title: Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors.
abstract: Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by beta-galactosidase alpha-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.
authors.affiliation: Department of Microbiology, University of Pennsylvania, 301 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA.
authors.forename: Claire Marie
authors.initials: CM
authors.lastname: Filone
authors.forename: Mark
authors.initials: M
authors.lastname: Heise
authors.forename: Robert W
authors.initials: RW
authors.lastname: Doms
authors.forename: Andrea
authors.initials: A
authors.lastname: Bertolotti-Ciarlet
mesh_terms: D000524:Alphavirus / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008561:Membrane Fusion; D012093:Replicon; D012295:Rift Valley Fever / Q000821:virology; D012296:Rift Valley fever virus / Q000235:genetics / Q000472:pathogenicity*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D014764:Viral Proteins