Molecular Regulation of Fusion: Voltage Dependence and Local Physical Interaction

描述（由申请方提供）：所有含有II类或III类融合蛋白（和一些I类）的病毒均从内体融合。对于这些病毒，内体是感染的初始位点。为了响应低pH内体环境， 经历构象变化，引起病毒和内体膜的合并，将病毒遗传物质释放到胞质溶胶中。如果融合蛋白上对感染至关重要的区域被定位，它们将为新的抗病毒药物和疫苗提供靶点。内体膜本身及其内部的性质，如膜电压，酸性脂质和氧化还原电位也可能对融合产生深远的影响;如果确定并修改调节特性，可能会产生阻止感染的新方法。我们的实验室已经证明，跨内体膜的电压可以控制许多具有II类或III类融合蛋白的病毒粒子的融合：跨膜的自然发生的负电压促进融合;正电压抑制融合。嵌合体实验强烈表明跨膜结构域（TMD）作为赋予电压敏感性的融合蛋白的区域。电压依赖性可能是因为TMD直接响应电压，或者因为外膜小叶中的酸性（带负电荷）脂质与TMD结合。外小叶中酸性脂质的浓度随着小叶之间的电压依赖性翻转而变化：通过实验性地将它们并入并测量对电压依赖性融合的后果来富集外小叶中酸性脂质的浓度，将确定酸性脂质结合是否引起电压依赖性融合。如果是这样，融合蛋白上的结合区域将通过改变蛋白质来鉴定。如果TMD是电压传感器，则测量合成TMD的位移电流将确定大的偶极矩是否是感测电压的关键。转移电子所需的能量（氧化还原电位）也可能在调节病毒融合中起重要作用：内体的氧化还原电位取决于其NADPH氧化酶（NOX）的水平。内体内NOX活性的抑制表明融合的病毒体的数量以与细胞-细胞融合相同的方式随氧化态而变化。将改变NOX活性，并通过共聚焦显微镜监测内体内的融合，以确定感染中氧化还原电位的相关性。将通过将亲脂性带电探针与融合蛋白偶联，并通过电生理学确定融合的电压依赖性是否改变，来开发监测病毒融合蛋白片段的膜插入的方法。这种方法的灵敏度将远远高于目前的方法。II类和III类蛋白质共享一些结构特征;因此插入研究可以产生统一这两类融合蛋白作用机制的基本原理。在临床上，确定病毒融合的内体控制机制将揭示哪些过程可以被中断以减少或预防感染。

DESCRIPTION (provided by applicant): All viruses that contain class II or class III fusion proteins (and some with class I) fuse from within endosomes. For these viruses, the endosome is the initial site of infection. In response to the low-pH endosomal environment, a fusion protein undergoes conformational changes that cause merger of the viral and endosomal membranes, releasing the viral genetic material into cytosol. If regions on the fusion protein critical for infection are located, they will provide targets for new anti-viral drugs and vaccines. Properties of the endosomal membrane itself and its interior such as membrane voltage, acidic lipids, and redox potentials could also exert profound effects on fusion; if regulatory properties are identified and could be modified, new methods of halting infection could result. Voltage across endosomal membranes has been shown by our laboratory to control fusion of a number of types of virions that have class II or class III fusion proteins: the naturally occurring negative voltag across a membrane promotes fusion; positive voltage inhibits it. The universality of voltage dependence of class II and III fusion proteins is now reasonably certain. Chimera experiments strongly suggest the transmembrane domain (TMD) as the region of the fusion protein that confers voltage sensitivity. Voltage dependence could arise either because a TMD directly responds to voltage, or because acidic (negatively charged) lipids in outer membrane leaflets bind to TMDs. The concentration of acidic lipids in outer leaflets varies with voltage-dependent flip-flop between leaflets: enriching the concentration of acidic lipids in outer leaflets by experimentally incorporating them and measuring the consequences to voltage-dependent fusion will determine if acidic lipid binding causes voltage-dependent fusion. If it does, the binding region on the fusion protein will be identified by altering the protein. If the TMD is the voltage sensor, measuring displacement currents of synthetic TMDs will determine whether a large dipole moment is the key for sensing voltage. The energy required to transfer electrons (redox potentials) may also have an important role in regulating viral fusion: The redox potential of an endosome depends on its level of NADPH oxidase (NOX). Inhibition of NOX activity within endosomes indicates that the number of virions that fuse varies with the oxidation state in the same manner it does in cell-cell fusion. NOX activity will be altered, and fusion within endosomes monitored by confocal microscopy, to determine the relevance of redox potentials in infection. Methods to monitor membrane insertion of segments of viral fusion proteins will be developed through coupling lipophilic, charged probes to a fusion protein and electrophysiologically determining if voltage dependence of fusion is altered. This method will have far greater sensitivity than current methods. Class II and III proteins share some structural features; insertion studies could thus yield fundamental principles that unify the mechanisms of action of fusion proteins in these two classes. Clinically, identifying the mechanisms for endosomal control of viral fusion will reveal which processes could be interrupted to reduce or prevent infection.