title: Cell surface nucleolin facilitates enterovirus 71 binding and infection.
abstract: Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human nucleolin as a novel binding receptor for EV71. Glycoproteins purified by lectin chromatography from the membrane extraction of human cells were treated with sialidase, followed by immunoprecipitation with EV71 particles. Among the 16 proteins identified by tandem mass spectrometry analysis, cell surface nucleolin attracted our attention. We found that EV71 interacted directly with nucleolin via the VP1 capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface nucleolin decreased EV71 binding, infection, and production in human cells. Furthermore, the expression of human nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71 infection and production in the cells. These results strongly indicate that human nucleolin can mediate EV71 binding to and infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens.
  
  
  OBJECTIVE
  Outbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting glycoproteins. Several EV71-interacting glycoproteins are identified, and the role of cell surface nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71 infection mechanism and anti-EV71 drug discovery but also provide a new strategy for virus receptor identification.
authors: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Taiwan, Republic of China.
authors: Pei-Yi
authors: PY
authors: Su
authors: Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan, Republic of China.
authors: Ya-Fang
authors: YF
authors: Wang
authors: Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan, Republic of China.
authors: Sheng-Wen
authors: SW
authors: Huang
authors: Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan, Republic of China Institute of Bioinformatics and Biosignal Transduction, College of Bioscience and Biotechnology, National Cheng Kung University, Taiwan, Republic of China.
authors: Yu-Chih
authors: YC
authors: Lo
authors: Institute of Oral Medicine and Department of Stomatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Taiwan, Republic of China.
authors: Ya-Hui
authors: YH
authors: Wang
authors: Institute of Oral Medicine and Department of Stomatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Taiwan, Republic of China.
authors: Shang-Rung
authors: SR
authors: Wu
authors: Institute of Oral Medicine and Department of Stomatology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Advanced Optoelectronic Technology Center and Center for Micro/Nano Science and Technology, National Cheng Kung University, Taiwan, Republic of China.
authors: Dar-Bin
authors: DB
authors: Shieh
authors: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan, Republic of China Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Taiwan, Republic of China.
authors: Shun-Hua
authors: http://orcid.org/0000-0002-4585-9279
authors: SH
authors: Chen
authors: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan, Republic of China.
authors: Jen-Ren
authors: JR
authors: Wang
authors: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan, Republic of China Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Taiwan, Republic of China.
authors: Ming-Der
authors: MD
authors: Lai
authors: Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Taiwan, Republic of China Center of Infectious Disease and Signaling Research, National Cheng Kung University, Taiwan, Republic of China affa@mail.ncku.edu.tw.
authors: Chuan-Fa
authors: http://orcid.org/0000-0003-2404-1248
authors: CF
authors: Chang
mesh_terms: D002845:Chromatography; D030016:Enterovirus D, Human / Q000378:metabolism* / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D009439:Neuraminidase; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D053585:Virus Attachment; D053586:Virus Internalization; D000097796:Nucleolin
chemical_list: D008565:Membrane Proteins; D009439:Neuraminidase; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin