jin Q, Zhao Z (2017) Enterovirus 71 protease 2Apro and 3Cpro differentially inhibit the cellular endoplasmic reticulum-associated degradation (ERAD) pathway via distinct mechanisms, and enterovirus 71 hijacks ERAD component p97 to promote its replication. PLoS Pathog 13:e1006674
        doi: 10.1371/journal.ppat.1006674

    49. Watters K, Palmenberg AC (2011) Differential processing of nuclear pore complex proteins by rhinovirus 2A proteases from different species and serotypes. J Virol 85:10874–10883
        doi: 10.1128/JVI.00718-11

    50. Watters K, Inankur B, Gardiner JC, Warrick J, Sherer NM, Yin J, Palmenberg AC (2017) Differential disruption of nucleocytoplasmic trafficking pathways by rhinovirus 2A proteases. J Virol 91:e02472

    51. Wobst HJ, Delsing L, Brandon NJ, Moss SJ (2017) Truncation of the TAR DNA-binding protein 43 is not a prerequisite for cytoplasmic relocalization, and is suppressed by caspase inhibition and by introduction of the a90v sequence variant. PLoS ONE 12:e0177181
        doi: 10.1371/journal.pone.0177181

    52. Wong J, Si X, Angeles A, Zhang J, Shi J, Fung G, Jagdeo J, Wang T, Zhong Z, Jan E, Luo H (2013) Cytoplasmic redistribution and cleavage of AUF1 during coxsackievirus infection enhance the stability of its viral genome. FASEB J 27:2777–2787
        doi: 10.1096/fj.12-226498

    53. Wu S, Wang Y, Lin L, Si X, Wang T, Zhong X, Tong L, Luan Y, Chen Y, Li X, Zhang F, Zhao W, Zhong Z (2014) Protease 2A induces stress granule formation during coxsackievirus b3 and enterovirus 71 infections. Virol J 11:192
        doi: 10.1186/s12985-014-0192-1

    54. Zhang YJ, Xu YF, Dickey CA, Buratti E, Baralle F, Bailey R, Pickering-Brown S, Dickson D, Petrucelli L (2007) Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43. J Neurosci 27:10530–10534
        doi: 10.1523/JNEUROSCI.3421-07.2007

    55. Zhao T, Zhang Z, Zhang Y, Feng M, Fan S, Wang L, Liu L, Wang X, Wang Q, Zhang X, Wang J, Liao Y, He Z, Lu S, Yang H, Li Q (2017) Dynamic interaction of enterovirus 71 and dendritic cells in infected neonatal rhesus macaques. Front Cell Infect Microbiol 7:171
        doi: 10.3389/fcimb.2017.00171

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    TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71

      Corresponding author: Zhaohua Zhong, zhongzh@hrbmu.edu.cn
      Corresponding author: Wenran Zhao, zhaowr@hrbmu.edu.cn
    • 1. Department of Cell Biology, Harbin Medical University, Harbin 150081, China
    • 2. Department of Microbiology, Harbin Medical University, Harbin 150081, China

    Abstract: 

    Enterovirus A71 (EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease (HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3C between the residues 331Q and 332S, while mutated TDP-43 (Q331A) was not cleaved. In addition, mutated 3C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2A rather than 3C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71infection.

    • Enterovirus A71 (EV-A71) is a globally important neurotropic virus in the genus of Enterovirus of Picornaviridae family (Lei et al. 2017; Wang C et al. 2017). EV-A71 is one of the causative pathogens that leads to the epidemic of hand, foot, and mouth disease (HFMD) commonly affecting young children, especially in Asian-Pacific region (Chang et al. 2016). From 2008 to 2013, there were about 9 million HFMD cases reported in China (Takahashi et al. 2016). Although typical EV-A71 infection is mild and selflimiting, severe neurological complications, such as brainstem encephalitis, aseptic meningitis, and acute flaccid paralysis can sometimes occur with severe consequence (Caine et al. 2016; Chen et al. 2019; Ju et al. 2020; Lee 2016; Teoh et al. 2016). The neuropathogenesis of EV-A71 infection is not completely understood. It is believed that the inflammatory response initiated by the cytokine storm in the central nervous system plays an important role (Feng et al. 2016; Huang et al. 2017; Zhao et al. 2017).

