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: 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=761246
• 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.

S.酿酒酵母被依赖于有丝分裂传播的RNA病毒慢性感染。我们已经发现，酵母同源物的nuc 1，本地化的内切核酸酶G，反对这些病毒在减数分裂细胞中的有害积累，通过一个明显的祖先细胞死亡途径。研究最广泛的酵母病毒是双链（ds）RNA病毒L-A及其常见的“杀手”卫星病毒。L-A基因组编码组装成支持L-A繁殖的病毒样颗粒的gag和gag-pol融合蛋白。各种dsRNA“杀手”病毒作为L-A的卫星存在。最常见的是“M”，它编码一种被称为K1的加工和分泌毒素，该毒素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。该提案将描述Nuc 1的先天免疫功能以及L-A和酵母宿主之间的神秘关系的调查。 酵母配子发生在产生它们的细胞残余物中的4个减数分裂产物的发育中达到高潮。我们发现，程序性细胞死亡（PCD）的这一残余发生作为一个内在方面的孢子形成，并通过液泡破裂执行。有趣的是，NUC 1是需要从减数分裂产物的基因组DNA片段经常席卷在这个PCD。然而，这种挑衅性的关联仍然令人困惑，因为PCD在没有NUC 1的情况下不受干扰地进行。值得注意的是，这也是后生动物细胞凋亡及其相关的DNA片段化因子（包括核酸内切酶G）的情况，其在PCD中没有已知的指导作用。事实上，尽管在生命的多个王国中，神经驱动的DNA片段化途径与不同形式的PCD普遍存在关联，但对它们可能发挥的任何适应性作用一无所知。 我们现在已经确定，NUC 1的功能在一个基本的凋亡模块，保护孢子酵母从致命的病毒积累。通过减数分裂程序化转换，Nuc 1从线粒体释放并以依赖于Por 1/2电压依赖性阴离子通道（VDAC）的方式在细胞溶质中积累，已知Por 1/2电压依赖性阴离子通道类似地介导细胞凋亡期间线粒体内容物的释放。在细胞质中，Nuc 1的功能是抑制L-A和Killer dsRNA病毒的积累。这种发育程序化的类嗜杀过程与成熟的酵母“先天免疫”SKI复合物（“超级杀伤者”）协作，以防止杀伤病毒编码的毒素在减数分裂后代中的致死性积累。本提案将从三个方面推进这些研究结果。我们将研究NUC 1的抗病毒机制，解决Por 1/2介导的Nuc 1释放的调节和性质，并确定配子发生过程中Nuc 1的其他靶点。这些将涉及持续的假设驱动的遗传，细胞生物学和生化实验，以及一个简单但强大的基因组筛选策略。