The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies inhibit HIV-1 infection, suggesting a need for more potent reagents. Here we evaluated full length single chain (FLSC) IgG1, a novel IgG-CD4-gp120(BAL) fusion protein with several characteristics that make it an attractive candidate for treatment of HIV-1 infections, including bivalency and a potentially increased serum half-life over FLSC, the parental molecule. FLSC IgG1 binds two domains on CCR5, the N-terminus and the second extracellular loop, lowering the levels of available CCR5 viral attachment sites. Furthermore, FLSC IgG1 synergizes with Maraviroc (MVC), the only licensed CCR5 antagonist. In this study, we used both microscopy and functional assays to address the mechanistic aspects of the interactions of FLSC IgG1 and MVC in the context of CCR5 conformational changes and viral infection. We used a novel stochastic optical reconstruction microscopy (STORM), based on high resolution localization of photoswitchable dyes to visualize direct contacts between FLSC IgG1 and CCR5. We compared viral entry inhibition by FLSC IgG1 with that of other CCR5 blockers and showed FLSC IgG1 to be the most potent. We also showed that lower CCR5 surface densities in HIV-1 infected primary cells result in lower FLSC IgG1 EC50 values. In addition, CCR5 binding by FLSC IgG1, but not CCR5 Ab 2D7, was significantly increased when cells were treated with MVC, suggesting MVC allosterically increases exposure of the FLSC IgG1 binding site. These data have implications for future antiviral therapy development.

中文翻译：

---

CCR5拮抗剂Maraviroc增强了融合蛋白FLSC IgG1与CCR5的结合。

CCR5趋化因子受体对于人类1型免疫缺陷病毒（HIV-1）感染至关重要，它是HIV-1进入和传播的主要受体，因此是抗病毒治疗的诱人靶标。研究表明，在病毒体接触后，CCR5表面密度及其构象变化限制了进入速率，进而限制了感染。并非所有的CCR5抗体都能抑制HIV-1感染，这表明需要更有效的试剂。在这里，我们评估了全长单链（FLSC）IgG1，这是一种新颖的IgG-CD4-gp120（BAL）融合蛋白，具有多种特征，使其成为治疗HIV-1感染的诱人候选物，包括双价性和血清半衰期可能增加。寿命超过FLSC，即亲本分子。FLSC IgG1结合CCR5的两个结构域，N末端和第二个细胞外环，降低了可用的CCR5病毒附着位点的水平。此外，FLSC IgG1与唯一获得许可的CCR5拮抗剂Maraviroc（MVC）协同作用。在这项研究中，我们使用显微镜和功能测定法来解决在CCR5构象变化和病毒感染的情况下FLSC IgG1和MVC相互作用的机理。我们使用了一种新型的随机光学重建显微镜（STORM），它基于可光转换染料的高分辨率定位来可视化FLSC IgG1和CCR5之间的直接接触。我们比较了FLSC IgG1与其他CCR5阻断剂对病毒进入的抑制作用，并显示FLSC IgG1最有效。我们还显示，HIV-1感染的原代细胞中较低的CCR5表面密度导致较低的FLSC IgG1 EC50值。此外，当用MVC处理细胞时，FLSC IgG1的CCR5结合而不是CCR5 Ab 2D7显着增加，表明MVC变构地增加了FLSC IgG1结合位点的暴露。这些数据对未来抗病毒治疗的发展具有影响。