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.

项目摘要 腺病毒（ADV）和其他基因组大小有限的人DNA病毒具有最大的基因表达 通过使用替代剪接和聚腺苷酸化来编码潜力。 ADV双链 DNA基因组已成为基因调节和RNA中精神分裂的强大系统 加工。主机有五个早期转录单元（E1A，E1B，E2，E3和E4） RNA聚合酶II，并通过细胞机械处理。每个病毒转录单元均由指定的 启动子并产生替代剪接产生的多个mRNA。产生的规范蛋白 通过研究病毒突变体的感染，已经从每个病毒转录物中分配了功能。这 E2区域编码参与病毒DNA复制的三种蛋白质。 E4区域编码多功能 涉及调节转录，剪接和病毒mRNA的剪接和翻译的蛋白质以及对抗 固有的细胞防御。我们结合了短读和直接读取测序技术来定义 ADV感染期间的病毒转录本，因此产生了病毒的最完整注释 转录组。我们的分析确定了许多融合多肽的非经典嵌合蛋白 不同的早期转录单元。在这里，我们专注于我们拥有的这些意外基因产品之一 指定的E4ORF6/DBP跨越E2和E4区域。我们的初步数据表明这本小说 嵌合蛋白具有牢固的表达，我们建议它可以在期间提供尚未确定的功能 ADV感染。在AIM 1中，我们将评估成绩单的产生方式以及其交互的重要性 合作伙伴。在AIM 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 生成或累积的所有试剂以及定义功能所需的所有试剂和技术专长 E4ORF6/DBP嵌合蛋白。我们的发现挑战了离散的早期转录单元的概念 他们自己指定的启动子和poly（a）站点，并建议研究单独的开放阅读框 不能反映病毒转录组的真实复杂性。我们的研究旨在发现分子机制 通过对非经典融合蛋白的深入了解来调节病毒宿主相互作用 扩展病毒蛋白组的多样性。该提案将生成一种询问的模型方法 具有复杂病毒蛋白质组病毒产生的非典型嵌合蛋白的功能。