HSV-2 leads to neoplastic transformation of normal diploid cells. However, the role that the virus plays in carcinogenesis is unclear and doubt has been voiced about the very existence of oncogenes. The proposed studies are based on the following recent findings from our laboratory: (i) the large subunit of HSV-2 ribonucleotide reductase (ICP10) has an associated protein kinase (PK) activity that appears to be coded by the minimal transforming DNA fragment (mtrIII) and is also localized in the plasma membrane, (ii) the PK minigene has properties similar to those of growth factor receptors (GFRs) and (iii) cell DNA contains mtrIII-homologous sequences. The working hypothesis is that HSV-2 mediated hamster cell transformation involves this PK function which by analogy to retroviral oncogenes has a cellular counterpart and originated from a truncated, as yet unidentified GFR or a PK in the GF pathways. The proposed studies seek to define the role of the PK function in transformation and assess the contribution of specific catalytic domains and of the transmembrane helical region. Deletion mutants that respectively lack the PK or RR domains were prepared since the last review. Others, in the non-catalytic region N-terminal to the PK (includes the transmembrane helix) will be prepared. Specific PK catalytic domains considered of functional significance for other PKs, and the transmembrane helical region will be mutated by site-directed mutagenesis and expressed in the constitutive expression vector. All mutants will be studied for: (i) ICP10 synthesis, (ii) PK activity, (iii) subcellular localization and (iv) transforming ability. Transformed lines will be studied for DNA maintenance and gene expression using Sl nuclease assay. The potential mechanism of transformation will be studied. Hybrid plasmids consisting of the PK minigene and the extracellular ligand binding domain of the epidermal growth factor receptor (EGFR) will be constructed and lines will be established. They will be studied for synthesis processing and cell surface localization of the hybrid protein. Ligand-mediated activation of the PK minigene will be determined and correlated with phosphatidylinositol turnover, nuclear oncogene (c-myc, fos, jun) activation (increased expression) and transforming potential. Characterization of this novel ICP10-PK and definition of its involvement in the neoplastic process should help elucidate problems relating to the mechanism of HSV-2 induced neoplastic transformation.
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