EBV-encoded dUTPase induces immune dysregulation: Implications for the pathophysiology of EBV-associated disease.

Epstein-Barr virus (EBV) encodes for several enzymes that are involved in viral DNA replication. There is evidence that some viral proteins, by themselves, can induce immune dysregulation that may contribute to the pathophysiology of the virus infection. In this study, we focused on the EBV-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and present the first evidence that the dUTPase is able to induce immune dysregulation in vitro as demonstrated by the inhibition of the replication of stimulated peripheral blood mononuclear cells (PBMCs) and the upregulation of several proinflammatory cytokines including TNF-alpha, IL-1beta, IL-8, IL-6, and IL-10 produced by unstimulated PBMCs treated with purified EBV-encoded dUTPase. Depletion of CD14-positive cells (monocytes) eliminated the cytokine profile induced by EBV dUTPase treatment. The data support the hypothesis that at least one protein of the EBV early antigen complex can induce immune dysregulation and may be involved in the pathophysiology of EBV-associated disease.

Stress-associated changes in the steady-state expression of latent Epstein-Barr virus: implications for chronic fatigue syndrome and cancer.

Antibodies to several Epstein-Barr virus (EBV)-encoded enzymes are observed in patients with different EBV-associated diseases. The reason for these antibody patterns and the role these proteins might play in the pathophysiology of disease, separate from their role in virus replication, is unknown. In this series of studies, we found that purified EBV deoxyuridine triphosphate nucleotidohydrolase (dUTPase) can inhibit the replication of human peripheral blood mononuclear cells in vitro and upregulate the production of TNF-alpha, IL-1beta, IL-6, IL-8, and IL-10. It also enhanced the ability of natural killer cells to lyse target cells. The EBV dUTPase also significantly inhibited the replication of mitogen-stimulated lymphocytes and the synthesis of IFN-gamma by cells isolated from lymph nodes and spleens obtained from mice inoculated with the protein. It also produced sickness behaviors known to be induced by some of the cytokines that were studied in the in vitro experiments. These symptoms include an increase in body temperature, a decrease in body mass and in physical activity. The data provide a new perspective on how an early nonstructural EBV-encoded protein can cause immune dysregulation and produce clinical symptoms observed in patients with chronic fatigue syndrome (CFS) separate from its role in virus replication and may serve as a new approach to help identify one of the etiological agents for CFS. The data also provide additional insight into the pathophysiology of EBV infection, inflammation, and cancer.

Epstein-Barr virus-encoded dUTPase modulates immune function and induces sickness behavior in mice.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis (IM). In addition, latent infections with EBV are associated with nasopharyngeal carcinoma (NPC) and Burkitt's Lymphoma (BL). Antibodies to several EBV-encoded early antigens (EA) are often observed in patients with NPC and BL, however, the role of EBV-encoded proteins in the etiology of these and other EBV-associated diseases is not completely understood. The EA complex encodes for at least six different viral enzymes including deoxyuridine triphosphate nucleotidohydrolase (dUTPase). dUTPase has recently been shown to modulate activation of human peripheral blood mononuclear cells in vitro (unpublished data). Therefore, these studies were designed to test whether dUTPase would modulate immune function in an in vivo model. Mice were injected with purified EBV dUTPase, and baseline immune function and sickness behaviors were measured. EBV dUTPase treatment inhibited replication of mitogen-stimulated lymphocytes obtained from treated mice. These lymphocytes were also less able to synthesize interferon-gamma after re-stimulation. In addition, treatment with dUTPase induced sickness behaviors. For example, as compared to control animals, dUTPase-treated animals lost body mass, had elevated body temperature, and displayed diminished locomotor activity. These data suggest that individual viral proteins may play a role in the pathophysiology of EBV associated disease.

Ca2+ transport in mitochondria from yeast expressing recombinant aequorin.

We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca(2+) transport in vivo and in vitro. When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl(2), the matrix free Ca(2+) concentration is 200 nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca(2+) and promptly increases the value to 1.2 microM. Elevated Ca(2+) concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca(2+) when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca(2+) release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502-40509]. When a respiratory substrate is not present, Ca(2+) enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca(2+) and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K(1/2) value for Ca(2+) that is near to 900 nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca(2+) in yeast mitochondria is a function of the matrix Ca(2+) content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca(2+) transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca(2+) in yeast.