      EV-A71 is a small, non-enveloped, single-stranded, positive-sensed RNA virus with a genome of 7.4 kb (Lei et al. 2017; Wang C et al. 2017). During viral replication, the genome of EV-A71 encodes a primary polyprotein, which is cleaved by the viral proteases 2A and 3C and matures into viral structural and non-structural proteins (Li B et al. 2017; Li J et al. 2017; Wang et al. 2013). The proteases of enteroviruses cleave not only viral polyproteins, but also cellular proteins such as MAVS, MDA5, and Gasdermin D (Feng et al. 2014; Lei et al. 2017; Wang et al. 2013). These virus proteases have been demonstrated to contribute to a large spectrum of disrupted cellular functions during EV-A71 infection, including inhibited interferon response, pyroptosis, suppressed endoplasmic reticulum-associated degradation pathway, and promoted ERK activity (Duan et al. 2017; Lei et al. 2017; Wang C et al. 2017; Wang T et al. 2017).

      TAR DNA-binding protein 43 (TDP-43) is a nuclear RNA- and DNA-binding protein with multiple functions involved in transcription, RNA processing, mRNA stability, and translation (Gao et al. 2019; McCluskey et al. 2009; Tudor et al. 2010). The cytoplasmic mislocalization and aggregation of TDP-43 in the motor neurons and the surrounding cells is a key pathological hallmark of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by the progressive loss of motor neurons, followed by muscle paralysis (Donde et al. 2020; Riku et al. 2014). The cleavage and cytoplasmic aggregation of TDP-43 is the pathological hallmark of the abnormal neurons of ALS, implicating the role of TDP-43 in the pathogenesis of ALS (Berning and Walker 2019; Shimonaka et al. 2016). Coxsackievirus B3 (CVB3), a species of enterovirus which can also cause neurological complications (Holmes et al. 2016), has been shown to cleave TDP-43 and induce its cytoplasmic accumulation in the stress granules (Fung et al. 2015). However, it is unknown if EV-A71 infection could impact the integrity and distribution of TDP-43. In this study, we found that TDP-43 was cleaved and mislocalized during EV-A71 infection.

    • HeLa cells were gifts from Professor Fengmin Zhang (Department of Microbiology, Harbin Medical University, Harbin, China). SH-SY5Y cells were obtained from American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Bioindustry, Israel), penicillin (100 U/mL), and streptomycin (10 lg/mL). EV-A71 BrCr strain was provided by the Center for Disease Control of Heilongjiang Province of China (Harbin, China). Virus was propagated in HeLa cells. Virus titer was measured by tissue culture infective dose (TCID50).

    • pEGFP-TDP-43 and pEGFP-TDP-43-mut, in which 331Q was mutated to 331A, were purchased from Viewsolid (Beijing, China). pEGFP-2A, pEGFP-3C, and pEGFP-3C-mut were constructed based on pEGFP-C1 (Clonetech) as described previously (Guo et al. 2014; Wu et al. 2014).

    • HeLa cells were grown in 12-well plate for 24 h to reach 60% confluency. Transfection solution was prepared with plasmids mixed with Lipofectamine 2000 (Thermo Fisher) in serum-free DMEM. Cells were incubated with transfection mixture for 4 h and then grown in the fresh medium containing 10% FBS and antibiotics. Cells were harvested at 24 h after transfection for further analysis. Each transfection was performed in triplicate.

