Expanding the Genetic Code of Viruses

扩展病毒的遗传密码

• 批准号: RGPIN-2018-06357
• 负责人: Russell, Rodney
• 金额: $ 2.62万
• 依托单位: Memorial University of Newfoundland
• 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=746271
• 关键词: Expanding; Genetic; Code; Viruses

The study of a viral protein, or any protein, for that matter, depends on the ability to visualize and purify that protein, which typically relies on the availability of an antibody that specifically recognizes that protein. Such antibodies facilitate the study of protein levels within a cell through Western blotting, protein-protein interactions by immunoprecipitation, and protein localization and colocalization with other proteins through immunofluorescence and confocal microscopy. Unfortunately, it is sometimes difficult to generate a “good” antibody for a protein of interest. To circumvent this issue, recombinant versions of proteins can be engineered containing various tags, such as GFP, HA or His. Although such methods are powerful and have facilitated the discovery of relevant findings for many proteins, the structural impact of such tags can affect the function and topology of a protein, resulting in the appearance of artefactual results.Over the past two decades there has been a push to expand the human genome through the use of unnatural amino acids (Uaa). To date over 100 Uaa containing side chains with diverse physiochemical and biological properties are in use. At this point Uaa technology has been applied to the study of bacteria, yeast, mammalian cells, stem cells, neurons, multicellular organisms, insects, plants, and very recently, in mice. The application of Uaa technology to the study of viruses is an exciting possibility that allows us to perform analyses of viral proteins in ways that we have not been able to in the past.The goal of this proposal is to establish a program for the study of viral proteins through the use of unnatural amino acid technology. Our lab has been studying the molecular biology of the hepatitis C virus (HCV) for ten years, and to establish and validate Uaa technology in the context of a virus, we have tested whether the fluorescent Uaa Anap can be incorporated into the HCV Core protein. Preliminary results showed that Core proteins efficiently incorporated Anap, and that these Uaa-containing proteins displayed the typical cellular distribution of unmodified Core proteins. With this system now established, we will carry out the following objectives:1. To use Uaa technology to study the HCV p7 protein.2. To generate an infectious version of HCV containing a Uaa within the p7 protein.3. Initiation of Uaa technology studies for broader virus-related applications.In summary, the establishment of unnatural amino acid technology in the context of viruses will enable fluorescence-based imaging of virus proteins without modification by peptides or large protein tags. More importantly, this strategy can be applied to other proteins of any virus, and given that numerous different Uaa with various side chains can be incorporated in this manner, we will be able to perform a broad spectrum of Click-Chemistry-based analyses on proteins of interest.

对病毒蛋白或任何蛋白质的研究取决于可视化和纯化该蛋白质的能力，这通常依赖于特异性识别该蛋白质的抗体的可用性。这些抗体有助于通过Western印迹、通过免疫沉淀的蛋白质-蛋白质相互作用以及通过免疫荧光和共聚焦显微镜的蛋白质定位和与其他蛋白质的共定位来研究细胞内的蛋白质水平。不幸的是，有时很难产生针对感兴趣蛋白质的“好”抗体。为了避免这个问题，蛋白质的重组版本可以被工程化，包含各种标签，如GFP，HA或His。虽然这些方法是强大的，并促进了许多蛋白质的相关发现的发现，这些标签的结构影响可能会影响蛋白质的功能和拓扑结构，从而导致假象的结果的出现。在过去的二十年中，一直在推动通过使用非天然氨基酸（Uaa）来扩展人类基因组。迄今为止，超过100种具有不同理化和生物学性质的含Uaa侧链正在使用。在这一点上，Uaa技术已被应用于研究细菌，酵母，哺乳动物细胞，干细胞，神经元，多细胞生物，昆虫，植物，最近，在小鼠。将Uaa技术应用于病毒研究是一种令人兴奋的可能性，它使我们能够以过去无法实现的方式分析病毒蛋白质。本提案的目标是建立一个通过使用非天然氨基酸技术研究病毒蛋白质的计划。我们的实验室已经研究了丙型肝炎病毒（HCV）的分子生物学十年，为了在病毒的背景下建立和验证Uaa技术，我们测试了荧光Uaa Anap是否可以并入HCV核心蛋白。初步结果表明，核心蛋白有效地纳入Anap，这些Uaa的蛋白质显示出典型的细胞分布的未修饰的核心蛋白。建立这一制度后，我们将实现以下目标：1.利用Uaa技术研究HCV p7蛋白.产生在p7蛋白内含有Uaa的HCV的感染性版本。启动Uaa技术研究，以实现更广泛的病毒相关应用。总之，在病毒背景下建立非天然氨基酸技术将使病毒蛋白质的荧光成像成为可能，而无需通过肽或大蛋白质标签进行修饰。更重要的是，这种策略可以应用于任何病毒的其他蛋白质，并且考虑到可以以这种方式掺入具有各种侧链的许多不同的Uaa，我们将能够对感兴趣的蛋白质进行广泛的基于点击化学的分析。