MOLECULAR BIOLOGY OF HERPES SIMPLEX VIRUSES

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Herpes simplex viruses (HSV), members of a family of oncogenic viruses, cause moderate to severe disease, particularly in neonates and in infection-or drug induced immunologically deficient individuals including cancer patients, and are associated as co-factors with cancer of the cervix. This application is a competing renewal of an OIG which consolidated four long standing projects centering on the molecular biology of these viruses. Current research objectives are as follows: Project 1 concerns lambda134.5, a recently discovered viral gene whose product is essential from viral replication in CNS. The lambda134.5 minus virus induces specifically in neuronal cells programmed cell death which precludes viral multiplication. Lambda134.5 in transformed premitotic neuroblastoma cells precludes apoptosis and complements viruses lacking this gene and also induces cells to differentiate. A homolog, MyD116, sharing with lambda134.5 only the 63 carboxyl terminal amino acids was isolated by others form myelogenous leukemic cells induced to differentiate by interleukin 6. Viruses carrying MyD116 in place lambda134.5 replicate normally and do not induce apoptosis in neuronal cell. Our objectives are to determine (i) the structural components of lambda134.5 and MYD116 proteins, (ii) where and lambda134.5 and MyD116 proteins function and in particular to test for ligand and effector functions of the amino terminal and carboxyl-terminal domains of the proteins, (iii) the receptors of lambda134.5 and Myd116 putative effector and ligand domains and (iv) the relationship of anti-apoptosis and differentiation functions of lambda134.5 and MyD116. In addition it was noted that while injection of > 100,000 LD50 doses of lambda134.5 minus virus is non destructive to mice, the virus is able to destroy lethal doses of malignant glioma cells implanted previously into the CNS. The intent is to determine whether avirulent viruses specific for CNS cancer cells can be designed. Project 2 centers on several regulatory proteins which are nucleotidylated and/or phosphorylated and on the recently identified substrates of two viral protein kinases (UL13 and US3). The substrate of US3 is the essential membrane protein UL34. In cells infected with US3 minus virus or with a virus in which the phosphorylation site in UL34 had been mutagenized, the UL34 protein becomes associated in immune precipitation tests with four phosphoprotein. The substrate of UL13 is the product of the regulatory gene alpha22. The objectives are to determine (i) the function and mechanism of guanylation and adenylation of viral proteins, (ii) the function of post-translation modification of the UL13 protein, (iii) the nature of the phosphoprotein quartet associated with the Ul34 protein and (iv) the role of phosphorylation of alpha22 in gene expression. The objective of Project 3 is to resolve the functions of the 5' untranscribed and transcribed noncoding domains of lambda (late) viral genes and in particular to determine the role of potential intrastrand base pairing on the dependence of late genes on viral DNA synthesis. The 3 projects rely, non exclusively, on the genetic engineering of viruses carrying specifically designed chimeric genes and on proteins carrying specific modifications.