    • Total RNA was extracted by TRIzol reagent (Thermo Fisher) according to the manufacturer's protocol. 1 lg of total RNA was reverse-transcribed using TransScript All-in-One SuperMix (TransGen, Beijing, China) in a total of 20 lL reverse transcription system. For quantitative realtime PCR (qRT-PCR), TransStart Top Green qPCR SuperMix was used on a LightCycler (Roche). PCR reaction was carried out for 35 cycles according to the cycling conditions: denaturation at 94 ℃ for 5 s, annealing at 55 ℃ for 15 s, and extension at 72 ℃ for 1 min. The relative RNA amount was measured with 2-CT threshold cycle method and normalized to the amount of GAPDH. Reactions were carried out in triplicate. Primers were synthesized by Genewiz (Suzhou, China). Primer sequences are as listed: for EV-A71, forward primer 5'-CCCCTGAATGCGGCTAAT-3' and reverse primer 5'-CAATTGTCACCATAAGCAGCCA-3'; for GAPDH, forward primer 5'-CTGGGCTACACTGAGCACC-3' and reverse primer 5'-AAGTGGTCGTTGAGGGCAATG-3'.

    • Cells were harvested and proteins were extracted by RIPA buffer (Biotopped, Beijing, China) containing 1% phenylmethanesulfonyl fluoride (PMSF) (Biotopped). Proteins were quantified by Bradford assay, separated by 12% polyacrylamide gel (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Kenilworth, NJ). The membrane was blocked with 0.1% Tween-20 in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) and then incubated with primary antibodies at 4 ℃ overnight. The membrane was washed three times with 0.1% Tween-20 in PBS and incubated with anti-rabbit IgG for 2 h at room temperature. Immunoblot was visualized with FluorChem R system (ProteinSimple, Santa Clara, CA). The results were analyzed by Image J and quantified by Alpha View 3.4.0 (ProteinSimple). Rabbit anti-enterovirus 3D polyclonal antibody was prepared by the Department of Microbiology of Harbin Medical University. Anti-GFP was purchased from Santa Cruz (SC-9996). Antibody against TDP-43 was purchased from Abcam (ab109535). Antibodies against caspase-3 (19677-1-AP), GAPDH (10494-1- AP), β-actin (66009-1-Ig), β-tubulin (10068-1-AP), and secondary antibodies were purchased from Proteintech (Wuhan, China).

    • HeLa cells cultured in the glass-bottom dish for 24 h. Cells were washed with PBS three times, fixed with 4% paraformaldehyde for 15 min and permeabilized with 1% Triton-X 100 for 20 min at room temperature (RT). After blocking with 1% BSA for 30 min at RT, cells were incubated with anti-TDP-43 antibody diluted by 1:500 at 4 ℃ overnight and then incubated with fluorescence-conjugated anti-rabbit IgG (H + L) antibodies (Proteintech) at RT for 1 h. Cells were viewed by fluorescence microscope (ECLIPSE Ni, Nikon, Japan) and CV1000 confocal system (Yokogawa, Japan).

    • All experiments were repeated three times. Data were expressed as mean ± SD and analyzed by Student's t test. P < 0.05 was defined as statistically significant.

    • It has been reported that TDP-43 was cleaved and aggregated in the cytoplasmic stress granules during the infection of CVB3 (Fung et al. 2015). Since both CVB3 and EV-A71 belong to genus Enterovirus and share similar features in viral structure and constituents, we postulated that EV-A71 infection may also lead to the cleavage of TDP-43. To this end, HeLa cells and SH-SY5Y were cultured in 12-well plates to 60%–70% confluence and infected with EV-A71 at 1 × 104 TCID50 for 24 h. As shown in Fig. 1, a~35 kDa fragment of TDP-43 was identified in both HeLa and SH-SY5Y cells infected with EV-A71 (Fig. 1A, 1B), while the addition of aurintricarboxylic acid (Sigma-Aldrich), which has been demonstrated to function as the inhibitor of the 3D RNAdependent RNA polymerase of EV-A71 (Hung et al. 2010), abolished the cleavage of TDP-43 (Fig. 1A). Since the cleavage and pathological aggregation of TDP-43 in neurodegenerative diseases are largely attributed to the activation of caspases (Rohn 2009; Zhang et al. 2007), the impact of pan-caspase inhibitor on the cleavage of TDP-43 was studied. As shown in Fig. 1C, EV-A71 infection led to the cleavage of TDP-43 in the presence or absence of pancaspase inhibitor Z-VAD-FMK (20 μmol/L, Beyotime, China), indicating that the TDP-43 cleavage is not dependent on the activated caspase during EV-A71 infection. These results suggested that the cleavage of TDP-43 was induced by EV-A71 replication.

