Cellular attachment, penetration and transport of non-enveloped dsRNA viruses

无包膜 dsRNA 病毒的细胞附着、渗透和运输

• 批准号: 8304894
• 负责人: Z Hong ZHOU
• 金额: $ 35.49万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8304894
• 关键词: Actins; Amino Acids; Animal Viruses; Animals; Aquareoviruses; Bacteria; Binding Sites; Biochemical; Biological; Biological Models; Bluetongue virus; Caliber; Capsid; Capsid Proteins; Cell-Matrix Junction; Cells; Cellular biology; Chimeric Proteins; Complement; Complex; Cryoelectron Microscopy; Culicoides (genus); Cytoplasmic Polyhedrosis Viruses; Data; Development; Disease; Double Stranded RNA Virus; Economics; Family; Family Relationship; Flowers; Goals; HIV; Hand; Herpesviridae; Human; Human Virus; Infection; Insecta; Integrins; Knowledge; Lead; Learning; Length; Link; Livestock; Mediating; Membrane; Membrane Proteins; Methods; Microtubules; Modeling; Molecular; Mutagenesis; Mutation; Orbivirus; PTPN11 gene; Penetration; Pharmaceutical Preparations; Positioning Attribute; Process; Proteins; Regulation; Reoviridae; Reovirus; Research Personnel; Resolution; Role; Rotavirus; Secondary to; Sialic Acids; Structure; Surface; System; Technology; Testing; Vaccines; Viral; Virion; Virus; Virus Diseases; base; cell attachment protein; disulfide bond; flu; human disease; innovation; insight; killings; member; novel strategies; particle; pathogen; plant fungi; positional cloning; receptor; receptor binding; reconstruction; research study; social; transforming virus; vaccine development

DESCRIPTION (provided by applicant): Many non-enveloped viruses cause devastating human diseases. The mechanisms of entry of these viruses into host cells are poorly understood, although there are likely analogies with well-studied enveloped viruses like flu, HIV and herpesvirus. We have studied non-enveloped dsRNA viruses with a single-layered (cytoplasmic polyhedrosis virus - CPV), a double-layered (aquareovirus), and a triple-layered (bluetongue virus - or BTV -- a member of the Orbivirus genus of the Reoviridae family) capsid. Because its cell biology is well studied, and because it has separate attachment and penetration proteins, BTV in particular serves as a good model system for studying cell entry and transport by such viruses. Thus, the long term goal of this project is to uncover, by cryo electron microscopy (cryoEM), the structural basis of non-enveloped virus infection, particularly the processes of viral attachment and entry, as well as cellular transport of progeny viral particles. Our 7A-resolution cryoEM structure of the pre-penetration BTV virion suggests the presence of a central three-helix bundle and 18 amphipathic helices on the surface of the BTV penetration protein (VP5), similar in some respects to Class I fusion proteins of enveloped viruses. This assignment of amino acids to secondary structures constitutes a set of hypotheses begging to be tested at atomic resolution. Moreover, by use of low pH, we have transformed the virus to its penetration state and visualized the blossoming of long 'barbs' that we hypothesize to be unfurled amphipathic helices. Further, we hypothesize involvement of a disulfide bond within VP5 in the unfurling mechanism. Our cryoEM structure also suggests that the cell attachment protein (VP2) has two binding sites, one for sialic acid and one for an unknown target molecule, perhaps an integrin. In addition, our preliminary data shows that non-structural protein NS1 has a role on virus release and forms highly ordered helical tubules in a Zn2+-dependent manner, thus providing an opportunity for structural studies to explain its role. The proposed studies will test these hypotheses by carrying out four specific aims: (1) By determining the atomic structure of the native BTV virion, we will test our hypothesis that the VP5 penetration protein of the non-enveloped BTV virus has a three-helix bundle at its core, 18 amphipathic helices on its surface, and a disulfide bond in a critical position. (2) From the atomic structure of the native BTV virion in the presence of sialic acid, we will test our hypothesis that the VP2 attachment protein has a sialic-acid binding site. (3) By determining the structure of the (blossomed) penetration state (low pH) of the BTV particle, we will test our hypothesized unfurling mechanism. Moreover, we will carry out structure-based mutagenesis studies to complement structural studies to establish mechanisms for triggering the unfurling. (4) From the atomic structure of BTV non-structural protein NS1 in helical tubules, followed by functional and structure-based mutagenesis experiments, we will learn how NS1 regulates virus release. These mechanisms and structures will be correlated with those of other enveloped and non-enveloped viruses, including the ones cited above. PUBLIC HEALTH RELEVANCE: Viruses in the Reoviridae family are non-enveloped, dsRNA viruses that infect a wide range of hosts, including human, other animals, plants, fungi, and bacteria; within this family, reoviruses and rotaviruses infect humans, and BTV kills livestock, with devastating economic and social consequences. The proposed structural and functional studies focus on BTV to provide structural answers to fundamental questions of cell entry and transport of these and other non-enveloped dsRNA viruses. The results will provide profound insight into the structural basis of non-enveloped virus infection and reveal targets for rational development of anti-viral drugs and vaccines effective against these human and animal viruses.

