This proposal is for continued funding of a program with the long-term objective of elucidating molecular properties and modes of action of parasite regulatory mediators used in regulation of host gene expression. The goal of the proposal is to identify molecular parameters necessary for activity of a cloned venom protein from Chelonus near curvimaculatus that is necessary for survival of the parasite in host Trichoplusia ni. The interrelated specific aims toward this goal are: I. Identify molecular determinants of a 33 kDa venom protein necessary for activity for parasite survival. II. Determine the steps in the biosynthesis and processing of the 33 kDa protein that confer upon it these molecular determinants of a mature, active form. III.Elucidate the molecular/cellular identity of the target that interacts with these molecular determinants. The experimental design and methods to be used in accomplishing these specific aims include: I. Comparison of conserved sequences among proteins homologous to the 33 kDa protein from related species, and assay of the functional necessity of these residues by domain swapping, deletion and site mutagenesis. The in vivo assay involves rescue of survival of endoparasites in host embryos into which the mutant protein, or vector constructs expressing the mutant protein, are injected. The in vitro assay measures the binding of the test protein to a target found in host tissue. II. Identification of the sites of posttranslational modification which are potentially necessary for activity, using epitope mapping and fragment analysis of in vitro and in vivo synthesized/radiolabelled protein. III. Purification and biochemical characterization of the target now detected as binding activity in the host embryo, and cellular localization of this target through immunohistochemical methods. The results will be of significance for fundamental research since they will constitute the first mutational analysis of any wasp venom protein. The current gap in the field of molecular analysis of wasp venoms is staggering in view of the thousands of parasitic wasp species which each have a unique venom, evolutionarily-tailored to the biochemical template of their host(s). Arthropod venoms are of great medical significance due to their allergenic, neurological and other clinical effects. The venom protein to be studied here has cross-immunogenicity with components of clinically important venom of higher Hymenoptera. Since the effects which wasps induce in insect hosts affect immune response mechanisms conserved among several orders of medically important insects, these studies will provide us with a means of direct intervention in a life process critical to the survival of insect disease vectors.
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