This revised competing renewal program project grant application (PPG) seeks support for continued investigation of the molecular biology and genetics of viral-glial interaction in the central nervous system (CNS). This project remains focused on the molecular pathogenesis of two CNS disorders. One, Progressive Multifocal Leukoencephalopathy (PML), is induced by the human neurotropic virus, JCV, and the other, Acquired type 1 (HIV-1). The clinical similarity between PML and ADC, the association of PML with HIV-1 infection, and the utilization of common regulatory pathways by these two viruses, in the CNS underscores, the rationale for the simultaneous study of these diseases and their etiological agents at the molecular level. During the last funding period, through highly interactive and synergistic collaboration, the participants in this PPG have identified several regulatory proteins that modulate JCV gene expression and replication in CNS cells, and have molecular cloned genes responsible for expression of these proteins. Using in vitro cell culture and in vivo animal models, several proteins regulating myelin genes have been identified, molecularly cloned, and characterized to help unravel the mechanism whereby JCV T-antigen causes dysmyelination of the brain. Analysis of HIV-1 gene expression in the CNS has led to the identification of a novel regulatory pathway in astrocytic glial cells that modulates HIV-1 gene expression in these cells. Finally, we have developed and utilized highly sensitive in situ PCR methods to identify HIV-1 gene expression in these cells. Finally, we have developed and utilized highly sensitive in situ PCR methods to identify a HIV-1 gene expression in various CNS cells in clinical specimens and have employed these methods to examine viral replication. In the current PPG we shall maximally utilize the established collaboration among the leaders of this program to: (1) characterize the interaction of JCV and host regulatory factors, and their effects on viral gene expression/replication, and glial cell function; (ii) elucidate the molecular interactions between the HIV-1 regulatory protein, Tat, Tat- induced cytokines, and cell cycle regulatory factors from human microglial cells and astrocytes; iii) derived potent transcription factor, Tat, and the cellular regulatory protein Puralpha. These three highly integrated, yet independent, projects will benefit from the Neuropathology, and Tissue Culture Core which will provide a central source for reliable distribution of clinical samples and preparations of primary and established cells from fetal and adult brain cell cultures to all projects and preparation of virus stocks for studies in other components of this program. This program project brings together basic scientists and physicians with expertise in the areas of neurovirology, molecular, retrovirology, molecular and cellular biology, and neuropathology to perform the proposed studies.
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