Otake K, Fujii Y, Nakaya T, Nishino Y, Zhong Q, Fujinaga K, Kameoka M, Ohki K, Ikuta K
Institute for Laboratory Animal Research, Nagoya University School of Medicine, Japan.
J Immunol. 1994 Dec 15;153(12):5826-37.
Our previous studies have shown that the HIV-1 Nef Ag is expressed, at least in part, on the surface of infected cells. We demonstrated this by using membrane immunofluorescence and flow cytometry with Nef murine mAbs. To identify the domain of Nef exposed on the cell surface, epitope mapping of these and a new mAb was performed by ELISAs by using several recombinant truncated Nef fusion proteins and synthetic peptides. The results showed that mAbs F1, E7, E9, and 4H4 recognized Nef epitopes located at amino acid residues 148-157, 192-206, 158-206, and 1-33, respectively. The intensity of cell surface Nef staining was stronger with mAbs E7 and E9 than with F1, and there was no staining by 4H4, which indicates that the carboxyl-terminal region of Nef is predominantly exposed on the surface of HIV-1-infected T cell lines and PBMC. This surface Nef domain displayed high affinity for the surface of uninfected CD4+ T cells, because the binding of a soluble form of recombinant Nef protein to the cell surface was specifically blocked by the E7 and E9 mAbs or by synthetic peptides that contained the carboxyl-terminal region of Nef. In addition, syncytium formation between infected and uninfected cells also was specifically reduced by the same mAbs or peptides. Thus, the cell surface domain of Nef seems to play an important role in the interaction between HIV-1-infected and CD4+ uninfected T cells.
我们之前的研究表明，HIV-1 Nef抗原至少部分在受感染细胞表面表达。我们通过膜免疫荧光法以及使用Nef鼠单克隆抗体的流式细胞术证明了这一点。为了确定细胞表面暴露的Nef结构域，利用几种重组截短的Nef融合蛋白和合成肽，通过酶联免疫吸附测定法对这些单克隆抗体和一种新的单克隆抗体进行了表位作图。结果显示，单克隆抗体F1、E7、E9和4H4分别识别位于氨基酸残基148 - 157、192 - 206、158 - 206和1 - 33处的Nef表位。单克隆抗体E7和E9对细胞表面Nef的染色强度强于F1，而4H4无染色，这表明Nef的羧基末端区域主要暴露在HIV-1感染的T细胞系和外周血单核细胞表面。这种表面Nef结构域对未感染的CD4 + T细胞表面显示出高亲和力，因为重组Nef蛋白的可溶性形式与细胞表面的结合被E7和E9单克隆抗体或含有Nef羧基末端区域的合成肽特异性阻断。此外，相同的单克隆抗体或肽也特异性减少了感染细胞与未感染细胞之间的合胞体形成。因此，Nef的细胞表面结构域似乎在HIV-1感染的T细胞与未感染的CD4 + T细胞之间的相互作用中起重要作用。
