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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.

During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly., Author Summary Viral polymerases replicate the genome of the virus, which is essential for the synthesis of the progeny. All double-stranded (dsRNA) viruses have virion-associated polymerases that catalyze RNA synthesis within an intact capsid. Picobirnavirus (PBV) is a small dsRNA virus, and it has been shown that the capsid of PBV possesses an unusual architecture suggesting an uncommon assembly pathway that it could potentially share with another group of small dsRNA viruses called partitiviruses. We have performed both structural and functional studies to look at how the PBV polymerase performs its function and how these molecules are placed within the capsid. The PBV RdRP structure was solved and revealed that the RdRP has an interesting loop structure in its interior. Additionally, activity assays showed that the RdRP possesses catalytic activity in the absence of other viral proteins. Removing the loop structure was found to change the way that the RdRP initiates RNA replication. Further experiments also showed that the RdRP did not interact with the viral capsid protein (CP) but had a strong affinity for a conserved terminal sequence of the PBV genome. This suggests that in PBV the RdRP may be encapsidated based upon both the RdRP and CP co-interacting with the viral genome.

The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains “thumb” and “fingers.” These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.