Belov, G.A., Altan-Bonnet, N., Kovtunovych, G., Jackson, C.L., Lippincott-Schwartz, J. and Ehrenfeld, E.
Notes: In this study, the Renilla luciferase gene from the phRL-CMV Vector was incorporated into polioviral RNA in place of a structural protein-coding region. Renilla luciferase activity was then monitored as a convenient indicator of viral replication. Specifically, these authors tested whether the guanine nucleotide exchange factors GBF1 and BIG2 could rescue brefeldin A (BFA)-induced inhibition of polioviral replication in HeLa cells. Cells were transfected with plasmids encoding either GBF1 or BIG2, together with the firefly-luciferase-encoding pGL4.13 Vector, which was used as a control to monitor transfection efficiency. Eighteen hours post-transfection, the cells were transfected with polioviral replicon RNA containing the Renilla luciferase gene. One hour later, normal growth medium containing EnduRen® Live Cell Substrate and either BFA or solvent alone, was added and Renilla luciferase activity was monitored at hourly intervals for 7 hours. Cells were then lysed and firefly luciferase activity was measured to assess transfection efficiency using the Dual-Glo® Luciferase Assay System System. In the presence of BFA, GBF1 was able to partially rescue viral replication. In the presence of BIG2 and BFA, no viral replication occurred. (3562)