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. IMPORTANCEOutbreaks 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.

Respiratory syncytial virus entry and how to block it

“…Of these, the data for nucleolin are the strongest, and nucleolin has been shown to interact with many other viruses, including parainfluenza type 3 (PIV-3) 106 , enterovirus 71 (reF. 107 ), Crimean-Congo haemorrhagic fever virus 108 , adeno-associated virus type 2 (AAV-2) 109 and HIV-1 (reF. 110 ).…”

The nucleolar interface of RNA viruses

“…Cell surface-expressed nucleolin is involved in cell entry by a number of RNA viruses. It was reported to have co-receptor activity for diverse cytoplasmic viruses including the paramyxovirus human parainfluenza virus type 3 (HPIV-3) (Bose et al, 2004); the picornavirus, enterovirus 71 (EV71) (Su et al, 2015); Crimean-Congo haemorrhagic fever virus (Bunyaviridae family) (Xiao et al, 2011); and the flavivirus, JEV (Thongtan et al, 2012). The paramyxovirus, respiratory syncytial virus was reported to use nucleolin as a cellular receptor, which is bound by the viral envelope glycoprotein (G) (Tayyari et al, 2011).…”

Intracellular Growth and Morphological Characteristics of <i>Legionella pneumophila</i> during Invasion and Proliferation in Different Cells

“…Accumulating evidence suggests that attachment and internalization of bacterium is related to the cell membrane surface receptors, cell morphology, and cell migration of host cells. [41][42][43] The differences in the invasiveness of Legionella observed among these cell lines may help us determine the key surface proteins and mechanisms involved in bacteria uptake. 39) Additionally, bacteria regulate their own proliferation and lysosome escape in host cell.…”