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: 10548132
• 负责人: Peter Shen
• 金额: $ 46.78万
• 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/10548132
• 关键词: Adenosine; Amino Acids; Award; Binding; Biochemical; Biogenesis; Biology; Categories; Category A pathogen; Cell physiology; Cells; Cellular biology; Charge; Code; Cryoelectron Microscopy; Cytoplasm; Cytosol; DNA Viruses; DNA biosynthesis; Double Stranded DNA Virus; Eukaryotic Initiation Factors; Event; Evolution; Face; Family member; Genetic Transcription; Goals; Guanosine Triphosphate; Human; In Vitro; Laboratories; Left; Mediating; Medical; Messenger RNA; Methylation; Modeling; Oncolytic; Oranges; Phosphorylation; Phosphotransferases; Phylogenetic Analysis; Plants; Poly A; 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; future pandemic; inorganic phosphate; insight; mRNA Translation; mRNA capping; mimetics; pandemic potential; priority pathogen; programs; protein function; prototype; receptors for activated C kinase; reconstitution; synergism; 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.

项目总结 痘病毒包括被归类为A类优先病原体的家庭成员，具有重要的未来 大流行的可能性，而其他家庭成员的修饰形式被广泛用作疫苗载体和 溶瘤疗法。除了它们更直接的医学意义外，痘病毒也有很长的历史 在发现基本的细胞过程方面处于前沿的宝贵的研究工具。其中包括 5‘-7-甲基GTP封端、2’-O-甲基化和3‘-聚腺苷的发现，我们现在知道它们发生在 几乎所有的真核生物以及病毒的mRNAs。这在一定程度上是因为痘病毒完全编码自己的 功能性DNA复制、转录和信使核糖核酸生物发生机制，使这些显著 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 完全依赖于获得宿主核糖体来合成病毒蛋白和复制。而当 病毒及其宿主细胞通过真核生物的活性控制翻译的机制 启动因子(EIF)是众所周知的，直到最近，核糖体在很大程度上被视为被动的密码- 缺乏内在监管能力的阅读机。在上一个奖项中，我们透露了痘病毒 在丝氨酸和苏氨酸残基上，B1能磷酸化许多核糖体亚单位蛋白(RPS) 没有在未感染的细胞或感染了其他病毒的细胞中被修改。这些包括磷酸化的 S278在小的40S RP，活化C激酶1受体(RACK1)的环状结构域中，其作用于 加强病毒mRNAs的翻译，这种病毒含有不同寻常的5‘-PolyA-Leaders。这样的领导人通常是由选举产生的 在哺乳动物宿主中，腺苷均聚体仅限于3‘未翻译的PolyA- Tail，但B1介导的磷酸化有效地模拟了在细胞内发现的带负电荷的氨基酸 双子叶植物和原生动物的RACK1环，其翻译系统自然地适应5‘波利亚。超越 对病毒操纵核糖体的洞察，这些发现提供了核糖体的第一个例子之一 翻译后修改，可以直接规范翻译，以及对 不同物种核糖体结构和功能的多样性。超越RACK1，它位于 MRNA退出通道，我们还在其他小的RPS中发现了额外的痘病毒特异性磷酸化事件， 包括位于信使核糖核酸进入通道的RPS28。生物化学和结构建模引导我们 假设这些修饰的RP形成一个相互连接的网络，该网络的磷酸化使 痘病毒重塑核糖体的mRNA通道，以更好地适应5‘-PolyA-Leader在其 哺乳动物的宿主。初步低温电子显微镜(CRYO-EM)和使用一种新的 开发的用痘病毒修饰的RPS的磷脂体重组的细胞系统进一步支持了这一点 假说，并暗示核糖体重塑或“物种再利用”达到前所未有的水平 这些磷酸化事件，这一更新申请建议研究精细的机制细节。