title: An enzymatic virus-like particle assay for sensitive detection of virus entry.
abstract: A viral entry assay where a beta-lactamase reporter protein fused to the influenza matrix protein-1 (BlaM1) is packaged as a structural component into influenza virus-like particles (VLPs) is described. The Bla reporter is released upon fusion with target cells and can be detected in live cells by flow cytometry, microscopy, or fluorometric plate reader for utility in high-throughput screening approaches. The production of BlaM1 VLPs and subsequent transfer of Bla activity to target cells requires the presence of influenza hemagglutinin (HA) and neuraminidase (NA). In addition, transfer of Bla by the VLPs can be blocked by an influenza neutralizing antibody, is impeded by a chemical inhibitor of influenza virus entry, and requires HA that is cleaved by a protease specific for its cleavage site. An analogous VLP system also was developed for Ebola (EBOV) and Marburg (MARV) viruses, demonstrating that this straightforward assay has broad application for studying the entry steps of enveloped viruses, especially those that require high levels of biosafety containment.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Tscherne
authors.forename: Balaji
authors.initials: B
authors.lastname: Manicassamy
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
mesh_terms: D000818:Animals; D002460:Cell Line; D004285:Dogs; D029043:Ebolavirus; D005434:Flow Cytometry / Q000379:methods; D005470:Fluorometry / Q000379:methods; D017930:Genes, Reporter; D029024:Marburgvirus / Q000235:genetics / Q000502:physiology; D008853:Microscopy / Q000379:methods; D009975:Orthomyxoviridae / Q000235:genetics / Q000502:physiology; D012328:RNA Viruses / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014763:Viral Matrix Proteins / Q000235:genetics*; D014773:Virology / Q000379:methods*; D022701:Virosomes; D053586:Virus Internalization; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: C488936:M1 protein, Influenza A virus; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D022701:Virosomes; D001616:beta-Galactosidase