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

In nature, synthesis of both minus- and plus-sense RNA strands of all the k nown double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other protei ns, the complex contains a putative polymerase possessing characteristic se quence motifs, However, none of the previous studies has shown template-dep endent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophag e phi 6 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 f ormed by newly synthesized, full-length minus-strands base paired with the plus: strand templates. P2-catalyzed replication is also shown to be very e ffective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.