Rotavirus species B NSP1-1 contributions to tropism and spread

项目总结 轮状病毒是腹泻病的重要原因。而轮状病毒A种(RVA)导致地方性流行 婴幼儿腹泻病，轮状病毒B种(RVB)引起散发性流行性腹泻 主要影响成人的疾病。在RVA和RVB之间，只有NSP1基因组片段被预测为 编码非同源蛋白。对于RVB，该片段编码两种未知功能的蛋白质。缺乏 对NSP1编码的蛋白质功能的了解，加上缺乏RVB组织培养模型，已经 阻碍了RVB生物学的研究。我们最近发现，RVB NSP1编码的较小的蛋白质 片段NSP1-1是一种融合相关的小跨膜(FAST)蛋白，其表达导致 以寄主物种特有的方式形成合胞体。我们还发现RVB NSP1-1的表达促进了 RVA复制。FAST蛋白是由其他呼肠孤病毒科表达的模块化病毒非结构蛋白 病毒并促进病毒复制和致病。为了克服缺乏RVB文化模式的问题，我们 将使用RVA反向遗传学技术在可存活的背景下阐明NSP1-1的生物学功能 嵌合轮状病毒。基于我们的初步发现，我们提出了两个综合的具体目标来测试 假设轮状病毒NSP1-1是促进细胞间直接传播的模块趋向性决定因素。 FAST蛋白含有N-末端、跨膜和C-末端结构域。我们的初步研究表明 RVB NSP1-1分享了这个结构域组织，并证明了人RVB NSP1-1介导了 灵长类细胞，而不是啮齿动物细胞。在具体目标1中，我们将检验NSP1-1是模数FAST的假设 以宿主物种特有的方式发挥作用的蛋白质。我们将从以下公式确定NSP1-1的容量 不同的轮状病毒物种或菌株，以中介来自不同宿主物种的培养细胞的融合。我们 将通过在人RVB NSP1-1之间交换结构域来识别负责物种特异性的结构域 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在人畜共患病中的传播。在具体目标2中，我们将检验NSP1-1增强 病毒复制和传播。我们将使用最近使用的工程RVB NSP1-1表达RVA报告病毒- 开发反向遗传学技术并在胎牛在场的情况下量化它们的复制和传播 血清。胎牛血清抑制轮状病毒通过标准途径进入，从而允许病毒传播 主要是通过直接的细胞-细胞融合。这些研究将揭示NSP1-1在病毒感染和 提出关于其在发病机制中的作用的假说。总而言之，这些发现将提供 洞察RVA和RVB之间的流行病学差异。此外，工程嵌合轮状病毒 来自不同物种的包装片段将促进我们对其他轮状病毒的未来研究 缺乏培养模型，并发现了广泛适用于分段dsRNA病毒工程的概念。

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.