Biochemical And Genetic Analyses Of Vaccinia Virus Temperature Sensitive Mutants Defective In Envelope Self-assembly

To elucidate the genetic endowment of vaccinia virus, it is essential to identify the maximum number of biological functions, inter-relate these functions to specific polypeptides and finally to locate them to precise loci on the viral genome. In such studies mutants are vital in illustrating the fundamental biological phenomenon. Recent developments in microanalytical technology have permitted systematic biochemical and genetic analyses of a group of thermo-labile mutants, derived from IHD-W strain of vaccinia, defective in envelope self-assembly.;The polypeptides of vaccinia virus were separated and analyzed by two-dimensional gel electrophoresis. Following labeling with {lcub}(\u2735)S{rcub}-methionine, {lcub}(\u2733)p{rcub}-phosphate, or {lcub}(\u273)H{rcub}-glucosamine, highly purified virions were dissociated and subjected to electrophoresis using either isoelectric focusing or non-equilibrium pH gradients in the first dimension and SDS-polyacrylamide slab gel in the second dimension. By this means at least 111 polypeptides, about 50% of which were basic proteins, were resolved in fluorograms. Authenticity of various single spots was established.;Another ts mutant (ts 6757) overproduces immature viral envelopes (IE) and thus provided an opportunity to isolate and partially characterize the IE. These IE, like the envelopes of normal immature virions, possess an external layer of spicules. Experimental evidence was obtained by several approaches suggesting that an early 65K polypeptide (p65E) constitutes the spicules.;Among five assembly-defective mutants relegated to group E which mimic closely in morphogenesis the effects of the antibiotic rifampicin, four have previously been shown to be defective in cleavage of one or more polypeptides. The one evincing no cleavage defect (ts 9251) was found to possess a novel EcoRI restriction site. Analysis of spontaneous revertants derived from ts 9251, employing the restriction enzyme and two-dimensional electrophoresis, provided compelling evidence that the ts lesion resides at a single base pair constituting the EcoRI restriction site in the gene which codes for a 37K polypeptide.;A recombination map including 5 group E mutations, a DNA-minus mutation and the locus for rifampicin-resistance, was derived from the data obtained by two and three factor crosses. The group E mutations, though phenotypically identical, were found widely spread on the genome. The morphogenetic basis of the envelope abberation, like polypeptide composition of the virion, therefore appears to be even more complex than it was thought before