RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro
Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus <i>Orthocurvulavirus</i> within the family <i>Orthocurvulaviridae</i>. The purpose of the study was to determine the biochemical requirements for RNA synt...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-08-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/13/9/1738 |
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| author | Alesia A. Levanova Eeva J. Vainio Jarkko Hantula Minna M. Poranen |
| author_facet | Alesia A. Levanova Eeva J. Vainio Jarkko Hantula Minna M. Poranen |
| author_sort | Alesia A. Levanova |
| collection | DOAJ |
| description | Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus <i>Orthocurvulavirus</i> within the family <i>Orthocurvulaviridae</i>. 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 <i>Escherichia coli</i> 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<sup>2+</sup> 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. |
| first_indexed | 2024-03-10T07:08:30Z |
| format | Article |
| id | doaj.art-4425c84f552e46818017d654c488ad9b |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T07:08:30Z |
| publishDate | 2021-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-4425c84f552e46818017d654c488ad9b2023-11-22T15:37:15ZengMDPI AGViruses1999-49152021-08-01139173810.3390/v13091738RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In VitroAlesia A. Levanova0Eeva J. Vainio1Jarkko Hantula2Minna M. Poranen3Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, 00790 Helsinki, FinlandNatural Resources Institute Finland, 00790 Helsinki, FinlandNatural Resources Institute Finland, 00790 Helsinki, FinlandMolecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, 00790 Helsinki, FinlandHeterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus <i>Orthocurvulavirus</i> within the family <i>Orthocurvulaviridae</i>. 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 <i>Escherichia coli</i> 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<sup>2+</sup> 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.https://www.mdpi.com/1999-4915/13/9/1738viral RdRpmycovirusdsRNA virusTNTase activityreplicase activitysemi-conservative transcription |
| spellingShingle | Alesia A. Levanova Eeva J. Vainio Jarkko Hantula Minna M. Poranen RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro Viruses viral RdRp mycovirus dsRNA virus TNTase activity replicase activity semi-conservative transcription |
| title | RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro |
| title_full | RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro |
| title_fullStr | RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro |
| title_full_unstemmed | RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro |
| title_short | RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro |
| title_sort | rna dependent rna polymerase from heterobasidion rna virus 6 is an active replicase in vitro |
| topic | viral RdRp mycovirus dsRNA virus TNTase activity replicase activity semi-conservative transcription |
| url | https://www.mdpi.com/1999-4915/13/9/1738 |
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