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

• Published in: Viruses 2021-08, Vol.13 (9), p.1738
• Main Authors: Levanova, Alesia A, Vainio, Eeva J, Hantula, Jarkko, Poranen, Minna M
• Format: Article
• Language: English
• Subjects: Binding sites; Cloning; DNA-directed RNA polymerase; Double-stranded RNA; dsRNA virus; E coli; Enzymatic activity; Enzymes; Fungal Viruses - genetics; Fungal Viruses - metabolism; Genome, Viral; Genomes; Heterobasidion; Liquid chromatography; mycovirus; Nucleotidyltransferases; Plasmids; Polymerization; Proteins; Replicase; replicase activity; RNA polymerase; RNA viruses; RNA Viruses - enzymology; RNA Viruses - genetics; RNA, Double-Stranded; RNA, Viral; RNA-Dependent RNA Polymerase - genetics; RNA-Dependent RNA Polymerase - metabolism; RNA-directed RNA polymerase; semi-conservative transcription; Severe acute respiratory syndrome coronavirus 2; Sodium; TNTase activity; Transcription; Viral Proteins - genetics; Viral Proteins - metabolism; viral RdRp; Viral Replicase Complex Proteins - metabolism; Virus Replication; Viruses
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v13091738

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus within the family . 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 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 Mn 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.