      Figure 1.  TDP-43 is cleaved in the cells infected with EV-A71. A–B HeLa cells and SH-SY5Y were cultured in 12-well plates to 60%–70% confluence and infected with EV-A71 at 1 × 104 TCID50 for 24 h. TDP-43 proteins were analyzed by Western blotting. HeLa cells were cultured in 12-well plates and infected or mock-infected EV-A71 for 24 h in the medium containing 10 lmol/L ATA (aurintricarboxylic acid, 3D inhibitor), the inhibitor of 3D polymerase of enteroviruses (A) or 20 μmol/L pan-caspase inhibitor Z-VAD-FMK (C). Cell lysates were subjected to Western blot analysis.

    • Enteroviral protease 2A and 3C are responsible for the cleavage of the viral polyprotein translated from the viral genome (Cai et al. 2013; Guo et al. 2014). These proteases also capable of cleaving cellular proteins such as MAVS, eIF4G, and AUF1 during viral infection (Feng et al. 2014; Wong et al. 2013; Wu et al. 2014). To determine whether viral proteases are responsible for the cleavage of TDP-43, HeLa cells were transfected with pEGFP-2A or pEGFP-3C. Control cells were transfected with pEGFP-C1, which was constructed to express EGFP as described previously (Wu et al. 2014). As shown in Fig. 2, the cleaved fragment (~35kDa) of TDP-43 was observed in the cells expressing EGFP-3C or infected with EV-A71, while cleaved TDP-43 was not observed in the cells overexpressing EGFP-2A (Fig. 2A), indicating that TDP-43 was cleaved by viral protease 3C rather than 2A. We also found that the cleaved TDP-43 was accumulating along with the expression of 3C protease (Fig. 2B).

      Figure 2.  TDP-43 is cleaved by the 3C protease of EV-A71. A HeLa cells were cultured in 12-well plates to 60% confluency and infected with EV-A71 or transfected with pEGFP-2A or pEGFP-3C for 24 h. Control cells were treated with PBS. Cell lysates were subjected to Western blot analysis. B Cells were transfected with pEGFP-3C for various times. Control cells were transfected with pEGFP-C1. Cell lysates were analyzed by Western blotting.

    • To confirm that 3C is the viral protease that causes the cleavage of TDP-43, HeLa cells were co-transfected with either pEGFP-3C or pEGFP-3C-mut and pEGFP-TDP-43. pEGFP-3C-mut was expressing a mutant 3C which lacks the protease activity due to the mutation of 147 cysteine to alanine (Fig. 3A). The cleaved EGFP-TDP-43 fusion protein (27 kDa + 43 kDa = 70 kDa), designated as EGFP-TDP-35 (27 kDa + 35 kDa = 62 kDa), was identified in he cells expressing EGFP-3C but not in the cells expressing EGFP-3C-mut (Fig. 3B). These data show that TDP-43 was cleaved by the protease 3C of EV-A71.

