Figure 2

Rapid assay for studying WNV assembly and release. (A) Schematic diagram delineating the steps for the rapid Ren-luc based virus release assay and comparing it to the classical radioimmunoprecipitation assay. 293T cells were transfected with WNV-CPrME along with the Ren/Rep plasmids at a ratio of 1:1 or with the pUC vector as control. (B) For radioimmunoprecipitation based assay, cells were metabolically labeled with [³⁵S]Met-Cyst protein labeling mix (PerkinElmer) in RPMI 1640 medium supplemented with 10% FBS but devoid of Met and Cys 24 h post transfection. Following ultracentrifugation, cell and virus lysates were immunoprecipitated using anti-WNV serum, run on an SDS PAGE gel followed by fluorography. Virus release was calculated as ratio of virion associated versus cell+virion associated E protein. (C) For ren-luc based virus release assay, culture supernatants were harvested 24 h post transfection and cells lysed and read for ren-luc activity (cell associated) using the Dual Glo luciferase assay substrate (Promega). Equal volume of the harvested supernatants were then used to infect 293T cells, cells lysed and read for luciferase activity (virion-associated) 24 h post infection. Virus release was calculated as ratio of virion associated versus cell+virion associated ren-luc activity.