title: Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of β-galactosidase.
abstract: Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Christine
authors.initials: C
authors.lastname: Burkard
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Louis-Marie
authors.initials: LM
authors.lastname: Bloyet
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Oliver
authors.initials: O
authors.lastname: Wicht
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Frank J
authors.initials: FJ
authors.lastname: van Kuppeveld
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Peter J M
authors.initials: PJ
authors.lastname: Rottier
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Cornelis A M
authors.initials: CA
authors.lastname: de Haan
authors.affiliation: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors.forename: Berend Jan
authors.initials: BJ
authors.lastname: Bosch
mesh_terms: D000818:Animals; D002415:Cats; D002460:Cell Line; D017930:Genes, Reporter; D015726:Giant Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D051379:Mice; D006517:Murine hepatitis virus / Q000502:physiology; D018116:Vesiculovirus / Q000502:physiology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D053585:Virus Attachment; D053586:Virus Internalization / Q000187:drug effects; D014779:Virus Replication; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D014759:Viral Envelope Proteins; D001616:beta-Galactosidase