Non-canonical chimeric proteins generated during Adenovirus infection

腺病毒感染期间产生的非典型嵌合蛋白

• 批准号: 10312411
• 负责人: Matthew D. Weitzman
• 金额: $ 22万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-09 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312411
• 关键词: Address; Adenovirus Infections; Adenovirus Protein; Adenoviruses; Affect; Alternative Splicing; Antiviral Agents; Cell Line; Cell physiology; Cells; Chimeric Proteins; Code; Complement; Complex; DNA; DNA Packaging; DNA Viruses; DNA biosynthesis; DNA-Binding Proteins; Data; Development; Family; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Genome; Goals; Host Defense; Individual; Infection; Knowledge; Messenger RNA; Modeling; Molecular; Morphogenesis; Morphology; Nuclear; Open Reading Frames; Outcome; Pathway interactions; Phase; Poly A; Polyadenylation; Positioning Attribute; Production; Proteins; Proteome; RNA; RNA Polymerase II; RNA Processing; RNA Splicing; Reagent; Research; Resolution; Seminal; Series; Signal Transduction; Site; System; Technical Expertise; Technology; Transcript; Transcriptional Regulation; Translations; Viral; Viral Genes; Viral Genome; Viral Packaging; Viral Proteins; Virus; Virus Diseases; Virus Replication; base; ds-DNA; gene product; human DNA; mutant; novel; polypeptide; promoter; protein function; transcriptome; viral DNA; viral RNA; virus host interaction

PROJECT SUMMARY Adenovirus (AdV) and other human DNA viruses with limited genome size have maximized gene expression and coding potential through the use of alternative splicing and polyadenylation. The AdV double-stranded DNA genome has served as a powerful system for seminal discoveries in gene regulation and RNA processing. There are five early transcription units (E1A, E1B, E2, E3 and E4) which are transcribed by host RNA polymerase II and processed by cellular machinery. Each viral transcription unit is driven by a designated promoter and gives rise to multiple mRNAs generated by alternative splicing. The canonical proteins produced from each viral transcript have been assigned functions through the study of infection with viral mutants. The E2 region encodes three proteins involved in viral DNA replication. The E4 region encodes multifunctional proteins involved in regulation of transcription, splicing and translation of viral mRNAs, as well as antagonizing intrinsic cellular defenses. We combined short-read and direct long-read sequencing technologies to define viral transcripts during AdV infection, and thus generated the most complete annotation of the virus transcriptome. Our analysis identified a number of non-canonical chimeric proteins that fuse polypeptides from different early transcriptional units. Here we focus on one of these unexpected gene products that we have designated E4orf6/DBP since it spans the E2 and E4 regions. Our preliminary data show that this novel chimeric protein is robustly expressed, and we propose that it could provide a yet unidentified function during AdV infection. In Aim 1 we will evaluate how the transcript is generated, and the importance of its interacting partners. In Aim 2 we will determine functions of the fusion protein, and its impact on infection. We have generated or accumulated all reagents and technical expertise necessary to define the functions of the E4orf6/DBP chimeric protein. Our findings challenge the concept of discrete early transcriptional units with their own designated promoter and poly(A) site, and suggest that studying individual open reading frames may not reflect the true complexity of the viral transcriptome. Our studies aim to uncover molecular mechanisms that regulate virus-host interactions by providing a deeper understanding of non-canonical fusion proteins that expand the diversity of the viral proteome. The proposal will generate a model approach for interrogating functions of non-canonical chimeric proteins generated by viruses with complex viral proteomes.

项目概要 腺病毒 (AdV) 和其他基因组大小有限的人类 DNA 病毒最大限度地提高了基因表达 以及通过使用选择性剪接和聚腺苷酸化的编码潜力。 AdV 双链 DNA 基因组已成为基因调控和 RNA 领域开创性发现的强大系统 加工。有五个由宿主转录的早期转录单位（E1A、E1B、E2、E3 和 E4） RNA 聚合酶 II 并由细胞机器加工。每个病毒转录单元由指定的驱动 启动子并通过选择性剪接产生多个 mRNA。产生的经典蛋白质 通过病毒突变体感染的研究，每个病毒转录本都被分配了功能。这 E2区编码参与病毒DNA复制的三种蛋白质。 E4区编码多功能 参与病毒 mRNA 转录、剪接和翻译调节以及拮抗的蛋白质 内在的细胞防御。我们结合了短读长和直接长读长测序技术来定义 AdV感染期间的病毒转录本，从而生成病毒最完整的注释 转录组。我们的分析鉴定了许多非经典嵌合蛋白，它们融合了来自 不同的早期转录单位。在这里，我们重点关注我们拥有的这些意想不到的基因产品之一 指定为 E4orf6/DBP，因为它跨越 E2 和 E4 区域。我们的初步数据表明，这部小说 嵌合蛋白被稳健表达，我们认为它可以在过程中提供尚未识别的功能 AdV 感染。在目标 1 中，我们将评估转录本的生成方式及其交互的重要性 合作伙伴。在目标 2 中，我们将确定融合蛋白的功能及其对感染的影响。我们有 产生或积累了定义功能所需的所有试剂和技术专业知识 E4orf6/DBP嵌合蛋白。我们的发现挑战了离散早期转录单位的概念 他们自己指定的启动子和poly(A)位点，并建议研究单个开放阅读框可能 不能反映病毒转录组的真实复杂性。我们的研究旨在揭示分子机制 通过更深入地了解非规范融合蛋白来调节病毒与宿主的相互作用 扩大病毒蛋白质组的多样性。该提案将生成一个用于询问的模型方法 由具有复杂病毒蛋白质组的病毒产生的非典型嵌合蛋白的功能。