title: Enhancing dengue virus maturation using a stable furin over-expressing cell line.
abstract: Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies.
authors.affiliation: Viral Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Swati
authors.initials: S
authors.lastname: Mukherjee
authors.affiliation: Department of Biological Sciences and Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Devika
authors.initials: D
authors.lastname: Sirohi
authors.affiliation: Viral Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Dowd
authors.affiliation: Department of Biological Sciences and Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Zhenguo
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Departments of Medicine, Molecular Microbiology, Pathology and Immunology, and The Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, Saint Louis, MO 63110, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Diamond
authors.affiliation: Department of Biological Sciences and Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Kuhn
authors.affiliation: Viral Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: piersontc@mail.nih.gov.
authors.forename: Theodore C
authors.initials: TC
authors.lastname: Pierson
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003715:Dengue / Q000235:genetics / Q000378:metabolism / Q000821:virology; D003716:Dengue Virus / Q000502:physiology* / Q000648:ultrastructure; D045683:Furin / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D009500:Neutralization Tests; D014709:Vero Cells; D014779:Virus Replication
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D045683:Furin
keywords: Antibody neutralization; Dengue virus; Flavivirus; Furin; Virion maturation; West Nile virus