title: Enterovirus D68 receptor requirements unveiled by haploid genetics.
abstract: Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory disease and is associated with cases of paralysis, especially among children. Heretofore, information on host factor requirements for EV-D68 infection is scarce. Haploid genetic screening is a powerful tool to reveal factors involved in the entry of pathogens. We performed a genome-wide haploid screen with the EV-D68 prototype Fermon strain to obtain a comprehensive overview of cellular factors supporting EV-D68 infection. We identified and confirmed several genes involved in sialic acid (Sia) biosynthesis, transport, and conjugation to be essential for infection. Moreover, by using knockout cell lines and gene reconstitution, we showed that both α2,6- and α2,3-linked Sia can be used as functional cellular EV-D68 receptors. Importantly, the screen did not reveal a specific protein receptor, suggesting that EV-D68 can use multiple redundant sialylated receptors. Upon testing recent clinical strains, we identified strains that showed a similar Sia dependency, whereas others could infect cells lacking surface Sia, indicating they can use an alternative, nonsialylated receptor. Nevertheless, these Sia-independent strains were still able to bind Sia on human erythrocytes, raising the possibility that these viruses can use multiple receptors. Sequence comparison of Sia-dependent and Sia-independent EV-D68 strains showed that many changes occurred near the canyon that might allow alternative receptor binding. Collectively, our findings provide insights into the identity of the EV-D68 receptor and suggest the possible existence of Sia-independent viruses, which are essential for understanding tropism and disease.
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Jim
authors.initials: J
authors.lastname: Baggen
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Hendrik Jan
authors.initials: HJ
authors.lastname: Thibaut
authors.affiliation: Biochemistry Division, Netherlands Cancer Institute, 1006 BE, Amsterdam, The Netherlands;
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Staring
authors.affiliation: Biochemistry Division, Netherlands Cancer Institute, 1006 BE, Amsterdam, The Netherlands;
authors.forename: Lucas T
authors.initials: LT
authors.lastname: Jae
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907;
authors.forename: Yue
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Hongbo
authors.initials: H
authors.lastname: Guo
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Jasper J
authors.initials: JJ
authors.lastname: Slager
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Jost W
authors.initials: JW
authors.lastname: de Bruin
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Arno L W
authors.initials: AL
authors.lastname: van Vliet
authors.affiliation: Biochemistry Division, Netherlands Cancer Institute, 1006 BE, Amsterdam, The Netherlands;
authors.forename: Vincent A
authors.initials: VA
authors.lastname: Blomen
authors.affiliation: Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, 3720 BA, Bilthoven, The Netherlands;
authors.forename: Pieter
authors.initials: P
authors.lastname: Overduin
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907;
authors.forename: Ju
authors.initials: J
authors.lastname: Sheng
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan Xander
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands;
authors.forename: Erik
authors.initials: E
authors.lastname: de Vries
authors.affiliation: Virology Division, Centre for Infectious Diseases Research, Diagnostics and Screening, National Institute for Public Health and the Environment, 3720 BA, Bilthoven, The Netherlands;
authors.forename: Adam
authors.initials: A
authors.lastname: Meijer
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907; mr@purdue.edu F.J.M.vanKuppeveld@uu.nl.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rossmann
authors.affiliation: Biochemistry Division, Netherlands Cancer Institute, 1006 BE, Amsterdam, The Netherlands; Cancer Genomics Centre, 3584 CG, Utrecht, The Netherlands.
authors.forename: Thijn R
authors.initials: TR
authors.lastname: Brummelkamp
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL, Utrecht, The Netherlands; mr@purdue.edu F.J.M.vanKuppeveld@uu.nl.
authors.forename: Frank J M
authors.initials: FJ
authors.lastname: van Kuppeveld
mesh_terms: D000818:Animals; D002460:Cell Line; D030016:Enterovirus D, Human / Q000378:metabolism*; D006238:Haploidy; D006801:Humans; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D011991:Receptors, Virus
keywords: enterovirus D68; haploid genetic screen; receptor; sialic acid