Verrier F C, Charneau P, Altmeyer R, Laurent S, Borman A M, Girard M
Département de Virologie Moléculaire, Institut Pasteur, Paris, France.
Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9326-31. doi: 10.1073/pnas.94.17.9326.
The beta-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4(+) target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4(+) CCR-5(+) HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the "nonsyncytium-inducing," primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the "syncytium-inducing" HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the beta-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1beta (MIP-1beta) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these beta-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and beta-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.
β趋化因子受体CCR-5对于原代巨噬细胞嗜性HIV-1分离株高效进入CD4(+)靶细胞至关重要。为了研究依赖CCR-5的细胞间融合，我们开发了一种检测系统，该系统基于用表达来自原代B亚型HIV-1分离株（BX08）或实验室T细胞系适应株（LAI）的gp120/gp41包膜（Env）的Semliki森林病毒重组体感染CD4(+) CCR-5(+) HeLa细胞。在该系统中，“非融合诱导型”原代巨噬细胞嗜性HIV-1 BX08分离株的gp120/gp41与“融合诱导型”HIV-1 LAI株的gp120/gp41的融合能力至少相当。BX08 Env介导的融合受到β趋化因子RANTES（活化时调节，正常T细胞表达和分泌）和巨噬细胞炎性蛋白1β（MIP-1β）以及抗CD4抗体的抑制，而LAI Env介导的融合对这些β趋化因子不敏感。相反，可溶性CD4显著降低了LAI介导的融合，但对BX08 Env介导的融合没有影响，这表明原代分离株的Env糖蛋白对CD4的亲和力降低。使用单克隆抗体探测了gp120/gp41中与CD4和CCR-5分子相互作用的结构域。对于此处测试的抗体，针对构象依赖性而非线性表位的抗体对融合的抑制作用最大。融合的有效抑制并不局限于gp120/gp41任何一个结构域中的表位。该检测方法足够灵敏，能够区分使用患者BX08血清样本的抗体介导和β趋化因子介导的融合抑制，这表明该系统可能有助于筛选人血清中具有生物学意义的抗体。
