Histones located on the plasma membrane of T-cells.

Several sulphated polysaccharides have been shown to block the infection of T-cells by the human immunodeficiency virus (HIV)-l [1,2]. Dextrin-2-sulphate (D2S) is a synthetically sulphated polysaccharide which inhibits infection of human T-cell lines by a variety of HIV-1 cell-free isolates [3]. Although its mechanism of action has not been established, this compound appears to prevent HIV-1 infection by binding to the cell surface. Most sulphated polysaccharides interfere with the binding of m i 2 0 to CD4 [2], but D2S appears to act by a different mechanism [4]. The aim of this study was to identify cell surface proteins which bind D2S and may be involved in the entry of HIV1 into cells. A plasma membrane fraction was prepared from a human Tcell line, HPB-ALL, by ultracentrifugation [5]. This was subjected to ligand blotting in which the membrane fraction was separated by SDS-polyacrylamide gel electrophoresis (SDSPAGE) using 15% (w/v) gels, electrotransferred onto nitrocellulose filters and incubated with 'H-D2S. It was found that 'H-D2S bound in a specific, displaceable manner to proteins of molecular weights 17 kDa and 29 kDa. Microsequencing was attempted on proteins present in these regions. In the 17 kDa region the N-termini of two proteins was successfully determined. These were PEPAKSAPAPKKGXKKXVTKA and ARTKQTARKSTGGKAPRKQLAT, which correspond to the Ntermini of histones H2B and H3, respectively. In addition, an antibody was raised against the N-terminal sequence of histone H2B (PEPAKSAPAPKKGSKKAVTKAQK). The antibody bound to purified histone H2B, but not to the cell surface or to chromatin. This may be explained by occlusion of the antibody binding site in complexed histones. Immunoblotting with this antibody showed that histone H2B was present in the membrane preparation. To exclude the possibility that the histones in the membrane preparation was due to contamination by nuclear proteins, HPBALL membranes were prepared using an affinity based method of purification using polyethyleneimine-resin [6]. This method is based on interaction of the negatively charged cell membrane with the positively charged resin. HPB-ALL cells were suspended in 15 mM sodium acetate, pH 5.0 containing 220 mM sucrose and incubated with polyethyleneimine-resin for 1 h at room temperature. Free binding sites were blocked by the addition of 1 mg/ml polyglutamic acid. The cells were then lysed in a hypotonic buffer (10 mM Tris-HCI, pH 7.4). The polyethyleneimine-resin with plasma membrane material bound to it was then washed extensively with the same buffer. SDSPAGE of the bound material revealed the presence of histones H2A, H2B, H3, and H4 by comparison with the electrophoretic mobility of purified calf thymus histones. Also, immunoblotting using the anti-histone-H2B antibody showed the presence of histone H2B.