Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli.

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Abstract
The poliovirus genome is replicated by a virus-encoded RNA-dependent RNA polymerase (RNA polymerase). The RNA polymerase is first synthesized as a larger precursor polypeptide, which is subsequently processed by a viral proteinase, 3Cpro, to give the mature polymerase molecule, 3Dpol. To further characterize the poliovirus RNA polymerase, we have constructed plasmids that expressed this protein in Escherichia coli. The plasmids consisted of fusions between the E. coli DNA encoding the first 13 amino acids of the trp operon leader protein and viral genes encoding the 3Cpro and 3Dpol polypeptides. E. coli harboring such plasmids gave significant, inducible levels of enzymatically active RNA polymerase as determined by the poly(A).cntdot.oligo(U) poly(U) polymerase assay. The purified RNA polymerase activity from E. coli corresponded to a protein with the approximate molecular weight of the mature 3Dpol protein. The availability of a recombinant, enzymatically active poliovirus RNA polymerase provides a system in which we can precisely delineate the role this enzyme plays in the regulation of poliovirus replication.