Rotavirus VP4: Structure and function

描述(申请人提供)：轮状病毒胃肠炎每年导致全球约60万名儿童死亡，在美国约有6万名儿童住院。自轮状病毒疫苗停用以来，它再次成为一种严重的儿童疾病，目前尚无免疫接种。轮状病毒进入细胞的过程是疾病干预的主要目标。为了启动感染，轮状病毒将一个大的转录活性颗粒跨过细胞膜进入细胞质。无包膜轮状病毒颗粒的外层是完成这种鲜为人知的转位的递送装置。外衣壳含有两种蛋白质，VP7和VP4，这两种蛋白质都积极参与进入并成为中和抗体的靶标。VP7会在钙流失时引起脱涂层。VP4是一种尖峰蛋白，被胰酶切割，为病毒的感染做好准备。VP4是膜穿透机制的核心。它包含一个头部结构域和一个膜相互作用域。我们纯化了未裂解的VP4，用蛋白酶启动纯化的蛋白，确定了头部结构域，并结晶了启动形式的膜相互作用结构域。使用这些晶体，我们将解决膜相互作用区域的初始构象的结构。该结构将用于改进VP4未切割构象的结构分析策略。我们还将确定具有重要功能的VP4变体的结构。我们开发了一种轮状病毒的“重新包装遗传学”系统，通过用纯化的、重组的VP4和VP7重新包裹亚病毒颗粒，将传染性提高4到5个数量级。该系统将允许分析工程突变，包括基于结构的突变，在细胞进入过程中对VP4功能的影响。突变研究将阐明唾液酸依赖、蛋白酶启动和膜穿透的决定因素。使用重覆颗粒的基于细胞培养的分析将专注于使用基于结构的突变来分离进入过程中的碳水化合物、脂肪和蛋白质结合，并通过引入可逆的二硫键交联来阻止与进入相关的构象变化。这些结构和功能研究将阐明轮状病毒的进入途径，提供无包膜病毒进入的模型，并确定抗VP4抗体中和轮状病毒的结构基础。

DESCRIPTION (provided by applicant): Each year, rotavirus gastroenteritis kills an estimated 600,000 children, worldwide, and hospitalizes approximately 60,000 children, in the U.S. Since the withdrawal of a rotavirus vaccine, it is again a major childhood illness for which no immunization is available. The process of cell entry by rotavirus is a major target for intervention against disease. To initiate infection, rotavirus translocates a large, transcriptionally active particle across a membrane and into the cytoplasm. The outer layer of the non-enveloped rotavirus particle is the delivery apparatus that accomplishes this poorly understood translocation. The outer capsid contains two proteins, VP7 and VP4, both of which participate actively in entry and are targets of neutralizing antibodies. VP7 causes uncoating on loss of calcium. VP4, a spike protein, is cleaved by trypsin to prime the virus for infection. VP4 is central to the mechanism of membrane penetration. It contains a head domain and a membrane interaction domain. We have purified uncleaved VP4, primed the purified protein with protease, determined the structure of the head domain, and crystallized the primed form of the membrane interaction domain. Using these crystals, we will solve the structure of the primed conformation of the membrane interaction domain. This structure will be used to refine strategies for the structural analysis of the uncleaved conformation of VP4. We will also determine the structures of functionally important VP4 variants. We have developed a "recoating genetics" system for rotavirus by recoating subviral particles with purified, recombinant VP4 and VP7, boosting infectivity by 4 to 5 orders of magnitude. This system will allow analysis of the effect of engineered mutations, including structure-based mutations, on the function of VP4 during cell entry. Mutational studies will clarify the determinants of sialic acid-dependence, protease priming, and membrane penetration. Cell culture-based assays using recoated particles will focus on separating carbohydrate, lipid, and protein binding during entry using structure-based mutations and on blocking entry-associated conformational changes by introducing reversible disulfide cross-links. These structural and functional studies will clarify the rotavirus entry pathway, provide a model for entry by non-enveloped viruses, and define the structural basis for rotavirus neutralization by antibodies against VP4.