Molecular Regulation of Fusion: Voltage Dependence and Local Physical Interaction

描述(由申请人提供)：所有含有II类或III类融合蛋白的病毒(以及一些含有I类融合蛋白的病毒)都从内吞体内融合。对于这些病毒来说，内体是感染的初始部位。为了响应低pH值的内体环境，一种融合蛋白 经历构象变化，导致病毒和内体膜合并，将病毒遗传物质释放到细胞质中。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。内膜本身及其内部的性质，如膜电压、酸性脂类和氧化还原电位，也会对融合产生深远的影响；如果调节性质被确定并可以修改，可能会产生新的阻止感染的方法。我们的实验室已经证明，跨膜电压可以控制具有II类或III类融合蛋白的多种类型的病毒粒子的融合：跨膜的自然产生的负电压促进融合；正电压抑制融合。第二类和第三类融合蛋白的电压依赖性的普遍性现在是相当确定的。嵌合体实验强烈表明跨膜结构域(TMD)是融合蛋白的电压敏感性区域。电压依赖性可能是因为TMD直接对电压作出反应，也可能是因为外膜小叶中的酸性(带负电荷)脂质与TMD结合。外叶中酸性脂类的浓度随着小叶之间的电压依赖触发而变化：通过实验结合外叶中酸性脂类的浓度并测量其对电压依赖融合的影响，将确定酸性脂结合是否导致电压依赖融合。如果是这样的话，将通过改变蛋白质来识别融合蛋白上的结合区。如果TMD是电压传感器，测量合成TMD的位移电流将决定是否大偶极矩是传感电压的关键。传递电子所需的能量(氧化还原电位)也可能在调节病毒融合方面发挥重要作用：内体的氧化还原电位取决于其NADPH氧化酶(NOX)的水平。抑制内体内的NOX活性表明，融合的病毒粒子的数量随着氧化状态的变化而变化，与细胞-细胞融合的方式相同。NOx的活性将被改变，并通过共聚焦显微镜监测内体内的融合，以确定氧化还原电位与感染的相关性。监测病毒融合蛋白片段膜插入的方法将通过将亲脂、带电的探针偶联到融合蛋白上，并通过电生理学确定融合蛋白的电压依赖性是否改变来开发。这种方法将比目前的方法具有更高的灵敏度。第二类和第三类蛋白质共有一些结构特征；因此，插入研究可以得出基本原理，将这两类融合蛋白的作用机制统一起来。在临床上，识别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防止感染。

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.