Essential ionic triggers for enveloped virus entry

为了引起疾病，所有病毒都必须进入它们的目标细胞。病毒做到这一点的一种常见方式是劫持一个名为内吞网络的细胞系统，该系统通常被细胞用来从外部环境中吸收营养。这个网络由称为内小体的微小隔间组成，许多病毒已经进化出了欺骗细胞的能力，使它们成为有用的货物，以便进入网络，从而进入细胞。然而，一旦病毒进入内体，它就面临着一个问题；它实际上被困住了，因此病毒必须从内体逃逸才能开始感染。这项建议的目的是详细了解病毒如何能够逃脱内小体，这项工作是基于我们实验室令人兴奋的新研究发现。病毒使用覆盖在病毒粒子外部的特殊尖峰蛋白来逃避内体，并导致一个称为融合的过程。为了引起融合，这些尖峰与内体中的特定化学物质相互作用，并戏剧性地改变形状。在改变形状后，融合棘突从内部与内体膜相互作用，并迫使内体膜与病毒包膜混合。当这两层膜融合在一起时，融合就发生了，病毒基因组被释放到细胞质中，继续病毒复制周期。简单地说，这些病毒尖峰改变形状的能力对于病毒引起融合、逃脱内体从而继续感染过程至关重要。尽管发挥了这一关键作用，但尖峰改变形状进而导致融合的机制尚不清楚。突显这种认识不足的是，我们最近首次表明，被称为BunyaVirus(包括许多出血热病毒和蚊子传播的病毒--由于全球变暖，这在英国增加了风险)的一类重要病毒中的一些病毒需要钾离子(K+)才能使尖峰变形并产生融合。这一发现代表了融合机制的一个关键和以前被忽视的要求。这项建议描述了一组实验，这些实验将首先揭示对K+的要求是否是更广泛的布尼亚病毒组中其他病毒的一般特征。接下来，我们将调查内体内是否有任何其他离子在病毒进入时具有与K+相同的作用，然后使用最先进的冷冻电子显微镜和X射线结晶学技术来揭示非活动(融合前)和激活形状的棘突的高分辨率结构。最后，我们将使用各种遗传技术来识别对生化信号做出反应以及对调节形状变化本身至关重要的尖峰部分。综上所述，这些实验将在理解融合机制方面取得重大进展，高度详细地揭示尖峰是如何对内体中的化学信号做出反应、改变形状并导致融合的。需要这些信息来提供必要的基础，以设计阻止尖峰融合发生的策略；能够做到这一点的药物将防止感染和疾病。

In order to cause disease, all viruses must gain entry to their target cells. A common way viruses do this is by hijacking a cellular system called the endocytic network, which is normally used by cells to take up nutrients from the external environment. This network consists of tiny compartments called endosomes and many viruses have evolved the ability to trick cells that they are useful cargos in order to enter the network, and thus gain entry to the cell. However, once a virus enters an endosome, it is faced with a problem; it is effectively trapped, and so the virus must escape from the endosome in order to start an infection. The aim of this proposal is to understand in detail how viruses are able to escape the endosomes, and this work is based on exciting new research findings from our laboratory. Viruses escape endosomes using specialised spike proteins that cover the virion exterior and cause a process called fusion. To cause fusion, these spikes interact with specific chemicals within endosomes and dramatically change shape. After changing shape, the fusion spikes interact with the endosomal membrane from the inside, and force this membrane to mix with the viral envelope. When these two membranes merge together, fusion has occurred and the viral genome is released into the cytoplasm to continue the viral replication cycle. In simple terms, the ability of these viral spikes to change shape is critical in order for a virus to cause fusion, to escape the endosome and thus to continue the infection process. Despite this critical role, the mechanism by which spikes change shape and then cause fusion is poorly understood. Highlighting this poor understanding, we recently showed for the first time that some viruses within an important class of viruses known as bunyaviruses (which include many haemorrhagic fever viruses and viruses that are transmitted by mosquitoes - an increasing risk in the UK due to global warming) require potassium ions (K+) to cause the spikes to change shape and produce fusion. This finding represents a critical and previously overlooked requirement of the fusion mechanism.This proposal describes a set of experiments that will first reveal whether the requirement for K+ is a general characteristic of other viruses within the broader bunyavirus group. Next, we will investigate whether any other ions within endosomes have the same effect as K+ during virus entry, and then use state-of-the-art cryo-electron microscopy and X-ray crystallography techniques to reveal the high-resolution structure of the spikes in the inactive (pre-fusion) and activated shapes. Finally, we will use a variety of genetic techniques to identify parts of the spikes that are critical for responding to the biochemical signals, and also for mediating the shape changes themselves. Taken together, these experiments will provide a major advance in the understanding of the fusion mechanism, revealing in high detail how spikes are able to respond to chemical signals within endosomes, to change shape and cause fusion. This information is required to provide an essential foundation on which to design strategies to block spike fusogenesis; drugs that can do this would prevent infection and disease.