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Please use this identifier to cite or link to this item: http://hdl.handle.net/1993/772

Title: Interactions of human immunodeficiency virus type 1 proteins with astrocytes
Authors: Ma, Meihui
Issue Date: 1-May-1997
Abstract: To understand the interaction between gp120 and astrocytes, which may be the initial event for HIV-1 infection to astrocytes or gp120 effects on astrocytes, we characterized, kinetically and biochemically, the binding sites for HIV-1 gp120 on human fetal astrocytes. A single binding site was observed with values for Kd of 26 nM and Bmax of 29.9 fmoles/4 $\times$ 10$\sp4$ cells. Specific receptors for gp120 such as CD4 on lymphocytes and galactocerebroside (GalC) on SK-N-MC cells have been identified. In this study, neither a polyclonal antibody against GalC (anti-GalC) nor a monoclonal antibody against the gp120 binding domain of CD4 (OKT4a) affected the $\sp{125}$I-gp120 binding to astrocytes. However, binding of gp120 was inhibited by 60% with OKT4a on HeLa CD4 cells and approximately 70% by anti-GalC on SK-N-MC cells. Furthermore, neither CD4 nor GalC were detectable on astrocytes using flow cytometry or immunocytochemistry. The mass of the gp120 binding molecule as determined by cell surface labelling and immunoprecipitation was approximately 260 kDa. Thus, specific binding sites for gp120 are present on human fetal astrocytes and these sites appear to be distinct from both CD4 and GalC. Tat1-72 (1-72 amino acids from first exon) and Tat1-86 (full length Tat) were labelled with $\sp{125}$I and incubated with human fetal astrocytes and human fetal neurons. We demonstrated that the uptake of Tat1-72 without the second exon was much lower than that of Tat1-86. This suggests an important role for the C-terminal region of Tat for its cellular uptake and $\sim$90% of the internalized Tat was localized in the nuclei. Uptake of \sp{125}$I-Tat1-72 could be inhibited by dextran sulfate and competitively by unlabelled Tat1-72 but not by overlapping 15 mer Tat peptides, suggesting that Tat internalization is charge and conformation dependent. To understand the mechanism by which Tat initiates the effects on astrocyte, we characterized the Tat binding properties on the astrocyte cell membrane. By immunocytochemical staining, we have demonstrated that Tat specifically bound to the astrocyte surface. This result was confirmed by $\sp{125}$I-Tat finding assay on astrocytes in the presence of excess unlabelled Tat or anti-Tat antibody. This binding of Tat on astrocytes d es not depend upon integrins $\alpha$v$\beta$3, $\alpha 5\beta$1 or $\alpha$v$\beta$5, which has been reported to be Tat binding proteins on other cell lines. In an attempt to determine the epitope of Tat involved in binding to astrocytes, we used 15 mer peptides overlapping by 10 amino acids each and spanning the entire sequence of Tat1-86 in an attempt to block the binding of Tat. None of the peptides were able to inhibit Tat binding. In contrast, Tat binding on astrocytes was enhanced by Tat28-42. By immunoprecipitation of $\sp{125}$I-astrocytes with Tat, 35 and 43 kDa proteins were detected on the astrocyte surface. These may represent novel Tat binding proteins on astrocytes. (Abstract shortened by UMI.)
URI: http://hdl.handle.net/1993/772
Appears in Collection(s):FGS - Electronic Theses & Dissertations (Public)

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