Mechanisms of Enzyme Regulation by Viperin in the Cellular Antiviral Response

总结 蝰蛇蛋白（病毒抑制蛋白;内质网相关，干扰素诱导）是一种自由基， SAM酶存在于所有6个生命王国中。它构成了一种古老的防御病毒的机制， 在包括人类在内的高等动物中保存的极少数自由基SAM酶之一。蝰蛇已经被证明 限制一些重要的人类病毒的感染性，包括甲型流感病毒、HIV、巨细胞病毒和 丙型肝炎蝰蛇素的抗病毒活性有两个方面。首先，它合成抗病毒核苷酸3 '-脱氧- 通过CTP的脱水制备3 '，4'-二脱氢-CTP（ddhCTP）。ddhCTP作为一种链终止抑制剂， 被一些但不是全部的病毒RNA依赖性RNA聚合酶掺入以破坏RNA病毒的复制 基因组其次，在高等动物中，蝰蛇蛋白也集中整合到更广泛的细胞抗病毒蛋白中。 通过广泛的蛋白质-蛋白质相互作用网络的反应。通过这些相互作用，毒蛇 下调对病毒复制重要的各种细胞途径，这解释了它如何能够限制 DNA病毒和逆转录病毒如巨细胞病毒和HIV的复制。 在上一个研究周期中，我们确定了蝰蛇蛋白调节的两个重要的生化途径：蛋白质 泛素化和胆固醇生物合成，并进一步确定了这些途径中的特定酶， 与蝰蛇蛋白相互作用我们现在的目标是在分子水平上了解蝰蛇蛋白和 其伴侣酶调节伴侣酶和蝰蛇蛋白的活性。这些研究旨在揭示 蝰蛇蛋白与不同蛋白质之间看似完全不同的相互作用的机制， 有助于其抗病毒特性。 我们的研究旨在回答以下问题： i）蝰蛇蛋白如何激活蛋白质泛素化系统中的E3泛素连接酶？ ii）蝰蛇蛋白如何调节参与胆固醇生物合成的酶？ （iii）。如何改变蝰蛇蛋白的结构/活性改变其抗病毒活性？ 为了回答这些问题，我们将重组蝰蛇蛋白与各种酶的复合物， 体外使用纯化的酶，并进行详细的酶动力学分析，结合生物物理 蛋白质和配体结合的测量，以研究激活或抑制的机制。 同时，我们将酌情使用晶体学和/或cryo-EM来确定 蝰蛇蛋白与这些酶的复合物。利用我们从这些研究中学到的东西，我们将测试我们的理解 在基于细胞的病毒感染模型中研究蝰蛇蛋白的抗病毒特性。

Summary Viperin (Virus Inhibitory Protein; Endoplasmic Reticulum-associated, Interferon iNducible) is a radical SAM enzyme found in all 6 kingdoms of life. It constitutes an ancient defense mechanism against viruses and is one of very few radical SAM enzymes conserved in higher animals, including humans. Viperin has been shown to restrict the infectivity of a number of important human viruses including influenza A, HIV, cytomegalovirus and hepatitis C. Viperin has two facets to its antiviral activity. First, it synthesizes the antiviral nucleotide 3’-deoxy- 3’,4’-didehydro-CTP (ddhCTP) by dehydration of CTP. ddhCTP acts as a chain-terminating inhibitor that is mis- incorporated by some, but not all, viral RNA-dependent RNA polymerases to disrupt the replication of RNA virus genomes. Second, in higher animals viperin is also centrally integrated into the broader cellular antiviral response through a wide-ranging network of protein-protein interactions. Through these interactions, viperin down-regulates various cellular pathways important for viral replication, which explains how it is able to restrict replication of DNA viruses and retroviruses such as cytomegalovirus and HIV. In the last grant cycle, we identified two important biochemical pathways that viperin modulates: protein ubiquitination and cholesterol biosynthesis, and further identified specific enzymes within those pathways that interact with viperin. Our goal now is to understand at the molecular level how interactions between viperin and its partner enzymes regulate the activity of both the partner enzyme and viperin. These studies aim to uncover the mechanisms that underpin viperin’s seemingly disparate interactions with different proteins and which contribute to its antiviral properties. Our studies aim to answer the following questions: i) How does viperin activate E3 ubiquitin ligases in the protein ubiquitination system? ii) How does viperin regulate enzymes involved in cholesterol biosynthesis? iii). How do changes to the structure/activity of viperin alter its antiviral activity? To answer these questions, we will reconstitute the complexes of viperin with the various in enzymes in vitro using purified enzymes and undertake detailed enzyme kinetic analyses, combined with biophysical measurements of protein and ligand binding, to study to study the mechanism(s) of activation or inhibition. Concurrently, we will use crystallography and/or cryo-EM, as appropriate, to determine structures for the complexes of viperin with these enzymes. Using what we learn from these studies, we will test our understanding of viperin’s antiviral properties in cell-based models of viral infection.