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

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Summary: (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: I. The chemokine receptor CXCR4 is a primary coreceptor for the HIV -1 virus. The predicted MW of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T lymphocyte line revealed multiple molecular weight isoforms of CXCR4. Three of the bands could either be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or co-precipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, anti-ubiquitin antibodies reacted with the 62 kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101 kDa CXCR4 isoform in immunoblots by three- to seven-fold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV -1 envelope-expressing cells. Similar increases in the intensity of the 101 kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Anti-ubiquitin antibodies reacted with multiple proteins above 62 kDa that were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for both proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary cells. Changes in expression and conformation of CXCR4 could affect the susceptibility of a given cell to infection by T-tropic HIV-1 strains, which only use CXCR4 as a coreceptor. Such changes may also indirectly influence infection by M-tropic HIV-1, which uses CCR5 as a coreceptor, since the two coreceptors compete with each other for interaction with CD4 (J. Virol. 74:5016-5023, 2000). In addition, changes in conformation may alter the effectiveness of vaccines and small drugs that target the coreceptors. Thus, it is crucial to understand the heterogeneity and functional diversity of CXCR4 and the other principal coreceptor, CCR5. We and others have recently observed a predominant CCR5 62 kDa species. Ubiquitination may play a role in the constitutive intracellular transport, turnover, and conformation of the HIV-1 coreceptors. Our earlier findings together with the current data suggest that mono-ubiquitination may enhance the role of CXCR4 and CCR5 as HIV-1 coreceptors by inducing constitutive association with CD4 in monocytes, perhaps by altering their conformation. On the other hand, polyubiquitination of CXCR4 may decrease its steady state surface expression in resting cells. These studies have been extended to compare unactivated and activated thymocytes and T cells. In a preliminary study we found that in human thymocytes and peripheral T cells changes in post-translational modifications as determined by changes in the predominant MW species in Western blots, are seen following cell activation. These changes correlated with enhanced fusion activity and increased association with CD4 molecules without a parallel increase in transcription or in the cell-surface density of the coreceptors. II. In studying the induction of cell mediated immunity to the protozoan Toxoplasma gondii we have discovered that a protein, C-18 (cyclophilin) from the parasite binds to both murine and human CCR5. In vitro studies demonstrated that recombinant C-18 is effective in neutralizing infectivity of both lab-adapted and multiple primary HIV R5-isolates from different clades but was inactive against X4 viruses. Unlike previously described chemokine analogs used as co-receptor antagonists, C-18 does not appear to trigger receptor internalization and as foreign molecule is unlikely to induce autoimmunity. It therefore could provide the basis for the development of a new microbially derived antagonist lacking these deleterious side effects.