Weeks B S, Desai K, Loewenstein P M, Klotman M E, Klotman P E, Green M, Kleinman H K
Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Mar 5;268(7):5279-84.
The HIV-1 transactivator protein Tat is essential for viral gene expression and replication. Tat is taken up by cells and transactivates the HIV-LTR promoter in the cell nucleus. The present studies show that cells adhere to both synthetic and recombinant Tat, and, using synthetic peptides, we localize the binding site to a region spanning amino acid residues 49-57 (peptide Tat49-57). Tat49-57 also inhibited cell attachment to solid phase full-length Tat peptide and to recombinant Tat protein. Using Tat peptide affinity chromatography, we identified a 90-kDa cell surface protein that binds to Tat. The 90-kDa protein could be eluted from the Tat column using the Tat49-57 peptide. A 90-kDa cell surface Tat binding protein was also identified by coprecipitation with Tat after incubation with radiolabeled cell membrane preparations. Co-precipitation of the 90-kDa protein was inhibited by competition with a Tat49-65 peptide, but not with Tat55-86. Our findings suggest that cellular attachment to Tat is mediated through a 90-kDa cell surface protein that binds to a Tat domain between amino acids 49 and 57.
HIV-1反式激活蛋白Tat对病毒基因表达和复制至关重要。Tat被细胞摄取并在细胞核中反式激活HIV-LTR启动子。目前的研究表明，细胞能黏附于合成型和重组型Tat，并且利用合成肽，我们将结合位点定位到一个跨越氨基酸残基49 - 57的区域（肽Tat49 - 57）。Tat49 - 57也抑制细胞黏附于固相全长Tat肽和重组Tat蛋白。利用Tat肽亲和层析，我们鉴定出一种与Tat结合的90 kDa细胞表面蛋白。该90 kDa蛋白可用Tat49 - 57肽从Tat柱上洗脱下来。在用放射性标记的细胞膜制剂孵育后，通过与Tat共沉淀也鉴定出一种90 kDa细胞表面Tat结合蛋白。90 kDa蛋白的共沉淀被Tat49 - 65肽竞争抑制，但不被Tat55 - 86抑制。我们的研究结果表明，细胞对Tat的黏附是通过一种90 kDa细胞表面蛋白介导的，该蛋白与氨基酸49和57之间的Tat结构域结合。
