Discovery of New Chemical Entities that Inhibit NEF

We will identify compounds which inhibit the function(s) of the human immunodeficiency virus(HIV) accessory protein Nef. In vivo experiments in rhesus monkeys indicate that the simianimmunodeficiency virus (SIV) nef is critical pathogenic determinant of this virus. It has recently beenshown that CD4 down-regulation is a common property of HIV-1 nef genes isolated directly fromperipheral blood lymphocytes of HIV-1 infected individuals. In addition, Nef protein from the HIV-1 NL43isolate has a dramatic effect on the development of CD4+ T cells when expressed in transgenic miceand this effect correlates with CD4 down-regulation in thymic T cells. Stable human T lymphoid cellsexpressing this Nef allele display block in interleukin 2 (IL-2) and NF-kappaB T cell receptor-mediatedtranscriptional activation. Since blocking IL-2 activation results in immunosuppression, this observationrepresents a potentially important functional property of Nef. This Nef property has not yet beencorrelated with CD4 down-regulation, nor has it been analyzed using other Nef isolates. We willtransiently express several HIV-1 and HIV-2 primary Nef isolates together with an IL-2 promoter/luciferase and NF-kappaB/luciferase reporter constructs, in human T cell lines, and investigate whetherthe ability to impair the IL-2 and NF-kappaB transcriptional induction in response to stimuli that mimicT cell activation is a general property of functional Nef alleles. Promoter/luciferase reporter assaysrepresent an excellent system for the accurate, automated screening of large number of compounds.If a block in IL-2 induction is a common property of primary Nef isolates, we will establish a cell-basedsystem to search for compounds which inhibit the Nef-mediated block in IL-2 induction, using the uniquehigh-throughput screening system developed at Oncogene Science.