Expanding the Genetic Code of Viruses

对病毒蛋白或任何蛋白质的研究，就这一点而言，取决于可视化和纯化蛋白质的能力，这通常依赖于特异性识别该蛋白质的抗体的可用性。这些抗体通过Western blotting、免疫沉淀的蛋白-蛋白相互作用、免疫荧光和共聚焦显微镜的蛋白质定位和与其他蛋白质共定位，促进了细胞内蛋白质水平的研究。不幸的是，有时很难为感兴趣的蛋白质产生“好的”抗体。为了避免这个问题，重组蛋白的版本可以包含各种标签，如GFP， HA或His。尽管这些方法是强大的，并且促进了许多蛋白质相关发现的发现，但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，导致人工结果的出现。******在过去的二十年里，通过使用非天然氨基酸（Uaa）来扩展人类基因组一直是一种推动力。迄今为止，已使用了100多种具有不同物理化学和生物特性的含有侧链的Uaa。目前，Uaa技术已被应用于细菌、酵母、哺乳动物细胞、干细胞、神经元、多细胞生物、昆虫、植物的研究，最近还应用于小鼠的研究。Uaa技术在病毒研究中的应用是一种令人兴奋的可能性，它使我们能够以过去无法做到的方式对病毒蛋白质进行分析。******本提案的目标是通过使用非天然氨基酸技术建立一个研究病毒蛋白质的程序。我们实验室研究丙型肝炎病毒（HCV）的分子生物学已有十年，为了在病毒背景下建立和验证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，Core蛋白能有效地结合Anap，且这些含有uaa的蛋白表现出未修饰的Core蛋白的典型细胞分布。随着这一体系的建立，我们将实现以下目标：******1。利用Uaa技术研究HCV p7蛋白。在p7蛋白中产生含有Uaa的HCV感染版本。为更广泛的病毒相关应用启动Uaa技术研究。******总之，在病毒背景下建立非天然氨基酸技术将使基于荧光的病毒蛋白质成像不需要肽或大蛋白质标签的修饰。更重要的是，这种策略可以应用于任何病毒的其他蛋白质，并且考虑到许多具有不同侧链的不同的Uaa可以以这种方式结合，我们将能够对感兴趣的蛋白质进行广泛的基于click -化学的分析。

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.