Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli

All positive-strand RNA viruses encode a RNA-dependent RNA polymerase which in most cases has been only identified on the basis of its sequence conser vation. Catalytic activity has been experimentally demonstrated in only a h andful of these viral proteins, including that from Rabbit hemorrhagic dise ase virus. Studies from our laboratory have reported that RHDV RNA polymera se produced in Escherichia coil was enzymatically active showing poly(A)-de pendent poly(U) polymerase as well as RNA polymerase activity on heteropoly meric substrates. In this work, we have investigated the in vitro activity of the recombinant 3D(pol) from RHDV, including ion requirements, resistanc e to inhibitors, substrate specificity as well as data on the initiation me chanism of the template-linked products derived from heteropolymeric RNA su bstrates. Our study demonstrates that in an in vitro reaction recombinant R HDV RNA polymerase generated the minus strand of the heteropolymeric RNA su bstrates by a "copy-back" mechanism that initiated at the template 3'-termi nal OH.