The den V gene from bacteriophage T4 codes for a small, pyrimidine dimer specific, endonuclease. Recent studies have shown that transfection of the gene into DNA excision repair deficient, Xeroderma Pigmentosum cells, can partially restore the excision repair ability of the cells and results in an increased resistance to UV light. In this study the den V gene has been inserted into Herpes Simplex Virus type 1 (HSV 1) in order to determine if HSV 1 can be used as a suitable vector for studying DNA repair genes. A 1.9 kb cartridge containing the den V gene, the 3' LTR of Rous Sarcoma Virus as the promoter, and the SV40 polyadenylation signals was inserted as the thymidine kinase locus of the virus. Properly initiated transcription form the construct, HDV 1, was verified by primer extension analysis. The Host cell reactivation of this virus and several other strains of HSV 1 were examined in normal and Xeroderma Pigmentosum cells. The results from these experiments suggest that both the viral DNA polymerase and thymidine kinase genes play important roles for the survival of UV irradiated HSV 1. / Thesis / Master of Science (MS)

Many viral transcriptional activators have been shown to activate genes of heterologous systems. To assess the ability of the herpes simplex virus ICP4 trans-activating protein to complement an adenovirus mutant lacking its own trans-activator, the E1a protein, I constructed an adenovirus type 5 vector containing a temperature sensitive ICP4 gene, under control of its own promoter, within the E1 region of the genome. The recombinant virus expresses ICP4 in human cells which are permissive (293) or nonpermissive (KB and R970-5) for E1a⁻ viral replication, and at levels which approximate those obtained in herpes simplex infection. The adenovirus encoded protein is functional in that it complements an ICP4 deletion mutant of herpes simplex virus, however it is incapable of complementing adenovirus E1a⁻ mutants for viral growth or DNA replication. At the level of activation of gene expression, ICP4 stimulates the expression of the adenovirus E2a gene but not that of other early genes. My results indicate that ICP4 does not possess all of the functions of the E1a proteins and, furthermore, that adenovirus early genes differ in their susceptibility to heterologous trans-activators. / Thesis / Master of Science (MS)

Herpes simplex virus (HSV) infections are common in all human populations and for most people they represent relatively mild lifelong infections. To facilitate the persistent infection of hosts, HSV has evolved immune evasion strategies which suppress various aspects of the immune response including the actions of complement and antibodies. Previously in our laboratory, an HSV immediate early protein called ICP47 was shown to inhibit the MHC class I antigen presentation pathway and thereby block recognition of virus infected cells by CD8+ cytotoxic T lymphocytes (CTL). This thesis explores the potential cellular targets of ICP47. Using immunoprecipitation I found ICP47 associates with the transporter associated with antigen presentation (TAP). By blocking the transport of peptide antigens into the endoplasmic reticulum, MHC class I molecules become unstable and are subsequently degraded before displaying HSV antigens on the cell surface. Thus, CTL destruction of cells infected with HSV is blocked. In addition, an interaction between an ICP47 bacterial fusion protein, called GSTICP47-1 and a cellular protein, calcyclin, was examined. The functions of calcyclin are largely unknown. However, based on its association with ICP47, it was possible that calcyclin might play a role in the class I pathway -perhaps as the peptide shuttle. Nevertheless, the results of several experiments were consistent with the notion that calcyclin and ICP47 may not interact in vivo and that calcyclin may not play a role in the MHC class I antigen presentation pathway. / Thesis / Master of Science (MS)

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