Human immunodeficiency virus type 1 (HIV-1) is the causative agent of acquired immune deficiency syndrome (AIDS), and the development of effective antiviral therapies for HIV-1 infected individuals is of high priority. This NCDDG seeks to study the development of alternative antiviral strategies based on gene therapy technique employing genes or gene products of HIV-1 to specifically inhibit HIV-1 replication and infection. This proposal will explore the possibility of generating intracellular immunity to HIV-1 using the putative negative regulatory gene of the virus, the nef gene. Several studies have shown that Nef has a negative effect on virus replication, probably at the transcriptional level, whereas other laboratories have failed to detect an observable inhibitory effect. The first goal of this proposal is to characterize Nef and its role in the viral life cycle and to assess possible negative regulatory effects of Nef on viral replication in vitro experiments. Areas of study include: (1) the generation of Nef-expressing cell lines and determination of Nef effects on HIV-1 replication and transcription; (2) the role of Nef in- initiation and maintenance of viral latency; (3) the functional characterization of selected nef mutants; and (4) the role of Nef in disease pathogenesis in an in vivo animal model, an immune-deficient mouse reconstituted with human hematopoietic cells (the mouse/human chimera). The second goal of the proposal is to explore the potential of Nef as a candidate gene therapy antiviral agent (if inhibition of virus replication or disease pathogenesis by Nef can be convincingly demonstrated). The initial experiments will be performed in vitro and, if successful, the approaches will then be adapted to the mouse/human chimera model. Nef antiviral constructs will be introduced into human peripheral blood lymphocytes or bone marrow precursor cells prior to inoculation into mouse/human chimeric animals. Treated mice (and control mice) will be challenged with HIV-1 following introduction of the Nef-containing human peripheral blood lymphocytes to test disease prevention effects of Nef. Similar experiments will test the therapeutic effects of Nef-expressing cells by inoculating expressor cells into HIV-infected chimeric animals. Successful reduction of virus effects in the mouse/human chimera model would encourage further testing of antiviral gene therapy approaches based on the nef gene.
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