Our initial (1985) neuropathologic/virologic studies on AIDS encephalopathy in children, identified the brain as a primary site of persistent HIV-1 infection. Recently, we used PCR technology and DNA sequencing to study the HIV-1 regulatory nef gene and its expression in pathologic brain tissue. Targeted PCR amplification showed that cultivation selects against tissue-associated nef quasispecies, suggesting that important subsets of pathogenic HIV-1 strains are not recoverable in 7 necessitating the study of HIV-1 genes in situ. In 92% of nef genes cloned directly from brain and spleen tissues of 14 pediatric AIDS patients the reading frame was open, suggesting that nef is expressed. By immunocytochemistry and in situ hybridization using monospecific nef probes, we obtained evidence that nef mRNA and protein are expressed in astrocytes, in CNS tissues from children who died with AIDS encephalitis. This observation is in striking contrast with expression in the same tissues of structural HIV-1 mRNAs and proteins, e.g., p24/25 gag, in multinucleated giant cells (MGCs; formed by fusion of infected macrophages with resident microglia), but not in astrocytes. These findings show that HIV-1 infection in astrocytes is restricted - to overexpression of nef - and establish dual cellular targets, for productive and restricted HIV-1 gene expression, that are easily distinguishable by morphology. Nef is expressed via the class of early, multiply-spliced mRNAs that also encode tat and rev. We hypothesize that restricted infection involves either absence of tat or rev expression, or downregulation of the LTR by host factors. We propose to analyze the molecular basis for restricted expression directly in astrocytes and MGCs, in postmortem CNS tissues from pediatric AIDS patients at different stages of disease, from macaques experimentally infected with SIV, and in our Xenograft model, where their natural interactions with other neural cells are present. To do this, we will perform in situ hybridization with oligonucleotides that span HIV-1 splicing junctions, to determine whether mRNAs coding for tat and rev are present, and use immunocytochemistry for tat, rev and nef, to determine the cellular localization of the expressed regulatory proteins. We will also use DNA footprinting/DNAse protection assays, with in cultures of macrophages and of primary astrocytes from fetal brain, to test and compare downregulation of the viral LTR by specific host (astrocyte or macrophage) regulatory proteins.
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