Using apathogenic and mammalian-ubiquitous reoviruses to safely discover genetic and phenotypic changes in viruses that promote adaptation to new host species and distinct niches within a host.

使用非致病性和哺乳动物普遍存在的呼肠孤病毒来安全地发现病毒的遗传和表型变化，从而促进对新宿主物种和宿主内独特生态位的适应。

• 批准号: RGPIN-2022-03418
• 负责人: Shmulevitz, Maya
• 金额: $ 2.48万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760782
• 关键词: Using; apathogenic; mammalian; ubiquitous; reoviruses

Viruses are continuously entering new hosts, and whether they thrive depends on their ability to complete all steps of replication while subduing anti-viral host responses. It is important to understand how genetic and phenotypic changes promote virus adaptation to new niches. Mammalian orthoreovirus (MRV) is a perfect virus to study adaptation since it infects a wide assortment of mammals without causing disease. MRV naturally infects intestinal epithelia; yet most of our understanding of MRV comes from viruses adapted for 70 years to non-enteric cells such as the L929 tumorigenic mouse fibroblasts. Using 4 "wild" MRVs from primary effluent, we discovered inconsistencies in knowledge from lab-adapted MRVs. For example, MRV are said to undergo the essential proteolytic capsid uncoating step within cells, mediated by lysosomal proteases. We discovered that wild MRVs poorly uncoat intracellularly and exhibit deficiencies in other yet-to-be identified replication steps in non-enteric cell cultures. After 5 passages on L929 cells and isolation of single clonogenic sub-isolates, polymorphisms in the reovirus outercapsid protein s3 enable intracellular uncoating. This proposal seeks to further apply MRV as a model system to better understand virus adaptation to cells from distinct tissues and mammalian sources. Aim 1 will establish the true molecular features of MRV. We will evaluate all major steps of MRV replication for lab-adapted versus wild MRVs, and in L929 cells versus normal polarized intestinal epithelial cells from porcine, bovine and rat origins. Given MRV naturally encounters digestive proteases during enteric infection, we will also establish the true kinetics of extracellular MRV uncoating and sensitivity to proteases from distinct mammals. Given that even lab-adapted MRV strains differ substantially in their ability to induce host IFN and NfkB signaling pathways, we will also establish the true host response to wild MRVs in the context of different host cell cultures. Finally, we will derive a mathematical model for replication of MRVs in intestinal cells that can be applied to characterize MRV fitness between hosts and virus variants. AIM 2 will discover the path(s) to adaptation of MRVs. Using the clonogenic sub-isolates of wild MRVs that adapted to uncoat intracellularly, we will now determine which polymorphisms in s3 enable intracellular uncoating, whether different wild MRVs take the same or different paths of adaptation to cell culture, whether the s3 polymorphisms were present in the wild quasispecies or arose in passage, what relationship s3 polymorphisms have on other s3 functions such as cell signaling, and what polymorphisms in other MRV genes arise to promote non-uncoating steps of adaptation. We will also passage wild MRVs on distinct intestinal cell cultures and apply our clonogenic isolation approach to discover what polymorphisms arise during adaptation to intestinal cultures from distinct mammalian species.

病毒不断地进入新的宿主，它们是否茁壮成长取决于它们完成所有复制步骤的能力，同时保持抗病毒宿主反应。重要的是要了解遗传和表型变化如何促进病毒适应新的生态位。哺乳动物正呼肠孤病毒（MRV）是研究适应性的完美病毒，因为它感染多种哺乳动物而不会引起疾病。MRV自然感染肠上皮细胞;然而，我们对MRV的大多数理解来自70年来适应非肠细胞的病毒，如L929致瘤小鼠成纤维细胞。使用4个“野生”的MRV从一级流出物，我们发现不一致的知识，从实验室适应的MRV。例如，据说MRV在细胞内经历由溶酶体蛋白酶介导的必需的蛋白水解衣壳脱壳步骤。我们发现野生型MRV在非肠细胞培养物中细胞内的脱膜能力较差，并且在其他有待鉴定的复制步骤中表现出缺陷。在L929细胞上传代5次并分离单个克隆源亚分离株后，呼肠孤病毒外衣壳蛋白s3的多态性使细胞内脱壳成为可能。该提案旨在进一步应用MRV作为模型系统，以更好地了解病毒对来自不同组织和哺乳动物来源的细胞的适应。 目的1建立MRV的真实分子特征。我们将评估实验室适应的MRV与野生型MRV，以及L929细胞与来自猪、牛和大鼠来源的正常极化肠上皮细胞中MRV复制的所有主要步骤。鉴于MRV在肠道感染期间自然遇到消化蛋白酶，我们还将建立细胞外MRV脱壳的真实动力学和对来自不同哺乳动物的蛋白酶的敏感性。鉴于即使是实验室适应的MRV毒株诱导宿主IFN和NfkB信号传导途径的能力也有很大差异，我们还将在不同宿主细胞培养物的背景下建立对野生型MRV的真实宿主应答。最后，我们将推导出MRV在肠细胞中复制的数学模型，该模型可用于表征宿主和病毒变体之间的MRV适应性。 AIM 2将发现MRV适应的路径。使用适于细胞内去包被的野生MRV的克隆源亚分离株，我们现在将确定s3中的哪些多态性能够实现细胞内去包被，不同的野生MRV是否采取相同或不同的适应细胞培养的途径，s3多态性是否存在于野生准种中或在传代中出现，s3多态性与其他s3功能如细胞信号传导有什么关系，以及其他MRV基因中出现的多态性促进了适应的非脱壳步骤。我们还将在不同的肠细胞培养物上传代野生MRV，并应用我们的克隆分离方法来发现在适应不同哺乳动物物种的肠培养物期间出现了什么样的多态性。