A luciferase-based budding assay for Ebola virus.
The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.
McCarthy
Sarah E
SE
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104, USA.
Licata
Jillian M
JM
Harty
Ronald N
RN
D002460:Cell Line; D029043:Ebolavirus / Q000254:growth & development* / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D009698:Nucleoproteins / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism; D019065:Virus Assembly
D009698:Nucleoproteins; D014758:Viral Core Proteins; C084819:nucleoprotein VP40, Ebola virus; D049409:Luciferases, Firefly