Rotavirus species B NSP1-1 contributions to tropism and spread

轮状病毒 B 种 NSP1-1 对趋向性和传播的贡献

• 批准号: 10040304
• 负责人: Kristen M Ogden
• 金额: $ 21.25万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-21 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040304
• 关键词: Adult; Affect; Animals; Architecture; Biological; Biological Process; Biology; C-terminal; Cell fusion; Cells; Cessation of life; Child; Coupled; Cultured Cells; Detection; Diarrhea; Disease; Double Stranded RNA Virus; Engineering; Epidemic; Epidemiology; Family; Family suidae; Future; Genes; Genome; Giant Cells; Human; In Vitro; Infant; Infection; Integral Membrane Protein; Intestines; Knowledge; Mediating; Methods; Modeling; Molecular; Molecular Virology; N-terminal; Nonstructural Protein; Open Reading Frames; Orthoreoviruses; Pathogenesis; Pathway interactions; Population; Primates; Proteins; Publishing; Reovirus; Reporter; Reporting; Research; Rodent; Rotavirus; SH2D3A gene; Source; Species Specificity; Specificity; System; Testing; Tropism; Viral; Viral Nonstructural Proteins; Viral Pathogenesis; Virulence; Virus; Virus Diseases; Virus Replication; Work; Zoonoses; age group; diarrheal disease; experimental study; fetal bovine serum; genetic manipulation; genetic technology; improved; insight; live cell imaging; prevent; protein function; protein structure function; reverse genetics; tissue culture; transmission process

PROJECT SUMMARY Rotaviruses are an important cause of diarrheal disease. While rotavirus species A (RVA) causes endemic diarrheal disease in infants and children, rotavirus species B (RVB) causes sporadic epidemic diarrheal disease primarily affecting adults. Between RVA and RVB, only the NSP1 genome segment is predicted to encode non-homologous proteins. For RVB, this segment encodes two proteins of unknown function. A lack of knowledge of NSP1-encoded protein function, coupled with the lack of an RVB tissue culture model, has impeded studies of RVB biology. We recently discovered that the smaller protein encoded by the RVB NSP1 segment, NSP1-1, is a fusion-associated small transmembrane (FAST) protein whose expression results in syncytia formation in a host species-specific manner. We also found that RVB NSP1-1 expression promotes RVA replication. FAST proteins are modular viral nonstructural proteins that are expressed by other Reoviridae viruses and contribute to viral replication and pathogenesis. To overcome the lack of an RVB culture model, we will use RVA reverse genetics technology to elucidate biological functions of NSP1-1 in the context of viable chimeric rotaviruses. Building on our preliminary findings, we propose two integrated specific aims to test the hypothesis that rotavirus NSP1-1 is a modular tropism determinant that promotes direct cell-cell spread. FAST proteins contain N-terminal, transmembrane, and C-terminal domains. Our preliminary studies suggest RVB NSP1-1 shares this domain organization and demonstrate that human RVB NSP1-1 mediates fusion of primate cells but not rodent cells. In Specific Aim 1, we will test the hypothesis that NSP1-1 is a modular FAST protein that functions in a host species-specific manner. We will determine the capacity of NSP1-1 from different rotavirus species or strains to mediate fusion of cultured cells derived from distinct host species. We will identify the domain responsible for species-specificity by exchanging domains between human RVB NSP1- 1 and p10, a FAST protein that mediates rodent cell fusion. These studies will identify regions of NSP1-1 that may limit rotavirus zoonotic transmission. In Specific Aim 2, we will test the hypothesis that NSP1-1 enhances viral replication and spread. We will engineer RVB NSP1-1-expressing RVA reporter viruses using recently- developed reverse genetics technology and quantify their replication and spread in the presence of fetal bovine serum. Fetal bovine serum inhibits rotavirus entry via standard pathways, thereby permitting virus spread primarily via direct cell-cell fusion. These studies will reveal functions of NSP1-1 during viral infection and generate hypotheses regarding its contributions to pathogenesis. Together, these discoveries will provide insight into epidemiological differences between RVA and RVB. Furthermore, engineering chimeric rotaviruses that package segments from divergent species will promote future studies of other rotaviruses for which we lack culture models and uncover concepts broadly applicable to segmented, dsRNA virus engineering.

项目摘要 轮状病毒是腹泻病的重要原因。轮状病毒物种A（RVA）引起了地方性 婴儿和儿童的腹泻病，轮状病毒物种B（RVB）引起零星流行性腹泻 疾病主要影响成年人。在RVA和RVB之间，仅预测NSP1基因组段 编码非同源蛋白。对于RVB，该段编码未知功能的两个蛋白质。缺乏 了解NSP1编码的蛋白质功能，再加上缺乏RVB组织培养模型的知识， RVB生物学的阻碍研究。我们最近发现，较小的蛋白质由RVB NSP1编码 段NSP1-1是一种融合相关的小型跨膜（快速）蛋白，其表达导致 以宿主特异性方式形成合胞体。我们还发现RVB NSP1-1表达促进 RVA复制。快速蛋白是模块化病毒非结构蛋白，由其他reoviridae表示 病毒并有助于病毒复制和发病机理。为了克服缺乏RVB文化模型，我们 将使用RVA反向遗传技术在可行的背景下阐明NSP1-1的生物学功能 嵌合轮病毒。在我们的初步发现的基础上，我们提出了两个集成的特定目的，以测试 轮状病毒NSP1-1是促进直接细胞细胞扩散的模块化的模块化决定因素。 快速蛋白包含N末端，跨膜和C末端结构域。我们的初步研究表明 RVB NSP1-1共享该领域组织，并证明人RVB NSP1-1介导 灵长类动物细胞，而不是啮齿动物细胞。在特定的目标1中，我们将测试NSP1-1是模块化的假设 以宿主特异性方式起作用的蛋白质。我们将确定NSP1-1的能力 不同的轮状病毒物种或菌株介导了源自不同宿主物种的培养细胞的融合。我们 将通过在人RVB NSP1之间交换域来确定负责物种特异性的领域 1和P10，一种介导啮齿动物细胞融合的快速蛋白。这些研究将确定NSP1-1的区域 可能会限制轮状病毒动物传播。在特定目标2中，我们将测试NSP1-1增强的假设 病毒复制和扩散。我们将使用最近的 - 开发了反向遗传技术，并量化其复制并在胎牛存在下传播 血清。胎牛血清通过标准途径抑制轮状病毒进入，从而允许病毒扩散 主要通过直接细胞融合。这些研究将揭示病毒感染期间NSP1-1的功能 就其对发病机理的贡献产生假设。这些发现将共同提供 洞悉RVA和RVB之间流行病学差异。此外，工程嵌合轮状病毒 来自不同物种的包装细分市场将促进对我们的其他轮状病毒的未来研究 缺乏文化模型和发现广泛适用于细分的DSRNA病毒工程的概念。