Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi 6

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Abstract
In nature, synthesis of both minus‐ and plus‐sense RNA strands of all the known double‐stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template‐dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double‐stranded RNA bacteriophage φ6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage‐specific, positive‐sense RNA substrates to produce double‐stranded RNA molecules, which are formed by newly synthesized, full‐length minus‐strands base paired with the plus‐strand templates. P2‐catalyzed replication is also shown to be very effective with a broad range of heterologous single‐stranded RNA templates. The importance and implications of these results are discussed.