Non-canonical chimeric proteins generated during Adenovirus infection

项目总结 腺病毒(ADV)和其他基因组大小有限的人类DNA病毒使基因表达最大化 以及通过使用交替剪接和聚腺苷酸化的编码潜力。ADV的双链结构 DNA基因组已成为基因调控和rna重大发现的强大系统。 正在处理。有5个早期转录单位(E1a、E1B、E2、E3和E4)由宿主转录 RNA聚合酶II，并由细胞机械处理。每个病毒转录单位由指定的 启动子并通过选择性剪接产生多个mRNAs。产生的典型蛋白质 通过研究病毒突变体的感染情况，对每个病毒转录本的功能进行了分配。这个 E2区编码三种与病毒DNA复制有关的蛋白质。E4区编码多功能 参与病毒mRNAs转录、剪接和翻译调控以及拮抗的蛋白质 固有的细胞防御系统。我们结合了短读和直接长读测序技术来定义 ADV感染期间的病毒转录本，从而生成了对病毒最完整的注释 转录组。我们的分析确定了一些非规范的嵌合蛋白，它们融合了来自 不同的早期转录单位。在这里，我们将重点放在我们拥有的这些意想不到的基因产物之一 命名为E4orf6/DBP，因为它跨越E2和E4区域。我们的初步数据显示，这部小说 嵌合蛋白得到了强健的表达，我们认为它可以提供一个未知的功能 ADV感染。在目标1中，我们将评估文字记录是如何生成的，以及其相互作用的重要性 合伙人。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 产生或积累所有必要的试剂和技术专门知识，以确定 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架可以 不能反映病毒转录组的真正复杂性。我们的研究旨在揭示分子机制 通过提供对非规范融合蛋白的更深层次的理解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。该提案将产生一种审讯的示范方法 病毒与复杂病毒蛋白质组产生的非规范嵌合蛋白的功能。

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.