      Figure 3.  TDP-43 is not cleaved by the mutated 3C protease. A The construct expressing the fusion protein of EGFP with a mutated 3C (designated as pEGFP-3C-mut), which lacks the protease activity, was generated. B Cells were co-transfected with either pEGFP-TDP-43 and pEGFP-3C or pEGFP-TDP-43 and pEGFP-3C-mut for 24 h. Cell lysates were analyzed by Western blotting in which anti-EGFP antibody was used.

    • The potential cleavage site of the protease 3C in TDP-43 was analyzed (Fig. 4A). To confirm the cleavage site of TDP-43, the construct expressing EGFP-TDP-43 or a mutant TDP-43 (Q331A) was generated. Cells were transfected with either pEGFP-TDP-43 or pEGFP-TDP-43-mut (Q331A) for 24 h and infected with EV-A71 for 24 h. As shown Fig. 4B, EV-A71 infection induced the cleavage of EGFP-TDP-43, designated as EGFP-TDP-35 (with a molecular weight of 62 kDa), while EGFP-TDP-35 was not observed in the cells expressing EGFP-TDP43-mut, demonstrating that TDP-43 was cleaved between 331Q and 332S. Endogenous TDP-43 and its cleaved form (TDP-35) were also observed in the cells expressing either wild type or mutant TDP-43, further demonstrating that the cleavage of TDP-43 was the result of EV-A71 infection.

      Figure 4.  TDP-43 is cleaved at a site which is close to its C-terminal. The constructs expressing EGFP fusion proteins containing TDP-43 or mutated TDP-43 were generated. The construct containing mutated TDP-43 was designated as pEGFP-TDP-43-mut, in which 331Q is mutated to 331A. The domains of TDP-43 protein and the potential cleavage site of protease 3C of the enteroviruses are indicated (A). Cells were transfected with either pEGFP-TDP-43 (wild type) or pEGFP-TDP-43-mut (Q331A) for 24 h and then infected with EVA71 for 24 h (B and C). Control cells were transfected with pcDNA3.1 (C). Cell lysates were subjected to Western blotting using anti-TDP-43 antibody (B) or anti-EGFP antibody (C).

      We also noted that a fragment of TDP-43 with a size smaller than TDP-35 appeared in the cells transfected with pEGFP-TDP-43 and then infected with EV-A71 (Fig. 4B, left lane). The occurrence of this fragment, which is obviously originated from the endogenous TDP-43, indicates that it is very likely that there is another cleavage site in TDP-43 except that between 331Q and 332S. However, this extra TDP-43 fragment (smaller than TDP-35) was not observed in the cells transfected with pEGFP-TDP-43-mut followed by the infection of EV-A71 (Fig. 4B, right lane), suggesting that virus-induced TDP-43 cleavage at this site may not as efficient as that between 331Q and 332S.

      The cleavage of TDP-43 in EV-A71-infected cells was also evaluated by Western blotting with anti-EGFP antibody (Fig. 4C). EFGP-TDP-35 was identified in the cells expressing EGFP-TDP-43, but not in the cells expressing EGFP-TDP-43-mut or control cells, which were transfected with pcDNA3.1 (Fig. 4C). Collectively, these results demonstrated that TDP-43 was cleaved between 331Q and 332S, which is close to its C-terminal.

    • Study has shown that TDP-43 was translocated into the cytoplasmin in CVB3-infected cells (Fung et al. 2015). Our previous study has demonstrated that EV-A71 infection induced the accumulation of proteins in the form of cytoplasmic stress granules. Thus, we asked the question whether the intracellular localization of TDP-43 is altered by EV-A71 infection. As shown in Fig. 5, TDP-43 was localized almost entirely in the nucleus of control cells (Fig. 5A) or in the cells expressing protease 3C of EV-A71 (Fig. 5B, upper panel), while cytoplasmic TDP-43 was identified in the cells expressing 2A (Fig. 5B, lower panel). These findings demonstrate that the cytoplasmic translocation of TDP-43 was induced by protease 2A rather than protease 3C, suggesting that the cleavage of TDP-43 does not contribute to its mislocalization in EV-A71-infected cells.

