Nodavirus RNA replication: mechanism and harnessing to vaccinia virus recombinants

In order to harness RNA replication for the amplification of mRNAs expressed from recombinant vectors and vaccines, we constructed a VV recombinant that expressed the RNA replicase encoded in the larger genomic segment of the nodavirus FHV. When both termini of the VV-derived transcript were correct, the encoded enzyme replicated its own mRNA, and replication dominated the RNA synthetic capacity of the cell. The smaller genomic segment of FHV could also be replicated by the enzyme when supplied in trans, either by coinfection with another VV recombinant or by transfection of an appropriate plasmid. However, two requirements had to be fulfilled for replication of the smaller FHV RNA segment. The first was the prior replication of the larger genomic segment, which was interpreted as a mechanism to achieve sufficient replicase synthesis before the onset of coat protein synthesis. The second was the presence in the smaller genomic RNA of an internal region between about nucleotides 525-620. Work is in progress to elucidate the reasons for these requirements for RNA 2 replication.