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An inducible, mutant virus, designated vvtetO:I7L/G1L, was used to study the morphogenic proteolysis step of the vaccinia virus life cycle. The vvtetO:I7L/G1L controlled the expression of two genes, I7L, a cysteine proteinase, and G1L, a putative metalloproteinase. These proteins are involved in the maturation of viral core proteins, p4a, p4b, and p25K, to form infectious virions. DNA extraction and genomic sequencing verified the correct insertion of the tetracycline operators. The multiplicity of infection (MOI) was optimized, and a MOI of 0.5 was best, with a 99.25% reduction in viral plaque formation compared to the wild type vaccinia virus. A growth curve over 12 hours was done and the vvtetO:I7L/G1L in the “on” state closely followed the growth kinetics of the wild type vaccinia virus and the vvtetO:I7L/G1L in the “off” state had significantly lower viral titers throughout the last 6 hours of the cycle. Viral core protein processing in the “on” and “off” states, and in rescue experiments with transfected plasmids containing the I7L or G1L genes, was examined by immunoblot assay. Protein processing was difficult to effectively visualize, with the best processing occurring at a MOI of 0.2, using Fugene as a transfection reagent. |
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