Latinovic Olga, Schneider Kate, Szmacinski Henryk, Lakowicz Joseph R, Heredia Alonso, Redfield Robert R
Institute of Human Virology, University of Maryland, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA.
Antiviral Res. 2014 Dec;112:80-90. doi: 10.1016/j.antiviral.2014.10.006. Epub 2014 Oct 24.
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.
CCR5趋化因子受体对人类免疫缺陷病毒1型（HIV-1）感染至关重要，它作为HIV-1进入和传播的主要共受体，因此是抗病毒治疗的一个有吸引力的靶点。研究表明，CCR5的表面密度及其在病毒体结合后的构象变化是进入以及随后感染的限速因素。并非所有CCR5抗体都能抑制HIV-1感染，这表明需要更有效的试剂。在此，我们评估了全长单链（FLSC）IgG1，这是一种新型的IgG-CD4-gp120（BAL）融合蛋白，具有多种特性，使其成为治疗HIV-1感染的有吸引力的候选药物，包括双价性以及与亲本分子FLSC相比可能延长的血清半衰期。FLSC IgG1结合CCR5上的两个结构域，即N端和第二个细胞外环，从而降低了可用的CCR5病毒附着位点的水平。此外，FLSC IgG1与唯一获得许可 的CCR5拮抗剂马拉维若（MVC）具有协同作用。在本研究中，我们使用显微镜和功能测定法来研究在CCR5构象变化和病毒感染的背景下FLSC IgG1与MVC相互作用的机制方面。我们使用了一种基于可光开关染料的高分辨率定位的新型随机光学重建显微镜（STORM）来可视化FLSC IgG1与CCR5之间的直接接触。我们将FLSC IgG1对病毒进入的抑制作用与其他CCR5阻滞剂进行了比较，结果显示FLSC IgG1是最有效的。我们还表明，HIV-1感染的原代细胞中较低的CCR5表面密度会导致较低的FLSC IgG1 EC50值。此外，当用MVC处理细胞时，FLSC IgG1与CCR5的结合显著增加，而CCR5抗体2D7与CCR5的结合则没有增加，这表明MVC通过变构增加了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重要意义。
