Assessing the interplay between the mono-ADP-ribosyltransferase ARTD10 and the Chikungunya viral macrodomain in modulating the antiviral immune response.

进入宿主细胞的病毒由先天免疫系统识别。为了有效地复制，病毒进化出了对抗免疫系统和劫持细胞过程的策略。最近的研究结果表明，翻译后修饰的单腺苷二磷酸核糖化(MAR化)是先天免疫反应的一部分。在这项提案中，我们计划定义MAR化在哺乳动物宿主与基孔肯雅病毒(CHIKV)之间的冲突中的作用。CHIKV近年来在全球范围内引起了大规模的疫情暴发，带来了严重的健康和经济负担。目前，既没有疫苗，也没有治疗方法。CHIKV只编码4种非结构蛋白，其中NSP3含有一个与MAR化密切相关的大结构域，即保守的蛋白质折叠。MAR化涉及ADP-核糖从NAD+转移到底物蛋白上，细胞内主要由ADP-核糖基转移酶类白喉毒素(ARTDS)催化。我们专注于ARTD10，它是对I型干扰素(IFN)的反应而诱导的。我们确定CHIKV的大结构域是一种脱甲基酶，这是有效复制病毒所必需的。此外，初步工作表明，nsP1-NSP3是ARTD10的底物，因此它们的活性可能受到MAR化的控制。这些发现使我们推测MAR化在先天性免疫中的作用，我们认为它以两种不同的方式发挥作用：(1)宿主依赖的CHIKV NSP的MAR化直接影响它们的功能，从而影响病毒的生命周期。(2)宿主因子的MAR化使它们能够促进抗病毒状态。这两个假说都暗示了病毒大结构域及其MAR水解酶功能的关键作用。因此，我建议澄清ARTD10催化的NSPS MAR化的生化和生理后果。这将涉及绘制修饰位点图，解决MAR化对单个NSP特定功能的影响，从而对病毒生命周期的影响。此外，我们的目标是鉴定受MAR化调控的宿主因子。为了确定这些，我们将确定ARTD10和CHIKV NSP3水解酶的共同底物。进一步的选择标准将是特定底物的MAR化是否受到干扰素的调节。我认为这些底物是重要的宿主因素，我们计划就它们与病毒复制的相关性进行调查。这将涉及蛋白质组学研究、单个细胞蛋白的MAR化分析、酶MAR化和去MAR化分析、细胞宿主因子的敲除和敲除以及对病毒生命周期的影响。总之，这些研究将确定MAR化如何用于抗病毒防御，以及CHIKV如何对抗这一先天免疫策略。这些研究的发现将与确定抗病毒治疗的新切入点相关。

Viruses entering host cells are recognized by the innate immune system. For efficient replication viruses evolved strategies to antagonize the immune system and to hijack the cellular processes. Recent findings suggest that mono-ADP-ribosylation (MARylation), a post-translational modification, is part of the innate immune response. In this proposal we plan to define the role of MARylation in the conflict between mammalian hosts and Chikungunya virus (CHIKV).CHIKV caused large epidemic outbreaks worldwide in recent years with severe health and economic burden. Presently neither a vaccine nor therapeutic treatments are available. CHIKV encodes only 4 non-structural proteins (nsPs), of which nsP3 harbors a macrodomain, a conserved protein fold, closely linked to MARylation. MARylation involves the transfer of ADP-ribose from NAD+ onto substrate proteins and is intracellularly mainly catalyzed by ADP-ribosyltransferases diphtheria toxin-like (ARTDs). We focus on ARTD10, which is induced in response to type I interferons (IFNs). We identified the CHIKV macrodomain as a de-MARylating enzyme, which is required for efficient viral replication. Moreover, preliminary work demonstrates that nsP1-nsP3 are substrates of ARTD10 and thus their activities potentially controlled by MARylation. These findings led us to hypothesize a role for MARylation in innate immunity, which we propose to function in two distinct ways: (I) Host-dependent MARylation of the CHIKV nsPs directly affects their function and thereby impacts on the viral life cycle. (II) MARylation of host-factors enables them to promote an antiviral state. Both hypotheses imply a critical role of the viral macrodomain and its MAR hydrolase function.Therefore, I propose to clarify the biochemical and physiological consequences of ARTD10-catalyzed MARylation of the nsPs. This will involve mapping the modification sites, addressing the impact of MARylation on specific functions of the individual nsPs, and in consequence on the viral life cycle. In addition, we aim at the identification of host factors regulated by MARylation. To determine those, we will identify common substrates of ARTD10 and the CHIKV nsP3 hydrolase. A further selection criterion will be whether the MARylation of a particular substrate is subject to IFN regulation. I assume that these substrates are important host factors that we plan to investigate regarding their relevance for viral replication. This will involve proteomic studies, the analysis of MARylation of individual cellular proteins, enzymatic MARylation and de-MARylation assays, the knock-down and knock-out of cellular host factors and the consequences on the viral life cycle. Together these studies will define how MARylation is used in antiviral defense, and how CHIKV antagonizes this innate immune strategy. The findings of these studies will be relevant to define novel entry points for antiviral therapies.