Pathogenesis Of Diseases Induced By Non-oncogenic Retrov

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Abstract

The overall goal of this project is to understand how retroviruses adversely affect the central nervous system. We are utilizing the mouse as an animal model and are currently focusing our attention on a virus, FrCasE, which causes a non-inflammatory spongiform encephalomyelopathy. Transcriptional profiling, using high density microarray technology, revealed that Endoplasmic Reticulum (ER) Stress responses may be involved in the pathogenesis of this disease. In vitro studies indicated that the ER stress was induced by the instability and misfolding of the viral envelope protein within the ER, leading to a prototypic Unfolded Protein Response. The Unfolded Protein Response induced by this virus is associated with the upregulation of ER chaperones, the stable binding of the one of these chaperones proteins (BiP) to the viral envelope protein and the shunting of this viral protein to the ubiquitin-proteasome degradative pathway. In addition, we have observed in cells infected by FrCasE the intracellular accumulation of reactive oxygen species and evidence suggesting the inhibition of the ubiquitin-proteasome system. Interestingly, another virus, F43, which is neuroinvasive but avirulent, failed to induce any of these responses. F43 differs from FrCasE only in its envelope protein. Previous work from us and others have demonstrated that the neurotoxicity of FrCasE and related neuropathogenic viruses is determined by the sequence of the viral envelope protein. Thus, the viral envelope protein specifies neurovirulence in vivo and also mediates the activation of cellular stress responses demonstrable in vivo and in vitro. It is for these reasons that we now suspect that the brain disease induced by FrCasE and related murine retroviruses represents a virus-induced protein folding disease. During the past year we have explored in more detail the molecular interactions associated with the ER stress responses induced by FrCasE. These include early events immediately downstream of the ER resident molecules that sense the accumulation of misfolded protein (ATF-6, PERK and IRE1). Virus infection induced the splicing of the mRNA of X-Box Protein1 by IRE1, a process that generates a powerful transcription factor driving the effectors of the Unfolded Protein Response. In addition, PERK mediated phosphorylation of the translation initiation factor Eif2alpha has been detected in virus-infected cells, and this is linked to translational suppression and the induction of the transcription factor CHOP. CHOP is a proapoptotic transcription factor that is induced when ER stress is not relieved by the elements of the Unfolded Protein Response. In vivo studies have identified the cells in the brainstem of infected mice that express CHOP. These cells also express the protein Olig2, a basic-helix-loop-helix transcription factor involved in lineage commitment of oligodendrocytes. These cells exhibit a form of cytopathology, characterized by cytoplasmic dissolution accompanied by ultrastructural signs of autophagy. Curiously, although CHOP expression has been linked to apoptosis, biochemical studies indicate the cytopathology of the CHOP+ cells is not associated with the classical apoptosis pathways. Curiously, FrCasE infects a variety of cells in the central nervous system, including microvascular endothelial cells, perivascular and parenchymal microglial cells and certain populations of neurons. However, it is only the infected oligodendrocytes that exhibit cytotoxicity, suggesting that these cells are particularly sensitive to the ER stress induced by this virus. Studies carried out in collaboration with Dr. David Ron of the Skirball Institute on the role of CHOP in the neuropathogenesis of FrCasE have been completed. Mice in which the CHOP gene was disrupted were found to exhibit a small delay in onset of clinical signs of neurologic disease. This delay was repeatable and statistically significant, but the absence of the CHOP gene did not appear to protect the mice from the spongiform neuropathology induced by this virus. Thus, we suspect that the upregulation of CHOP likely represents a late event in the cellular degeneration caused by this virus. In collaboration with Dr. Chesebro, LPVD, RML, we have extended these studies to the TSE diseases. Scrapie-infected mice also exhibit the upregulation of CHOP protein in the brain in mice with advanced disease, as well as at earlier times during the course of infection. The lineage of the CHOP+ cells has yet to be determined, but these cells appear to be localized in the brain to sites with the highest accumulation of PrPres and the greatest levels of astrocytosis. In collaboration with Dr. Nico Dantuma, Karolinska Institute Sweden, we are exploring the role of the ubiquitin-proteasome system in TSE diseases. Dr. Dantuma generated mice that carry a transgene encoding Green Fluorescent Protein linked to an uncleavable ubiquitin molecule. This protein is degraded rapidly in these mice, steady- state levels being at or below the level of detection. This transgene can be used as a sensor of proteasome function, since treatment of these mice either by peripheral injection or intraventricular infusion with proteasome inhibitors causes the level of GFP to increase in peripheral organs or the brain respectively. Although these are long-term studies, results from the initial groups of animals indicate that GFP is increased in scrapie-infected mice and appears to be most localized to sights of highest PrPres accumulation. Whether this is a consequence of proteasome dysfunction, or perhaps to the release of the ubiquitin tag by deubiquitinating enzymes, are issues that are currently being examined. These studies are important because ER stress and dysfunction of the protein-degradative machinery of the cell have been found to be associated with a variety of acquired and heritable degenerative diseases of humans, including Parkinsons disease, Huntington?s and Alzheimer?s disease. Understanding the nature of the cellular stress responses induced by the neurovirulent murine retroviruses as well as the TSE agents, and the connection between these responses and the cytopathology induced by these infectious agents, promises to uncover important biochemical pathways to which therapeutic intervention can be directed.