NODAVIRUS RNA REPLICATION - MECHANISM AND HARNESSING TO VACCINIA VIRUS RECOMBINANTS

In order to harness RNA replication for the amplification of mRNAs exp ressed from recombinant vectors and vaccines, we constructed a VV reco mbinant that expressed the RNA replicase encoded in the larger genomic segment of the nodavirus FHV. When both termini of the VV-derived tra nscript were correct, the encoded enzyme replicated its own mRNA, and replication dominated the RNA synthetic capacity of the cell. The smal ler 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 requirement s had to be fulfilled for replication of the smaller FHV RNA segment. The first was the prior replication of the larger genomic segment, whi ch was interpreted as a mechanism to achieve sufficient replicase synt hesis before the onset of coat protein synthesis. The second was the p resence 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.