      Figure 5.  The cytoplasmic translocation of TDP-43 is caused by protease 2A of EV-A71. HeLa cells were transiently transfected with pEGFP-C1 (A), pEGFP-2A (B), and pEGFP-3C (B), respectively, for 24 h. Cells were harvested for immunofluorescence staining using anti-TDP-43 antibody (Alexa-fluor-594). Cell nuclei were stained with DAPI. Fluorescence images were taken by fluorescence microscope (A) and CV1000 confocal system (B). Yellow squares in the upper panel of B indicate the cells expressing EGFP-3C. Yellow squares in the lower panel of B indicate the cells expressing EGFP-2A. The larger square in the upper right or lower right panel of B is the magnified image enclosed by the smaller square in the same panel. The distribution of TDP-43 was indicated by arrows in the upper right and lower right panels of B. Arrows in the upper-middle of B indicate the nuclei of the cells expressing EGFP-3C, while arrows in the lower-middle of B indicate the cytoplasm of the cells expressing EGFP-2A.

    • To reveal virus-host interaction is essential for understanding the pathogenesis of viral diseases and developing potential treatment. The pathogenesis of neurological injury caused by EV-A71 infection is incompletely understood (Huang et al. 1999; Teoh et al. 2016). As an RNA and DNA-binding protein, TDP-43 is widely involved in a variety of cellular processes. The pathological aggregation of TDP-43 is the key molecular event of ALS and frontotemporal lobar degeneration (FTLD) (Hart and Gitler 2012; Riku et al. 2016). The cleavage and cytoplasmic aggregation of TDP-43 have been observed in the cells infected with CVB3, another neurotropic enterovirus (Fung et al. 2015). In this study, we demonstrated that TDP-43 was cleaved by the protease 3C of EV-A71 and translocated into the cytoplasm, implicating the potential role of TDP-43 in pathogenesis of EV-A71 infection.

      Similar to the report in which TDP-43 was found to be cleaved and aggregated in the cytoplasmic stress granules during CVB infection (Fung et al. 2015), this study shows that TDP-43 was cleaved by protease 3C and translocated into the cytoplasm induced by protease 2A of EV-A71. However, we did not study whether TDP-43 or the cleaved TDP-43 was participating in the formation of stress granules in EV-A71-infected cells.

      The proteases 2A and 3C of enteroviruses are essential for viral replication by cleaving the viral polyprotein into viral structural and non-structural proteins. Virus protease 2A and 3C also cleave cellular proteins such as MDA5, RIG-I, MAVS, and TRIF to suppress innate immunity (Feng et al. 2014; Lei et al. 2010; Lei et al. 2011; Wang et al. 2013). Previous studies show that the cleavage site for protease 3C of enteroviruses is often between Q and G or Q and S in the target sequence of AXXQG or AXXQS (Sun et al. 2016). In this study, we demonstrated that TDP-43 was cleaved by 3C protease between the residue 331Q and 332S in the sequence of AALQS. When this sequence was mutated (331Q was mutated to 331A), 3C protease failed to cleave TDP-43.

      As a highly conserved nuclear DNA/RNA binding protein, TDP-43 is involved in all aspects of RNA metabolism including the control of transcription, splicing, mRNA transport, and translation (Fiesel et al. 2012; Freibaum et al. 2010). It is believed that TDP-43 may function as a part of ribonucleoprotein complexes (Colombrita et al. 2012; Ule 2008). Evidence suggests that nuclear RNA-binding proteins are utilized to promote efficient viral replication. Heterogeneous ribonucleoprotein C (hnRNP C) is able to stimulate poliovirus RNA synthesis (Ertel et al. 2010), and hnRNP A1 promotes the translation of EV-A71 through the interaction with type I internal ribosome entry site (IRES) (Levengood et al. 2013; Tolbert et al. 2017). TDP-43 has been described to interact with hnRNP A1, A2/B1, A3, and C2 (Buratti et al. 2005; Romano et al. 2014).

