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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA039691-06
Application #
3179021
Study Section
Virology Study Section (VR)
Project Start
1985-12-01
Project End
1995-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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Smith, C C; Luo, J H; Aurelian, L (1996) The protein kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) fused to the extracellular domain of the epidermal growth factor receptor is ligand-inducible. Virology 217:425-34
Hunter, J C; Smith, C C; Bose, D et al. (1995) Intracellular internalization and signaling pathways triggered by the large subunit of HSV-2 ribonucleotide reductase (ICP10). Virology 210:345-60
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Wymer, J P; Aprhys, C M; Chung, T D et al. (1992) Immediate early and functional AP-1 cis-response elements are involved in the transcriptional regulation of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). Virus Res 23:253-70
Luo, J H; Aurelian, L (1992) The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10). J Biol Chem 267:9645-53
Chung, T D; Luo, J H; Wymer, J P et al. (1991) Leucine repeats in the large subunit of herpes simplex virus type 2 ribonucleotide reductase (RR;ICP10) are involved in RR activity and subunit complex formation. J Gen Virol 72 ( Pt 5):1139-44
Smith, C C; Wymer, J P; Luo, J et al. (1991) Genomic sequences homologous to the protein kinase region of the bifunctional herpes simplex virus type 2 protein ICP10. Virus Genes 5:215-26

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