Vaccinia virus growth induces the formation of plaques two days after infection, providing a rapid in vivo test for the ?development of the virus. Since inhibiting the activity of an essential protein encoded by vaccinia virus inhibits plaque formation, the effects of alterations of that essential protein can be rapidly tested by this assay. In particular, substituted amino acids in conserved regions of the vaccinia virus (vv) DNA polymerase (dp) should inhibit plaque formation, except in situations where the substitution preserves the protein structure that allows enzyme function. The nature of that structure can then be inferred from the type of substituted residue. In this project allowable substitutions in the conserved region were selected by linkage to an aphidicolin resistance mutation. To test the effects of allowed substitutions on the function of the vvdp, in vitro measurements of nucleotide binding and polymerization are necessary. Overexpression of the enzyme would facilitate performing enzyme assays. Attempts by myself and others to express the vvdp in bacteria were not successful. However, it was found that polymerase expression could be enhanced in the normal tissue culture system used to grow the virus. My approach was to overexpress the enzyme by inserting its gene into the DNA of an engineered vaccinia virus, vT7lacOI, whose genome contains the T7 RNA polymerase (T7rp) gene. Such viruses that contain an ectopic copy of the dp gene in the hemagglutinin region of the vv genome are designated vdp2 . When cells infected with vdp2 are induced by a chemical agent, they are activated to overexpress the ectopic gene. Electrophoretograms of radiolabeled proteins obtained from induced infected cells showed an intense band at the position expected for vvdp, while uninduced cells showed only a faint band. The intensity of the induced band was greater when the cells were infected at a low multiplicity. Expanding the vdp2 stock to infect larger numbers of cells required several steps since about 35 viruses were produced per infected cell. Also, since the size of the plaques formed by the recombinant virus was more restricted than that of the wild type (wt) virus due to syncytia formation, the spread of vdp2 to neighboring cells was less than that found for the wt virus. Within these biological limits, conditions that maximize the production of the dp will be determined.
