Mitochondrially driven viral innate immunity in budding yeast

芽殖酵母中线粒体驱动的病毒先天免疫

• 批准号: RGPIN-2019-07230
• 负责人: Meneghini, Marc
• 金额: $ 2.33万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=739373
• 关键词: Mitochondrially; driven; viral; innate; immunity

S. cerevisiae is chronically infected with RNA viruses that rely on mitotic transmission for their persistence. We have discovered that the yeast homolog of mitochondrially localized endonuclease G, Nuc1, opposes the deleterious accumulation of these viruses in meiotic cells through an apparent ancestral cell death pathway. The most widely studied yeast viruses are the double stranded (ds) RNA virus L-A and its frequent "Killer" satellite viruses. The L-A genome encodes gag and gag-pol fusion proteins that assemble into virus-like-particles that support L-A propagation. Varied dsRNA "Killer" viruses exist as satellites of L-A. The most common is "M", which encodes a processed and secreted toxin called K1 that kills neighboring cells lacking immunity, which is conferred by the Killer genome itself. This proposal will describe investigations of an innate immune function for Nuc1 as well as of the mysterious relationship between L-A and the yeast host.     Yeast gametogenesis culminates in the development of 4 meiotic products within the remnant of the cell that produced them. We showed that programmed cell death (PCD) of this remnant occurs as an intrinsic aspect of sporulation and is executed through vacuolar rupture. Intriguingly, NUC1 is required for the fragmentation of genomic DNA from meiotic products frequently swept up in this PCD. This provocative association has remained puzzling however, as PCD proceeds unperturbed in the absence of NUC1. Notably, this is also the case with metazoan apoptosis and its associated DNA fragmentation factors (including endonuclease G), which have no known instructive role in PCD. Indeed, despite the ubiquitous association of mitochondrially driven DNA fragmentation pathways with diverse forms of PCD in multiple kingdoms of life, nothing is known of any adaptive roles they may play.    We have now determined that NUC1 functions in a rudimentary apoptotic module that protects sporulating yeast from lethal viral accumulation. Via a meiotically programmed transition, Nuc1 is released from mitochondria and accumulates in the cytosol in a manner dependent on the Por1/2 voltage dependent anion channel (VDAC), which is known to similarly mediate release of mitochondrial content during apoptosis. In the cytosol, Nuc1 functions to repress the accumulation of L-A and Killer dsRNA viruses. This developmentally programmed apoptotic-like process collaborates with the well-established yeast "innate immunity" SKI complex ("Super-Killer") to prevent a lethal accumulation of the Killer virus encoded toxin in the meiotic progeny. This proposal will advance these findings in three ways. We will examine the NUC1 anti-viral mechanism(s), address the regulation and nature of Por1/2 mediated Nuc1 release, and identify additional targets of Nuc1 during gametogenesis. These will involve continued hypothesis driven genetic, cell biological, and biochemical experiments as well as a simple but powerful genomic screening strategy.

酿酒酵母长期感染依赖有丝分裂传播的RNA病毒。我们已经发现，线粒体定位的内切酶G的酵母同源物Nuc1通过一条明显的祖先细胞死亡途径来反对这些病毒在减数分裂细胞中的有害积累。目前研究最广泛的酵母病毒是双链核糖核酸病毒L-A及其常见的“杀手”卫星病毒。L-A基因组编码Gag和Gag-Poll融合蛋白，这些融合蛋白组装成支持L-A繁殖的病毒样颗粒。各种dsRNA“杀手”病毒以L-A的卫星形式存在。最常见的是“M”，它编码一种名为K1的加工和分泌的毒素，这种毒素可以杀死缺乏免疫力的邻近细胞，这是由杀手基因组本身赋予的。这项建议将描述对Nuc1先天免疫功能的研究，以及L-A与酵母宿主之间的神秘关系。酵母配子发生的最终结果是在产生它们的细胞残留物内形成4种减数分裂产物。我们表明，这种残留物的程序性细胞死亡(PCD)是孢子形成的一个内在方面，并通过液泡破裂来执行。有趣的是，NUC1是从这种PCD中经常被扫过的减数分裂产物中分离基因组DNA所必需的。然而，这种挑衅性的联系仍然令人费解，因为PCD在NUC1缺席的情况下不受干扰地进行着。值得注意的是，后生动物细胞凋亡及其相关的DNA碎片因子(包括核酸内切酶G)也是如此，它们在PCD中没有已知的指导作用。事实上，尽管线粒体驱动的DNA碎片通路与多种形式的PCD在多个生命王国中普遍存在关联，但人们对它们可能扮演的适应性角色一无所知。我们现在已经确定NUC1在一个基本的凋亡模块中发挥作用，该模块可以保护产孢酵母免受致命病毒的积聚。通过减数分裂程序的转换，Nuc1从线粒体中释放出来，并以依赖于Por1/2电压依赖性阴离子通道(VDAC)的方式积聚在细胞质中，VDAC类似地介导了细胞凋亡过程中线粒体内容的释放。在胞浆中，Nuc1的功能是抑制L-A病毒和Killer双链RNA病毒的积聚。这一发育编程的凋亡样过程与成熟的酵母“先天免疫”滑雪复合体(“超级杀手”)合作，以防止杀手病毒编码的毒素在减数分裂后代中的致命积累。这项提案将从三个方面推进这些发现。我们将研究NUC1Nuc1的抗病毒机制(S)，阐述Por1/2介导的Nuc1释放的调节和性质，并确定Nuc1在配子发生过程中的其他靶点。这些将包括持续的假设驱动的遗传、细胞生物学和生化实验，以及简单但强大的基因组筛选策略。