Our recent studies have shown a link between Adenovirus-5 (Ad-5) and elevated lipids, which prompted the hypothesis that Ad-5 infection might augment hepatic and/or adipose tissue lipid metabolism. To test our hypothesis, mice were infected with Ad-5 and screened for changes in lipogenesis and plasma markers associated with the metabolic syndrome. We observed increased expression of sterol regulatory element binding protein 1 (SREBP-1) in infected liver tissues, but not in adipose tissues and this correlated with elevated plasma and hepatic triglyceride levels. Elevated expression of adiponectin was seen in Ad-5 infected adipose tissues and this correlated with phosphorylated AMPK in infected liver tissues. These data suggested that the AMPK pathway was activated in livers of Ad-5 infected mice. Indeed, we observed reduced expression of PEPCK, a downstream target of AMPK in livers of Ad-5 infected mice. As PEPCK is an enzyme essential for gluconeogenesis, we hypothesized that Ad-5 infection would reduce blood sugar. Indeed, infected mice exhibited a transient decline in plasma glucose. The increase in SREBP1 levels in Ad-5 infected hepatic tissues was evaluated by looking at Ad-5 infected HepG2 cells. Ad-5 is thought to mimic insulin’s actions in which the PI3K pathway is activated. We hypothesized that Ad5-induced SREBP-1 expression levels are mediated through the induction of PKC λ/ζ/ι, and not through Akt because it has been shown that PKC λ/ζ/ι mediates insulin-dependent lipogenesis. To test our hypothesis, HepG2 cells were infected with Ad-5 and screened for downstream targets of the PI3K pathway. Through western blot analyses, we observed increased levels of phosphorylated PKC λ/ζ/ι. These results prompted the use of PKC pan inhibitor to see whether Ad-5 induced SREBP-1 levels would be down regulated. Indeed, with the presence of the PKC pan inhibitor, SREBP-1 expression levels were reduced. Together, these studies suggest that Ad-5 induces changes in gene expression, glucose, and lipid metabolism; which prompts the hypothesis that this common respiratory pathogen may be associated with the Metabolic Syndrome, and this may preclude its use as a vector for gene therapy.

This study demonstrates the use of an adenovirus vector system to study the effect of a DNA repair gene on untransformed human fibroblasts. The bacteriophage T4 pyrimidine dimer DNA glycosylase (den V) gene has been inserted into the E3 region of human adenovirus type 5. The resulting recombinant virus Ad Den V was determined to be producing correctly initiated RNA from the RSV 3' LTR promoter used in the den V expression cartridge inserted into the virus. The effect of the den V gene product on human fibroblasts 'liras examined by assaying for the percent host cell reactivation (%HCR) of Vag production for UV irradiated Ad Den V in comparison to that for a control virus. It was shown that the %HCR was significantly greater for Ad Den V as compared to the control virus in xeroderma pigmentosum (XP) cells. UV survival of adenovirus in XP cells exhibited a two component nature. Introduction of the den V gene into XP group A cells increased the D0 value of the first component of the viral survival curve to a level similar to that of XPC cells, which showed no change in this component irrespective of the presence of the den V gene. It has been suggested that the den V gene is able to partially complement the deficiency in some XP cells because of its small size, allowing it to gain access to the DNA damage site where as the cellular repair enzyme complex can not. Since XPC cells are proficient in their alteration of DNA secondary structure prior to DNA excision repair, these results are consistant with the hypothesis that the first component of UV viral survival curves reflects the pathway involved in accessing the damaged sites. The manuscript of a paper has been included as an appendix. The work theorizes on the origin of mammalian immune system diversity and bacteriophage lambda, and their possible relationship to prokaryotic DNA repair genes. / Thesis / Master of Science (MS)