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Biochemical characterization of respiratory syncytial virus RNA dependent RNA polymerase complex

Balakrishnan, Anand and Hekmat-Nejad, Mohammad and Price, Edmund and Cantwell, John and Wartchow, Charles and Shaul, Jacob and Luu, Catherine and Spraggon, Glen and DiDonato, Michael and Vo, Michael (2020) Biochemical characterization of respiratory syncytial virus RNA dependent RNA polymerase complex. ACS infectious diseases, 6 (10). pp. 2800-2811. ISSN 2373-8227

Abstract

RNA dependent RNA polymerases (RdRP) from non-segmented 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 a 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 a non-radiometric assay for measuring RNA polymerization activity of RSV RdRP that is amenable to compound screening and profiling.

Item Type: Article
Keywords: RNA dependent RNA polymerases, Ribonucleoprotein complexes, RNA replication, mRNA transcription, viral polymerases, polymerase inhibitors.
Date Deposited: 24 Oct 2020 00:45
Last Modified: 24 Oct 2020 00:45
URI: https://oak.novartis.com/id/eprint/42311

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