RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Funding Information: This research was funded by the Academy of Finland, grant number 331627 (to M.M.P.), 309896 (to E.J.V.), Jane and Aatos Erkko Foundation, grant number 170046 (to M.M.P.), and Sigrid Juselius Foundation (to M.M.P.).Riitta Tarkiainen is acknowledged for excellent technical assistance. The facilities and expertise of the HiLIFE Biocomplex unit at the University of Helsinki, a member of Instruct-ERIC Centre Finland, FINStruct, and Biocenter Finland are gratefully acknowledged. We also acknowledge the DNA Sequencing and Genomics facility of the University of Helsinki for sequencing. Funding Information: Funding: This research was funded by the Academy of Finland, grant number 331627 (to M.M.P.), 309896 (to E.J.V.), Jane and Aatos Erkko Foundation, grant number 170046 (to M.M.P.), and Sigrid Juselius Foundation (to M.M.P.). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.