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: 9215409
• 负责人: Derek Walsh
• 金额: $ 39.19万
• 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 2021-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9215409
• 关键词: Affect; Africa; Antiviral Response; Attenuated Vaccines; Binding; Biology; Cells; Clinical; Color; Complex; Cowpox virus; Cytoplasm; DNA Viruses; DNA biosynthesis; Dangerousness; Data; Dependence; Development; Disease Outbreaks; Double Stranded DNA Virus; Elements; Eukaryotic Initiation Factors; Event; Evolution; Exhibits; FRAP1 gene; Face; Frequencies; Fright; Future; Genes; Genetic Transcription; Genetic Translation; Genome; Head; Health; Herpesviridae; Human; Immune response; Individual; Infection; Laboratories; Lesion; Life; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolic; Minor; Modern Medicine; Modernization; Modification; Molluscum Contagiosum; Monkeypox; Oncolytic; Oxidation-Reduction; Phosphorylation; Phosphotransferases; Polymerase; Population; Poxviridae; Poxviridae Infections; Protein Biosynthesis; Proteins; Proteomics; Raptors; Recording of previous events; Recruitment Activity; Recycling; Refractory; Regulation; Regulatory Pathway; Reporting; Research; Ribosomal Proteins; Ribosomes; Rodent; Role; Series; Signal Transduction; Site; Smallpox; Smallpox Vaccine; Smallpox Viruses; Stimulus; Structure; System; Taterapox virus; Therapeutic; Translating; Translations; Vaccinated; Vaccination; Vaccines; Vaccinia virus; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; Zoonoses; base; cancer therapy; combat; cytotoxicity; gene therapy; inhibitor/antagonist; insight; live cell imaging; new therapeutic target; novel strategies; pathogen; prototype; sensor; small molecule; therapeutic development; transcription factor; translation factor; transmission process; vector; vector vaccine

PROJECT SUMMARY Humans have a profound, double-edged relationship with poxviruses. On one hand, the devastating effects of smallpox are unparalleled by any other pathogen in recorded history, and its eradication is a milestone in modern medicine. On the other, poxviruses are now used as highly effective gene therapy and vaccine vectors as well as oncolytics in the treatment of cancer. Moreover, molluscum contagiosum is widespread and causes prolonged, untreatable lesions, while emerging poxviruses are a serious concern. Indeed, smallpox evolved from a rodent Taterapox virus and zoonotic poxvirus infections resulting in human-to-human transmission are being reported at an increasing frequency. In some cases smallpox vaccination does not provide protection, while live smallpox vaccines such as Vaccinia Virus (VacV) pose serious, life-threatening complications for many individuals. As such, whether it be infection by existing or future poxviruses, zoonotic infections or complications from therapeutic vectors, it is important to understand how these unusual pathogens replicate. Unlike most other double-stranded DNA viruses, poxviruses replicate in the cytoplasm of infected cells within viral factories (VFs). Encoding >200 genes that include their own polymerases, transcription factors and redox system, poxviruses exhibit remarkable self-sufficiency. Despite this, poxviruses remain absolutely dependent on gaining access to host ribosomes in order to synthesize viral proteins, representing an exploitable weakness. Indeed, we have shown previously that VacV activates the host cap-dependent translation machinery and that this can be targeted using small molecules to suppress virus replication without cytotoxicity. Our preliminary data identifies a series of new and unexpected modifications induced by VacV. This includes a viral protein that remodels mammalian Target of Rapamycin (mTOR), a key regulator of ribosome recruitment and host immune responses, displacing regulatory subunits to render mTOR constitutively active and beyond host control. In addition, mass spectrometry and dual-color live cell imaging revealed that VacV phosphorylates the small ribosomal subunit, RACK1 at unique sites not induced by other viruses or stimuli, and recruits RACK1 to VFs as they form. Moreover, we find that this modification is required for selective synthesis of late VacV proteins, but not proteins of other viruses, and is induced by a VacV kinase. Finally, proteomic analysis of ribosome complexes isolated from primary human cells further revealed that VacV induces highly selective modifications to other ribosomal proteins and to the subunit composition of ribosomes themselves. Our data suggests that this “ribosome specialization” is important for poxvirus protein synthesis, and is dispensable to the host. Understanding how these modifications facilitate VacV protein synthesis will provide important insights into fundamental aspects of poxvirus biology as well as mechanisms of selective mRNA translation. In addition, identifying factors involved in selective viral versus host protein synthesis has the potential to uncover new therapeutic targets and approaches to combat poxvirus infection.

项目总结 人类与痘病毒有一种深刻的、双刃剑的关系。一方面，这场灾难的破坏性影响 在有记录的历史上，天花是任何其他病原体都无法比拟的，它的根除是 现代医学。另一方面，痘病毒现在被用作高效的基因治疗和疫苗载体。 以及肿瘤溶解剂在癌症治疗中的作用。此外，传染性软体动物病的流行和引起 长期的、无法治疗的损害，而新出现的痘病毒是一个严重的问题。事实上，天花是进化出来的 来自啮齿动物的Taterapox病毒和人畜共患病的痘病毒感染导致人与人之间的传播 以越来越高的频率被报道。在某些情况下，接种天花疫苗并不能提供保护， 虽然天花活疫苗如痘苗病毒(VacV)会对 很多人。因此，无论是由现有的或未来的痘病毒感染，人畜共患感染或 对于治疗媒介的并发症，重要的是了解这些不寻常的病原体是如何复制的。 与大多数其他双链dna病毒不同，痘病毒在体内感染细胞的细胞质中复制。 病毒工厂(VFS)。编码200个基因，包括它们自己的聚合酶、转录因子和氧化还原 系统中，痘病毒表现出惊人的自给自足能力。尽管如此，痘病毒仍然是绝对依赖的 在获得宿主核糖体以合成病毒蛋白方面，代表了一种可开发的 软弱。事实上，我们之前已经证明了VacV激活了依赖于宿主帽的翻译 可以使用小分子来抑制病毒复制，而不需要 细胞毒性。我们的初步数据确定了VacV引起的一系列新的和意想不到的修改。 这包括一种病毒蛋白，它可以重塑哺乳动物的雷帕霉素靶标(MTOR)，mTOR是雷帕霉素的关键调节因子 核糖体募集和宿主免疫反应，取代调节亚基提供mTOR 体质活跃且不受宿主控制。此外，质谱学和双色活细胞成像 发现VacV使核糖体小亚基RACK1在独特的位置上磷酸化，而不是由其他基因诱导 病毒或刺激，并在他们形成时招募RACK1到VFS。此外，我们发现这一修改是必要的 用于选择性合成晚期VacV蛋白，而不是其他病毒的蛋白，并由VacV诱导 激活剂。最后，从原代人类细胞中分离出的核糖体复合体的蛋白质组学分析进一步揭示了 VacV可诱导对其他核糖体蛋白及其亚基组成的高度选择性修饰 核糖体本身。我们的数据表明，这种“核糖体特化”对痘病毒蛋白很重要。 合成，对宿主来说是可有可无的。了解这些修饰如何促进VacV蛋白 合成将对痘病毒生物学的基本方面以及机制提供重要的见解 选择性信使核糖核酸的翻译。此外，识别参与选择性病毒与宿主蛋白的因素 合成有可能发现新的治疗靶点和抗痘病毒感染的方法。