Rotavirus species B NSP1-1 contributions to tropism and spread

项目摘要 轮状病毒是导致腹泻的重要原因。虽然A型轮状病毒（RVA）引起地方性 轮状病毒B种（RVB）引起婴儿和儿童的腹泻病， 主要影响成年人的疾病。在RVA和RVB之间，只有NSP 1基因组片段被预测为 编码非同源蛋白质。对于RVB，该片段编码两种功能未知的蛋白质。缺乏 对NSP 1编码蛋白功能的了解，加上缺乏RVB组织培养模型， 阻碍了RVB生物学的研究。我们最近发现，由RVB NSP 1编码的较小蛋白质 片段NSP 1 -1是一种融合相关的小跨膜（FAST）蛋白，其表达导致 合胞体以宿主物种特异性的方式形成。我们还发现，RVB NSP 1 -1表达促进RVB NSP 1 -1的表达。 RVA复制。FAST蛋白是由其他呼肠孤病毒科病毒表达的模块化病毒非结构蛋白 病毒，并有助于病毒复制和发病机制。为了克服RVB文化模式的不足，我们 将使用RVA反向遗传学技术来阐明NSP 1 -1的生物学功能， 嵌合轮状病毒。基于我们的初步研究结果，我们提出了两个综合的具体目标，以测试 轮状病毒NSP 1 -1是一个促进细胞间直接扩散的模块向性决定子。 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 NSP 1 -1共享该结构域组织，并证明人RVB NSP 1 -1介导了RVB NSP 1 - 1与RVB NSP 1 -1的融合。 灵长类动物细胞而不是啮齿类动物细胞。在具体目标1中，我们将检验NSP 1 -1是模块化FAST的假设 以宿主物种特异性方式起作用的蛋白质。我们将根据以下内容确定NSP 1 -1的容量： 不同的轮状病毒物种或毒株来介导来自不同宿主物种的培养细胞的融合。我们 将通过在人RVB NSP 1-NSP 3之间交换结构域来鉴定负责种特异性的结构域。 1和p10，一种介导啮齿动物细胞融合的FAST蛋白。这些研究将确定NSP 1 -1的区域， 可能限制轮状病毒的人畜共患传播。在具体目标2中，我们将测试NSP 1 -1增强 病毒复制和传播。我们将利用最近的技术设计表达RVB NSP 1 -1的RVA报告病毒- 开发了反向遗传学技术，并量化了它们在胎牛存在下的复制和传播。 血清的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 主要通过直接细胞-细胞融合。这些研究将揭示NSP 1 -1在病毒感染过程中的功能， 产生关于其对发病机制的贡献的假设。总之，这些发现将提供 了解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未来对其他轮状病毒的研究， 缺乏培养模型和发现广泛适用于分段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.