描述（由申请人提供）：许多非发育的病毒会导致毁灭性的人类疾病。这些病毒进入宿主细胞的机制知之甚少，尽管可能有类似的包裹病毒，例如流感，艾滋病毒和疱疹病毒。我们已经研究了非发育的DSRNA病毒，并使用了单层（细胞质多角质病毒-CPV），双层（aquareovirus）和三层（Bluetongue病毒 - 或BTV）（或BTV），这是Revoviridae家族的Orbivirus Gentus）Capsid。由于其细胞生物学经过了充分的研究，并且具有单独的附着和渗透蛋白，因此BTV特别是研究该病毒细胞进入和运输的良好模型系统。因此，该项目的长期目标是通过冷冻电子显微镜（Cryoem）揭示非发育病毒感染的结构基础，尤其是病毒附着和进入的过程，以及后代病毒颗粒的细胞运输。 我们的7A分辨率冷冻结构BTV病毒粒子表明，在BTV渗透蛋白（VP5）的表面上存在中央三螺旋束和18座两倍的螺旋，在某些方面与包裹病毒的I类融合蛋白相似。将氨基酸分配到二级结构的分配构成了一组乞讨在原子分辨率上测试的假设。此外，通过使用低pH，我们将病毒转化为其穿透状态，并可以看到长长的“倒钩”的开花，我们假设我们假设是两亲的螺旋螺旋。此外，我们假设二硫键在VP5中的参与中参与了解体机制。我们的冷冻结构还表明，细胞附着蛋白（VP2）具有两个结合位点，一个用于唾液酸，一个用于未知靶分子，也许是整联蛋白。此外，我们的初步数据表明，非结构性蛋白质NS1在病毒释放中起作用，并以Zn2+依赖性方式形成高度有序的螺旋小管，从而为结构研究提供了解释其作用的机会。拟议的研究将 通过执行四个特定目的来检验这些假设：（1）通过确定天然BTV病毒素的原子结构，我们将测试我们的假设，即非发育BTV病毒的VP5渗透蛋白在其核心上具有三螺旋束，其表面上的18放置螺旋螺旋在其表面上是18次，并且在其表面上具有二氨基键的键。 （2）来自天然BTV病毒体的原子结构 在存在唾液酸的情况下，我们将检验假设VP2附着蛋白具有唾液酸结合位点。 （3）通过确定BTV粒子的（开花）穿透性状态（低pH）的结构，我们将测试我们假设的解体机制。此外，我们将进行基于结构的诱变研究，以补充结构研究，以建立触发突出的机制。 （4）来自螺旋小管中BTV非结构蛋白NS1的原子结构，然后进行基于功能和结构的诱变实验，我们将学习NS1如何调节病毒释放。这些机制和结构将与其他包裹和非发育病毒（包括上面引用的机制）相关。 公共卫生相关性：依伏迪科家族中的病毒是非发育的，DSRNA病毒感染了广泛的宿主，包括人，其他动物，植物，真菌和细菌；在这个家庭中，静脉病毒和轮状病毒感染了人类，而BTV则杀死了牲畜，造成了经济和社会后果。拟议的结构和功能研究的重点是BTV，为这些细胞进入和运输的基本问题和其他非发育DSRNA病毒提供了结构答案。结果将为非发育病毒感染的结构基础提供深刻的见解，并揭示了有效针对这些人类和动物病毒有效的抗病毒药物和疫苗合理发展的靶标。