Elucidating the mechanisms of alphavirus subgenomic RNA translation

阐明甲病毒亚基因组 RNA 翻译机制

• 批准号: 10678281
• 负责人: Kenneth Harold Dinnon
• 金额: $ 6.91万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678281
• 关键词: 2019-nCoV; Ablation; Acute; Alphavirus; Alphavirus Infections; Amino Acid Substitution; Amino Acids; Antiviral Agents; Arsenites; Attenuated; Binding; Binding Proteins; Biological Models; Biology; COVID-19 pandemic; Cells; Chimeric Proteins; Code; Complex; DNA-Directed RNA Polymerase; Defect; Development; Disease Outbreaks; Double-Stranded RNA; Drug Targeting; Drug usage; Eastern Equine Encephalitis Virus; Excision; FDA approved; Fever; G3BP1 gene; Genetic Transcription; Goals; Homeostasis; Human; In Vitro; Infection; Innate Immune Response; Internal Ribosome Entry Site; Knowledge; Learning; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Methods; Mutation; Neurologic; Nonstructural Protein; Organelles; Peptide Initiation Factors; Phosphorylation; Poly A; Poly(A)-Binding Proteins; Polyadenylation; Prokaryotic Initiation Factor-2; Protease Inhibitor; Protein Biosynthesis; Proteins; RNA; Replication-Associated Process; Replicon; Repression; Research; Resistance; Reticulocytes; Reverse engineering; Ribosomes; Role; Semliki forest virus; Sindbis Virus; Stress; Structural Protein; Sucrose; System; Testing; Therapeutic; Transfection; Translating; Translation Initiation; Translations; Vaccines; Viral; Viral Nonstructural Proteins; Viral Proteins; Viral Structural Proteins; Virus; Virus Replication; Work; arthropod-borne; biological adaptation to stress; chikungunya; density; druggable target; forest; gene therapy; insight; interest; medical countermeasure; mutant; mutation screening; next generation; novel; novel therapeutic intervention; pandemic pathogen; pandemic potential; pathogen; pre-pandemic; prevent; protein kinase R; reconstitution; recruit; response; scaffold; side effect; stress granule; stress reduction; stressor; targeted treatment; vector; vector vaccine; viral transmission

PROJECT SUMMARY/ABSTRACT Alphaviruses are arthropod-transmitted viruses that cause acute febrile illness in humans, with some cases causing long-term skeletomuscular and neurological sequelae. Currently there are no FDA-approved vaccines or therapeutics for alphavirus infection, and continued global outbreaks of chikungunya and Eastern equine encephalitis virus highlight the need for medical countermeasures. Effective antiviral treatments target key viral replication processes without host side-effect are built on the foundational knowledge of viral replication as evidenced by the rapid development of RNA polymerase and viral protease inhibitors towards SARS-CoV-2 during the COVID-19 pandemic. While alphaviruses have been studied for decades, there are many foundational gaps in our understanding of their biology. Like all viruses, alphavirus infection of host cells induces a multifaceted innate immune response, of which one result is the activation of the integrated stress response (ISR) through viral double-stranded RNA sensing by host protein kinase R (PKR) and subsequent phosphorylation of translation initiation factor 2 (eIF2a), causing shutoff of global protein translation. However, alphaviruses have evolved a mechanism by which their structural proteins are uniquely still efficiently translated from the viral subgenomic RNA (sgRNA) during ISR activation. While many canonical translation initiation factors have been ruled dispensable for sgRNA translation, the mechanism of sgRNA translation is yet to be described. Interestingly, the resistance of the sgRNA to the ISR is only observed in infected cells, suggesting viral factors are necessary. Other work has shown that viral nonstructural protein nsP3 interacts with components of stress granules, organelles formed during ISR activation to sequester translation machinery until relief of the stress and return to homeostasis, suggesting a role for nsP3 in modulating translation during alphavirus infection-induced ISR. Together, this leads to the hypothesis that alphaviruses evade the ISR through recruitment of non-canonical initiation factors or the ribosome directly, and that ISR resistance is mediated in part through interactions of the nsP3 and host proteins. Using Sindbis virus (SINV) as a model system, this proposal seeks to identify how alphaviruses evade the ISR to maintain translation of viral structural proteins with hopes of identifying druggable targets to prevent alphavirus replication and spread. Aim 1 seeks to identify translation initiation factors responsible for sgRNA translation during ISR activation and eIF2a phosphorylation. Aim 2 will address the role of viral factors, specifically the hypervariable domain (HVD) of nsP3, in modulating the ISR and sgRNA translation through use of deep mutational scanning methods. Successful completion of this proposed work will provide mechanistic insight for an under-characterized phenomenon observed for decades in alphavirus research, allowing for novel therapeutic interventions for alphavirus infections.

项目摘要/摘要 甲病毒是节肢动物传播的病毒，可引起人类急性发热性疾病，其中一些 导致长期骨骼肌和神经系统后遗症的病例。目前，没有FDA批准的 甲病毒感染的疫苗或治疗剂，以及基孔肯雅热和东部 马脑炎病毒突出了医疗对策的必要性。有效的抗病毒治疗目标 没有宿主副作用的关键病毒复制过程建立在病毒复制的基础知识之上 针对SARS-CoV-2的RNA聚合酶和病毒蛋白酶抑制剂的快速发展证明了这一点 在COVID-19大流行期间。虽然甲病毒已经研究了几十年，但仍有许多基本的 我们对它们生物学的理解存在着差距。像所有病毒一样，甲病毒感染宿主细胞诱导了一种新的病毒， 多方面的先天免疫反应，其中一个结果是激活综合应激反应 (ISR)通过宿主蛋白激酶R（PKR）和随后的病毒双链RNA传感， 翻译起始因子2（eIF 2a）的磷酸化，导致整体蛋白质翻译的关闭。然而，在这方面， 甲病毒已经进化出一种机制，通过这种机制，它们的结构蛋白可以被独特而有效地翻译 从病毒亚基因组RNA（sgRNA）在ISR激活。虽然许多经典的翻译起始因子 尽管SgRNA的翻译已经被排除在外，但SgRNA翻译的机制仍有待描述。 有趣的是，sgRNA对ISR的抗性仅在感染的细胞中观察到，这表明病毒因素 是必要的。其他研究表明，病毒非结构蛋白nsP 3与应激组分相互作用， 在ISR激活过程中形成的颗粒、细胞器，以隔离翻译机器，直到应力缓解， 恢复到稳态，表明nsP 3在甲病毒感染诱导的转录过程中调节翻译的作用。 侦察监视器总之，这导致了甲病毒通过募集非典型的RNA来逃避ISR的假设。 起始因子或核糖体直接作用，ISR抗性部分是通过转录起始因子或核糖体的相互作用介导的。 nsP 3和宿主蛋白。 使用辛德毕斯病毒（SINV）作为模型系统，该提案旨在确定甲病毒如何逃避 ISR维持病毒结构蛋白的翻译，希望能确定可药物化的靶点， 甲病毒复制和传播。目的1旨在鉴定负责sgRNA的翻译起始因子。 在ISR激活和eIF 2a磷酸化过程中的翻译。目标2将阐述病毒因子的作用， 特别是nsP 3的高变结构域（HVD），通过使用 深度突变扫描方法成功完成这项拟议工作将提供机械 在甲病毒研究中观察了几十年的一种特征不足的现象， 甲病毒感染的治疗干预。