Minigenome System to Study Respiratory Syncytial Virus Gene Expression.

Minigenome assays have been essential tools in the understanding of viral transcription and RNA replication for respiratory syncytial virus (RSV). Here, we describe the RSV minigenome assay for determining transcription by the viral polymerase in the absence of infection. We detail three different methods of detecting viral RNA synthesis: a firefly luciferase assay for rapid and sensitive measurement of RSV polymerase activity; a dual luciferase assay for assessing contributions of cis-acting signals for RSV transcription; and a real-time quantitative PCR method for determination of specific effects on the transcription of individual viral genes and the polar transcription gradient of RSV.