Molecular genetics of vaccinia virus: demonstration of marker rescue

Abstract

Two genomic variants of vaccinia virus isolated from serially propagated stocks were used to demonstrate marker rescue. The smaller (S variant) virus contains a 6.3 megadalton (MDal) deletion of unique DNA sequences present in the 123-MDal larger (L variant) virus. The deletion was mapped at 6.85 MDal from the left terminus of the genome, just outside of the inverted terminal repetition. Rescue of the unique deleted DNA sequences by infectious S variant virus was obtained in CV-1 cells by using the calcium orthophosphate precipitation technique of intact or restriction endonuclease-treated L-variant DNA. Restriction fragments that overlapped the deletion allowed marker rescue, but restriction of the L-variant DNA within the unique deleted sequences gave negative results. Restriction endonuclease analysis of the DNA obtained from twice-plaque-purified recombinant virus derived from the rescue of overlap donor fragments gave a restriction pattern identical to that of L-variant virus, indicating that the donor DNA was inserted into the rescuing virus by double recombination. No amplification of the unique sequences was observed from intact L-variant DNA in the absence of infectious S-variant virus, suggesting that deproteinized vaccinia DNA is noninfectious and that the donor DNA was neither integrated into the host DNA nor present as an episomal structure. By using 1 microgram of intact L-variant DNA per CV-1 monolayer in a 6-cm Petri dish, approximately 1--5% of the plaques contained the L-variant genotype, and the dose--response curve was essentially linear from 0.1 to 2 microgram of DNA.