The objective of the proposed program is to develop and utilize a new cellular system for evaluating the effects of HIV, and each of its proteins, on specific cells of the human central nervous system. The basis for this new cellular system is the use of a temperature-sensitive transforming gene; this new technique allows clonal lines of specific neurons or glia to be produced that are transformed at 34 degrees C, but undergo differentiation at 38 degrees C. This will allow us to test the hypothesis that HIV effects on the CNS are mediated, at least in part, by specific HIV protein products such as gp 120 (and if so, by what molecular mechanism) as opposed to other factors such as toxic monokines or viral replication. Whereas CNS cell presently used for neurological HIV research suffer from several limitations, and fail to mimic human in vivo, differentiated neural cells, the proposed system will offer a combination of features not presently available, by combining the advantages of primary culture with those of established cell lines. The specific steps of this program are: (1) Immortalization of human neural cells with the temperature- sensitive large T antigen gene of SV 40; characterization as neuronal, astroglial, oligodendroglia, or other. (2) Construction of inducible expression vectors for HIV proteins SU (gp120), TM(gp41) (and gp 160 separately), CA (p24), and VIP (p23). (3) Stable transfections of the constructs into the human cell lines at 34 degrees. (4) Differentiation of the cells at 38 degrees, and induction of expression of the HIV proteins, followed by analysis of the resultant effects on cell viability, heat-shock protein production, expression of the differentiated phenotype (e.g., myelin-basic protein production by oligodendrocytes), response to trophic factors (e.g., vasoactive intestinal peptide (VIP)), and effects on co-cultured non-expressing cells.
