HIV-1 mediated membrane fusion as target for anti-viral therapy

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(1) Goals of project: - To develop biological and biochemical assays for monitoring HIV-1 envelope-mediated cell fusion and formation of the tri- molecular complex between gp120/CD4/co-receptor. - To study the expression and function of HIV-1 co-receptors on primary human cells known to be targets for HIV-1 infection, and to study the effects of pro-inflammatory cytokines on the function of the HIV-1 co- receptors and infectivity of primary human cells. - Development of agents capable of blocking infection by cell-free or cell-associated HIV-1. (2) Experimental approach: - Biological (Ca++ flux, chemotaxis, HIV-fusion) and biochemical assays were developed to measure the association of the CD4/gp120 complex on human cells with the HIV-1 co-receptors for T-tropic and M-tropic envelopes (CXCR4 and CCR5). They included co-immunoprecipitations of CD4/co-receptors from cell lines and from primary human cells (i.e., monocytes and macrophages), and Western blots of whole cell and membrane extracts using our rabbit anti- CXCR4 and anti-CCR5 reagents. - Studies were conducted on multiple cell types: Langerhans cells (LC), dendritic cells (DC), Thymocyte subsets, CD34+ progenitors, peripheral blood T cells, monocytes (MO) and macrophages (MDM). - Develop new reagents for immunoprecipitation and Western blot analyses of the key HIV-1 co-receptors (CXCR4, CCR5). - Apply new biochemical assays including "Proteomics" to identifying the post-translational modifications of HIV-1 co-receptors that affect their function in primary human cells. (3) Major Findings: - Studies examine the effects of proinflammatory cytokines on co-receptor expression and function: Expression of surface CXCR4 on cultured LC was upregulated by IL-4 and TGFb and inhibited by IFN a/b/g The changes in surface expression correlated with susceptibility of cLCs to infection with X4- HIV strains. These findings suggest that cytokine dysregulation may contribute to the emergence of X4-HIV variants and progression to AIDS. -Treatment of macrophages with IFNg or IL-6 resulted in enhanced infection with X4 viruses - In biochemical studies on monocyts and macrophages (MO/MDM) it was found that the post translational modification of CXCR4 is significantly different in MO vs. MDM. In Mo all the CXCR4 molecules appeared monomeric, while predominantly high MW species of CXCR4 were found on the surface of MDM. The transition from low to high CXCR4 MW species correlated with loss of fusion with T-tropic (X4-) envelopes. -Coreceptor competition for association with CD4 may change the susceptibility of human cells to infection with T-tropic and M-tropic isolates.