A biosensor assay for studying ligand-membrane receptor interactions: Binding of antibodies and HIV-1 Env to chemokine receptors
A biosensor assay for studying ligand-membrane receptor interactions: Binding of antibodies and HIV-1 Env to chemokine receptors
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DOI：
10.1073/pnas.190274097
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发表时间：
2000-10-10
影响因子：
11.1
通讯作者：
Doms, RW
中科院分区：
文献类型：
--
作者：
Hoffman, TL;Canziani, G;Doms, RW
HIV包膜（Env）蛋白通过结合CD4和合适的辅助受体介导进入细胞。这会引发Env的结构变化，导致病毒和细胞膜之间的融合。主要的HIV-1共受体是7个跨膜结构域趋化因子受体CCR5和CXCR4。病毒株使用的共受体类型是病毒趋向性和发病机制的重要决定因素，病毒-受体相互作用可以成为治疗靶点。然而，来自许多病毒株的Envs与CXCR4和CCR5的相互作用具有低亲和力，因此使用标准的细胞表面结合技术直接研究这种重要的相互作用即使不是不可能，也是很困难的。我们已经开发了一种方法，使研究配体结合膜蛋白，包括env辅助受体的相互作用，使用光学生物传感器。将CCR5、CXCR4等膜蛋白掺入逆转录病毒颗粒中，纯化后附着在生物传感器表面。构象敏感抗体和Env与这些受体的结合很容易被检测到。从原型HIV-1株IIIB衍生的Env与CXCR4相互作用的平衡解离常数约为500 nM，这解释了使用标准平衡结合技术测量这种相互作用的困难。逆转录病毒假型容易产生。稳定、均匀的结构，可用于在天然脂质环境中呈现广泛的单一和多个膜跨越蛋白，用于生物传感器研究，从而避免了洗涤剂增溶、纯化和重构的需要。该方法应具有普遍适用性，并可用于将env受体结合常数与病毒的趋向性和发病机制联系起来。
The HIV envelope (Env) protein mediates entry into cells by binding CD4 and an appropriate coreceptor. which triggers structural changes in Env that lead to fusion between the viral and cellular membranes. The major HIV-1 coreceptors are the seven transmembrane domain chemokine receptors CCR5 and CXCR4. The type of coreceptor used by a virus strain is an important determinant of viral tropism and pathogenesis, and virus-receptor interactions can be therapeutic targets. However, Envs from many virus strains interact with CXCR4 and CCR5 with low affinity such that direct study of this important interaction is difficult if not impossible using standard cell-surface binding techniques. We have developed an approach that makes it possible to study ligand binding to membrane proteins, including Env-coreceptor interactions, using an optical biosensor. CCR5, CXCR4, and other membrane proteins were incorporated into retrovirus particles, which were purified and attached to the biosensor surface. Binding of conformationally sensitive antibodies as well as Env to these receptors was readily detected. The equilibrium dissociation constant for the interaction between an Env derived from the prototype HIV-1 strain IIIB for CXCR4 was approximately 500 nM, explaining the difficulty in measuring this interaction using standard equilibrium binding techniques. Retroviral pseudotypes represent easily produced. stable, homogenous structures that can be used to present a wide array of single and multiple membrane-spanning proteins in a native lipid environment for biosensor studies, thus avoiding the need for detergent solubilization, purification, and reconstitution. The approach should have general applicability and can be used to correlate Env-receptor binding constants to viral tropism and pathogenesis.