Cloning and expression of the Nodamura virus RNA-dependent RNA polymerase

Our laboratory studies the mechanism of viral RNA replication, using Nodamura virus (NoV) as a model, due to its genetic simplicity, tremendous levels of RNA amplification, and ability to replicate in a wide variety of host cells. NoV contains a bipartite positive-strand RNA genome. The larger segment (RNA1) encodes the RNA-dependent RNA polymerase (RdRp) that catalyzes replication of the viral genome, while RNA2, encodes the viral capsid protein precursor, which is dispensable for RdRp function. We are developing an in vitro replication system to study the specific mechanism by which the viral RdRp recognizes its RNA template. As a first step, we will express recombinant RdRp with an N-terminal 6-HIS tag in E. coli from an IPTG-inducible T7 promoter. We used PCR to introduce an NcoI site, a start codon, and a 6-HIS tag to the 5’-end of the RdRp ORF and to introduce a 3’-terminal HindIII site after the RdRp stop codon. The PCR products were subcloned into pGEM-T-Easy vectors and confirmed by DNA sequencing. The 5’ and 3’ terminal fragments isolated from these subclones will be ligated to an internal fragment containing the majority of the RdRp ORF and introduced into the pET45b(+) vector. The vector will be used to transform competent T7-EXPRESS E. coli cells and pilot studies will be performed to determine the optimal expression conditions. The expressed protein will be purified by affinity chromatography and assayed for RdRp activity. The purified protein will allow us to determine the replication initiation mechanism used by this enzyme.