Infected astrocytes and microglia have been shown to express the early gene product, Tat, which has been implicated in a variety of pathways leading to pathogenesis within the CNS via direct as well as indirect mechanisms. Thus Tat is an attractive target for the design of therapeutics for treatment of HIV-1 induced CNS disorders. We propose to utilize as a starting platform two naturally occurring proteins, which interact with Tat, YB-1 and Vpr. We have recently identified YB-1 as a transcription factor which interacts with Tat, the activation response element TAR, as well as the GC rich region present in the HIV-1 LTR. YB-1 activates HIV-1 gene expression in CNS cells and we have designed mutant forms of the protein, which are functionally inactive and can inhibit Tat-mediated transactivation of the viral LTR promoter. We propose a thorough analysis of YB-1 mutants to strategically design therapeutic proteins, which can interact with Tat, but inhibit rather than activate viral transcription. Vpr activates a variety of cellular promoters, is involved in translocation of viral core particles to the nucleus of infected cells, and also is able to dysregulate the cell cycle. We present evidence for the interaction of Tat with Vpr and present a strategy to design transdominant negative mutants based on Vpr which can inhibit Tat function. In order to develop more potent antagonists of Tat function using YB-1 and Vpr, we propose to analyze the inhibitory properties of YB-1 and Vpr fusion proteins containing the transcriptional repressor element, ERF-1, an ETS family protein which exhibits strong repressor activity. We will test the efficacy of these strategies in primary CNS cell cultures of purified microglia, astrocytes, as well as in mixed CNS cell cultures. In order to delivery therapeutic genes targeting Tat protein, we will utilize the recently developed HSV-1 gene delivery system permitting the expression of foreign/therapeutic genes in CNS cells. Toward this end, we will utilize the tissue specific promoters, macrosialin and glial fibrillary acidic protein (GFAP), in order to target gene expression to microglia and astrocytes, respectively.