      Although we show that TDP-43 was cleaved by the protease 3C of EV-A71, how the cleaved TDP-43 would impact viral replication and cellular functions remains to be studied. Our data show that neither knockdown of TDP-43 with siRNA nor TDP-43 overexpression altered the replication of EV-A71 (Supplementary Figure S1 and Figure S2). Although we did not investigate the cellular impact of TDP-43 cleavage during EV-A71 infection, it is logical to predict that processes associated with RNA metabolism could be affected due to the critical role played by TDP-43 in this aspect.

      EV-A71 infection primarily impacts the neural system of young children. However, up to now, there is no evidence about the cleavage and the cytoplasmic translocation of TDP-43 in the patients infected with EV-A71. Theiler's murine encephalomyelitis virus (TMEV), which belongs to the Cardiovirus genus of the Picornaviridae family, is the rodent pathogen of the central nervous system (Gerhauser et al. 2019). One report has shown that the infection of TMEV induced the cleavage of TDP-43 with the production of the cleaved fragment, TDP-35. Although the cleavage site in TDP-43 was not investigated (Masaki et al. 2019), we predict that TDP-43 is likely cleaved at the same site as in the case of EV-A71 infection, since TMEV also encodes a viral protease 3C with cysteine protease activity (GenBank: AAA47930.1). Moreover, the cytoplasmic mislocalization of TDP-43 was also seen in the cells infected with TMEV (Masaki et al. 2019). Collectively, the previous report and our results suggest that the cleavage and cytoplasmic mislocalization of TDP-43 might be a common feature for the infection of picornaviruses. In vivo study is needed to further evaluate the significance of the altered TDP-43 in the pathogenesis of EV-A71.

      This study shows that the cytoplasmic localization of TDP-43 was induced by protease 2A rather than 3C of EVA71. It has been demonstrated that protease 2A of enteroviruses cleaves nucleoporins in the nuclear pore complex (NPC), leading to the selective removal of the N-terminal phenylalanine-glycine (FG)-rich domains of the nucleoporins (Park et al. 2015; Watters and Palmenberg 2011). Although the overall structure of NPC remains relatively unaffected in the cells infected with enteroviruses, the permeability of nuclear pore is increased (Watters et al. 2017; Wobst et al. 2017). Thus, it is likely that the cytoplasmic localization of TDP-43 is the consequence of the increased permeability of the nuclear pores induced by 2A protease of EV-A71. However, it is unknown concerning the integrity of TDP-43 (cleaved or non-cleaved) which one is localized in the cytoplasm.

      In summary, the present study demonstrated that TDP-43 was cleaved by the protease 3C in EV-A71-infected cells, while viral protease 2A induced the cytoplasmic localization of TDP-43. Our data imply that the cleavage and mislocalization of TDP-43 might contribute to the pathogenesis of EV-A71 infection.

    • This study was supported by the National Natural Foundation of China (81672007 and 81971920 to Wenran Zhao, 81871652 to Zhaohua Zhong, and 81772188 to Yan Wang) and Health and Family Planning Commission of Heilongjiang Province (2017-158 to Xiaoman Wo). We are grateful to the help of Dr. Ying Wu (School of Life Sciences, Northeast Forestry University, Harbin, China) in viewing the images of fluorescence microscope.

    • WZ and ZZ conceived the experiments, analyzed the data. WZ wrote the manuscript. XW, YY, and YX performed the majority of the laboratory work. Yao Wang and YC prepared and performed the immunofluorescence visualization. SZ and Yan Wang prepared of plasmids. LL and XZ prepared Enterovirus A71 for this study.

    • There is no conflicting interest in this work.

    • This article does not contain any studies with human or animal subjects performed by any of the authors.

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