Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex.

Abstract

RNA-dependent RNA polymerases (RdRPs) from nonsegmented negative strand (NNS) RNA viruses perform both mRNA transcription and genome replication, and these activities are regulated by their interactions with RNA and other accessory proteins within the ribonucleoprotein (RNP) complex. Detailed biochemical characterization of these enzymatic activities and their regulation is essential for understanding the life cycles of many pathogenic RNA viruses and for antiviral drug discovery. We developed biochemical and biophysical kinetic methods to study the RNA synthesis and RNA binding activities of respiratory syncytial virus (RSV) L/P RdRP. We determined that the intact L protein is essential for RdRP activity, and in truncated L protein constructs, RdRP activity is abrogated due to their deficiency in RNA template binding. These results are in agreement with the observation of an RNA template-binding tunnel at the interface of RdRP and capping domains in RSV and vesicular stomatitis virus (VSV) L protein cryo-EM structures. We also describe nonradiometric assays for measuring RNA binding and RNA polymerization activity of RSV RdRP, which are amenable to compound screening and profiling.