The respiratory syncytial virus polymerase can perform RNA synthesis with modified primers and nucleotide analogs

Respiratory syncytial virus (RSV) is significant for public health, capable of causing respiratory tract disease in infants, the elderly and the immunocompromised. The RSV polymerase is an attractive target for antiviral drug development, but as yet, there is no high throughput assay for analyzing RSV polymerase activity, specifically. In this study, using a primer elongation assay as a basis, we analyzed the tolerance of the RSV polymerase for modifications at the 5' end of the primer, and nucleotide analogs. The RSV polymerase was found to accept primers containing 5' biotin or digoxygenin modifications, and nucleotide analogs that are reactive or fluorescent, including 5-ethynyl UTP, 8-azido ATP, 2-amino PTP, and thieno-GTP. These findings provide a menu of options for developing non-isotopic high throughput assays for RSV polymerase RNA synthesis activity, and yield insight regarding the molecular biology of the polymerase complex.