title: Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain).
abstract: Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1(K98E,E145A,L169F) with three substitutions in the VP1 protein-K98E, E145A and L169F-productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection.
authors.affiliation: Temasek Lifesciences Laboratory, National University of Singapore, Singapore 117604, Singapore.|Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore.
authors.forename: Carla Bianca Luena
authors.initials: CB
authors.lastname: Victorio
authors.affiliation: Temasek Lifesciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Yishi
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Temasek Lifesciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Qimei
authors.initials: Q
authors.lastname: Ng
authors.affiliation: Temasek Lifesciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Tao
authors.initials: T
authors.lastname: Meng
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore.
authors.forename: Vincent Tk
authors.initials: VT
authors.lastname: Chow
authors.affiliation: Temasek Lifesciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Kaw Bing
authors.initials: KB
authors.lastname: Chua
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids; D000818:Animals; D018955:CD36 Antigens / Q000235:genetics / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D029821:Enterovirus A, Human / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D041681:NIH 3T3 Cells; D059386:Reverse Genetics; D014709:Vero Cells
chemical_list: D000596:Amino Acids; D018955:CD36 Antigens; D036022:Capsid Proteins; D051907:Lysosomal Membrane Proteins; C489452:Scarb2 protein, mouse; C442501:VP1 protein, Foot-and-mouth disease virus; C442459:VP2 protein, Foot-and-mouth disease virus