Human immunodeficiency virus type I (HIV-1) can infect cells within the central nervous system (CNS) and lead to a variety of neurological disease states. Mechanisms of HIV-1-induced CNS dysfunction are poorly defined but remain of critical importance in combating the acquired immunodeficiency syndrome (AIDS) pandemic. Microglial cells, of monocyte/macrophage origin, appear to harbor much of the productively replicating HIV-1 in the CNS. Although, the levels of HIV-1 infection of astrocytic glial cells in vivo, the functional state of the virus (i.e., latency) within these cells and the molecular mechanisms characterizing HIV-1-specific replication in astrocytes remain controversial. In this proposal, a comprehensive and focused approach to the study of HIV-1 interactions with astrocytic glial cells is presented. Of note, our preliminary results demonstrate a novel mechanism(s) of tar- independent HIV-1 replication in astrocytic glial cell lines. Further characterization of this phenomenon, utilizing full viral constructs with both varying site-specific mutations in tar and in DNA-binding protein motifs within the 5' HIV-1 long terminal repeat (LTR) will be conducted. As well, the biphasic effect, by Tat, on tar-negative HIV-1 replication will be evaluated using viral constructs with mutations in the first exon of tat, in addition to mutations in tar. both transient transfections and stably transfected cell lines, using HIV-1 viral constructs containing a resistance marker gene, will be evaluated. Primary astrocyte explants shall be used as well; to assess this mechanism in primary human cells. to further investigate the mechanisms of HIV-1 replication in astrocytic glial cells, both the Rev protein, which rescues genomic unspliced HIV-1 RNA from the nucleus of infected cells, and Nef, whose function is controversial, will be evaluated in these cells. To evaluate HIV-1 infection of astrocytes in vivo, a newly developed technique, in situ polymerase chain reaction (PCR), which allows amplification and detection of the HIV-1 genome within intact cells, will be used to study frozen CNS tissue from various HIV-1-infected individuals. In addition, these CNS tissues will be primarily co- cultured with astrocytic cells in attempts to obtain HIV-1 viral strains with a specific-cellular tropism for astrocytes. Finally, a recently described molecular model for HIV-1 proviral latency, characterized by an aberrant pattern of HIV-1 RNA, will be assessed in astrocytic glial cells. A newly developed quantitative reverse transcriptase-PCR technique will e utilized in these studies. In sum, these studies seek to further our understanding of the molecular patterns of HIV-1 growth in astrocytic glial cells, in cell cultures and in vivo. Thus, the role(s) of astrocytes in HIV-1-induced CNS disease states will be explored.
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