Poxvirus manipulation of the host cell protein synthesis machinery

痘病毒操纵宿主细胞蛋白质合成机器

• 批准号: 10316611
• 负责人: Peter Shen
• 金额: $ 48.28万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-11-23 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10316611
• 关键词: Adenosine; Amino Acids; Award; Binding; Biochemical; Biogenesis; Biology; Category A pathogen; Cell physiology; Cells; Cellular biology; Charge; Code; Cryoelectron Microscopy; Custom; Cytoplasm; Cytosol; DNA Viruses; DNA biosynthesis; Double Stranded DNA Virus; Eukaryotic Initiation Factors; Event; Evolution; Face; Family member; Future; Genetic Transcription; Genetic Translation; Goals; Guanosine Triphosphate; Human; In Vitro; Laboratories; Lead; Left; Mediating; Medical; Messenger RNA; Methylation; Modeling; Oncolytic; Oranges; Phosphorylation; Phosphotransferases; Phylogenetic Analysis; Plants; Polyadenylation; Post-Translational Protein Processing; Poxviridae; Protein Biosynthesis; Protein Subunits; Proteins; RPS3 gene; Reading; Recording of previous events; Research; Ribosomes; Serine; Signaling Molecule; Site; Smallpox; Smallpox Vaccine; Structure; Surface; System; Tail; Testing; Therapeutic; Threonine; Translations; Vaccination; Vaccinia virus; Variola major virus; Viral; Viral Proteins; Virus; Virus Replication; Zoonoses; inorganic phosphate; insight; mRNA capping; pandemic disease; priority pathogen; programs; protein function; prototype; receptors for activated C kinase; reconstitution; tool; vector vaccine

PROJECT SUMMARY Poxviruses include family members that are categorized as Class A Priority Pathogens with significant future pandemic potential, while modified forms of other family members are widely used as vaccine vectors and oncolytic therapeutics. Beyond their more direct medical significance, poxviruses also have a long history as invaluable research tools at the forefront in the discovery of fundamental cellular processes. These include the discovery of 5’ 7-Methyl GTP capping, 2’-O-Methylation and 3’ polyadenylation that we now know to occur on almost all eukaryotic as well as viral mRNAs. This is in part because poxviruses encode their own fully functional DNA replication, transcription and mRNA biogenesis machinery, which enables these remarkably self-sufficient DNA viruses to replicate in the cytoplasm. Despite this, like all other viruses, poxviruses remain entirely dependent on gaining access to host ribosomes to synthesize viral proteins and replicate. While the mechanisms by which both viruses and their host cells control translation through the activity of eukaryotic initiation factors (eIFs) are well understood, until recently ribosomes were largely viewed as passive code- reading machines that lacked intrinsic regulatory capacity. In the prior award, we revealed that the poxvirus kinase, B1 phosphorylates a number of ribosomal subunit proteins (RPs) at Serine and Threonine residues that are not modified in uninfected cells or cells infected with other viruses. These include phosphorylation of S278 in a loop domain of the small 40S RP, Receptor for Activated C Kinase 1 (RACK1) that serves to enhance translation of viral mRNAs that harbor unusual 5’ polyA-leaders. Such leaders are normally selected against in their mammalian hosts where adenosine homopolymers are restricted to the 3’ untranslated polyA- tail, but B1-mediated phosphorylation effectively mimics negatively charged amino acids that are found in the RACK1 loop of dicot plants and protists whose translation systems naturally accommodate 5’ polyA. Beyond insights into viral manipulation of ribosomes, these findings provided one of the first examples of ribosomal post-translational modifications that can regulate translation directly as well as unexpected insights into the structural and functional diversification of ribosomes across different species. Beyond RACK1, which lies at the mRNA exit channel, we also identified additional poxvirus-specific phosphorylation events in other small RPs, including RPS28 that lies at the mRNA entry channel. Biochemical and structure modeling lead us to hypothesize that these modified RPs form an interconnected network whose phosphorylation enables poxviruses to remodel the mRNA channel of the ribosome to better accommodate 5’ polyA-leaders in their mammalian hosts. Preliminary cryo electron microscopy (cryo-EM) and functional studies using a newly developed cell system reconstituted with phosphomimetics of poxvirus-modified RPs further supports this hypothesis and suggests an unprecedented level of ribosome remodeling or “species repurposing” arises from these phosphorylation events, which this renewal application proposes to study in fine mechanistic detail.

项目摘要 痘病毒包括被归类为具有重要未来的A类优先病原体的家庭成员 大流行潜力，而其他家庭成员的修改形式被广泛用作疫苗向量和 溶瘤理论。除了他们更直接的医学意义之外，痘病毒还具有悠久的历史 在发现基本细胞过程的最前沿的宝贵研究工具。这些包括 发现5'7-甲基GTP上限，2'-O-甲基和3'聚腺苷酸化，我们现在知道会发生在 几乎所有的真核和病毒mRNA。这部分是因为痘病毒完全编码自己的 功能性DNA复制，转录和mRNA生物发生机制，可以显着实现这些 自给自足的DNA病毒在细胞质中复制。尽管如此，像所有其他病毒一样，痘病毒仍然存在 总体上取决于获得宿主核糖体的访问来合成病毒蛋白并复制。而 病毒及其宿主细胞通过真核的活性控制翻译的机制 启动因子（EIF）已得到充分了解，直到最近核糖体被视为被动代码 - 阅读机器缺乏固有的监管能力。在先前的奖项中，我们透露了痘病毒 激酶，B1在丝氨酸和苏氨酸残基上磷酸化许多核糖体亚基蛋白（RPS） 在未感染的细胞或感染其他病毒的细胞中未经修饰的。这些包括 S278在小40S RP的环域中 增强具有异常5'Polya-Leader的病毒mRNA的翻译。通常选择这样的领导者 反对在其哺乳动物的宿主中，腺苷均聚物仅限于3'未翻译的polya- 尾巴，但B1介导的磷酸化有效地模仿了在 Dicot植物和原生生物的RACK1循环，其翻译系统自然可容纳5'Polya。超过 对核糖体病毒操纵的见解，这些发现提供了核糖体的第一个例子之一 可以直接调节翻译以及意外见解的翻译后修改 核糖体跨不同物种的结构和功能多样化。超越架子1，位于 mRNA出口通道，我们还确定了其他小RPS中的其他痘病毒特异性磷酸化事件， 包括位于mRNA进入频道的RPS28。生化和结构建模导致我们进入 假设这些修饰的RP形成了一个相互联系的网络，其磷酸化可以实现 斑病毒重塑核糖体的mRNA通道以更好地容纳5'Polya-Leader 哺乳动物宿主。初步冷冻电子显微镜（冷冻EM）和功能研究使用新近 开发的细胞系统与痘病毒修饰的RP的磷光学重构进一步支持 假设并提出了前所未有的核糖体重塑或“重新利用物种”的水平 这些磷酸化事件，该更新应用提案以精细的机械细节进行研究。