Expression, purification and characterization of recombinant RNA-dependent RNA polymerase from Heterobasidion RNA virus 6

Abstract

RNA-dependent RNA polymerases (RdRps) are enzymes that catalyze nucleotide polymerization on RNA template in the presence of divalent metal ions. These enzymes are central for the life cycle of viruses with RNA genomes. A double-stranded RNA (dsRNA) mycovirus Heterobasidion RNA virus 6 (HetRV6) commonly infect forest pathogenic fungi from the species complex Heterobasidion annosum sensu lato. In general, the infection caused by HetRV6 is cryptic, but sometimes have negative or mutualistic impact on its host. The understanding of the HetRV6 life cycle, including RNA synthesis, can shed light on the biology of HetRV6 virus and explain its divergent impacts on the host. Furthermore, a recombinant HetRV6 RdRp can potentially be harnessed to produce dsRNA molecules applicable for silencing of the target genes in plants, fungi, insects and mammals via RNA interference (RNAi).

The aim of the current study was to express, purify and characterize the in vitro enzymatic activities of RdRp from the HetRV6 virus. To this end, a complementary DNA sequence of the RdRp gene was cloned into Escherichia coli expression vector, from which the recombinant polymerase was expressed and purified using affinity, ion-exchange and size-exclusion chromatography. The purified HetRV6 RdRp was a soluble and active in vitro enzyme, which possesses polymerase and terminal nucleotidyl transferase (TNTase) activities in the absence of any primers or accessory viral or host proteins. The impact of the buffer composition, pH, temperature, divalent cations and nucleoside triphosphate concentrations was studied and the optimal reaction conditions were identified. The polymerase is fully dependent on Mn2+ ions and does not produce dsRNA in its absence, while Mg2+ ions at concentration 1‒5 mM enhance the polymerization activity. HetRV6 polymerase is active on heterologous templates. However, the requirements for successful initiation and elongation should be still studied in more detail to fully understand how the enzyme can be used for efficient dsRNA synthesis for biotechnological applications. The discovered TNTase activity can potentially be used for in vitro RNA labeling.

The completed work is a useful starting point to further explore the properties of the HetRV6 RdRp in terms of possible RNA modifying tools, its structural organization and role in infection and host-microbe interactions.