The long term objective of this proposal is to study the role of L-cycloserine (LCS), a compound we have shown to be a potent inhibitor of sphingolipid biosynthesis, on the infectivity and reproduction of HIV. L-CS, as a result of its inhibition of sphingolipid biosynthesis, also causes a reduction in cell membrane fluidity. Furthermore, galactocerebrosides and sulfatides have been shown to bind gp120. These properties make L-CS a candidate drug for possible interference with HIV replication. Our preliminary results support this hypothesis; L-CS selectively down modulates CD4 but not CD3 or CD8 expression in CEM cells and in human peripheral blood mononuclear cells (PMBSCs). HIV infected CEM cells, cultured with L-CS, show a marked reduction in syncytia formed and reduced HIV-1 production as measured by p24 concentration. In this proposal, we will extend our studies on the feasibility and efficacy of L-CS as an anti-retroviral drug. (1) We will investigate further the role of LCS in the down regulation of CD4 in CEM cells to determine if LCS induces CD4 endocytosis and whether this is accompanied by serine phosphorylation and/or dissociation of p56 lck. CD4 will be detected by immunofluorescence. Secondly, we will determine if CD4 mRNA production is affected in LCS treated cells: CD4 mRNA will be determined by Northern blot analysis. (2) We will extend our findings on LCS reduction of HIV-1 infectivity in CEM cells by the determination of similar reduction by laboratory and primary isolate expression in monocytic, lymphocytic and neuronal cell lines including primary PBMCs, fetal liver and brain cells. The surrogate markers to be used will be syncytia formation P24pr production and viral RNA analysis. (3) A dimer and an acetylated derivative of L-CS will be evaluated for inhibition of sphingolipid biosynthesis in vivo in mice and for HIV propagation. (4) Lipid analyses will be performed on LCS- treated and control cells to determine the degree of reduction and changes in sphingolipid and phospholipid. (5) Possible synergistic action between L-CS and ACT, ddI and hydroxyurea will be determined. If these proposed experiments are successful, as preliminary experiments indicate, then we will examine the ability of LCS, its dimer or the acetylated compound to inhibit virus in HIV infected SAID-h mice.
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