Picornavirus Genome Replication

小核糖核酸病毒基因组复制

• 批准号: 10331323
• 负责人: CRAIG E. CAMERON
• 金额: $ 44.92万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331323
• 关键词: 3-Dimensional; Achievement; Address; Affect; Amino Acid Substitution; Anabolism; Atomic Force Microscopy; Binding; Binding Proteins; Binding Sites; Biochemistry; Biogenesis; Biological Assay; Cell Line; Cells; Coat Protein Complex I; Collaborations; Complex; Consensus; Development; Docking; Exhibits; Family Picornaviridae; Funding; GBF1 gene; Genetic; Genome; Goals; Grant; Guanine Nucleotide Exchange Factors; Human; Human poliovirus; Image; Infection; Integration Host Factors; Investigation; Knowledge; Laboratories; Life Cycle Stages; Lipid Bilayers; Lipids; Membrane; Membrane Proteins; Microfluidics; Modeling; Molecular; Molecular and Cellular Biology; Morbidity - disease rate; NMR Spectroscopy; Nonlytic; Null Lymphocytes; Organelles; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphotransferases; Picornaviridae Infections; Process; Production; Proteins; Proteomics; Public Health; RNA Viruses; Role; SERPINA4 gene; Site-Directed Mutagenesis; Structural Models; Structure; Structure-Activity Relationship; Tertiary Protein Structure; Testing; Time; Uncertainty; Universities; Vaccines; Viral Proteins; Virus; Virus Diseases; Work; base; inhibitor; insight; interest; molecular dynamics; mortality; novel; phosphatidylinositol 4-phosphate; recruit; scaffold; three-dimensional modeling

Project Abstract This is an application for renewal of a grant to study picornavirus genome replication. Studies of poliovirus (PV) continue to establish paradigms for the molecular and cellular biology of all positive-strand RNA viruses capable of causing morbidity and/or mortality in humans. PV replicates its genome in association with membranes. In fact, the virus creates its own genome-replication organelle (RO) with a unique lipid composition, including an abundance of the phosphoinositide (PIP), phosphatidylinositol-4-phosphate (PI4P). During the past five years, many laboratories have been in search of the mechanism by which PI4P biosynthesis is induced by various picornaviruses, including PV. In general, these studies tested the hypothesis that a single viral protein hijacks a single cellular PI4 kinase (PI4K), leading to kinase relocalization and synthesis of PI4P. Because of the long-established connection between the enteroviral 3A(B) protein and the guanine nucleotide exchange factor, GBF1, most of the early studies focused on 3A(B) and concluded that this viral protein is responsible for hijacking a PI4K, often by an indirect mechanism. However, this once-held consensus opinion has now returned to uncertainty. Many years ago, our laboratory obtained genetic evidence of a possible role of 3CD in the biogenesis of PV RO. During the previous funding period, we made a definitive connection between 3CD and RO biogenesis by showing that 3CD is both necessary and sufficient for induction of PI4P biosynthesis in cells. We demonstrated that the normal cellular GBF1-Arf1-PI4K axis is employed. We identified two derivatives of 3CD with amino acid substitutions in the 3C domain (3CmD) or 3D domain (3CDm) that are defective for induction of PI4P biosynthesis at discrete steps in this pathway. In both instances, the derivatives exhibit perturbations to PIP-binding activity of 3CD. In addition to PI4P, 3CD also induces PI(4,5)P2 (PIP2) biosynthesis in cells. PIP2 induction does not arise from the 3CD-dependent increase in PI4P but appears to be a distinct process based on the observation that both 3CmD and 3CDm proteins remain competent for PIP2 induction. PV 3CD is a PIP-binding protein and a regulator of multiple PIP biosynthetic pathways. Our proposed studies aim to address how and why. During the next funding period, we will pursue the following specific aims: (1) Define the structure-function relationships of the PIP-binding domains of 3C and 3D alone and in the context of 3CD; (2) Elucidate the mechanism of induction of PI4P biosynthesis by 3CD alone and in the context of infection; and (3) Elucidate the mechanism of induction of PIP2 biosynthesis by 3CD alone and in the context of infection.

项目摘要 这是续签赠款的应用，以研究Picornavirus基因组复制。脊髓灰质炎病毒的研究 （PV）继续为所有阳性链RNA病毒的分子和细胞生物学建立范例 能够引起人类的发病率和/或死亡率。 PV复制其基因组与 膜。实际上，该病毒用独特的脂质创建了自己的基因组复制细胞器（RO） 组成，包括丰富的磷酸肌醇（PIP），磷脂酰肌醇4-磷酸（PI4P）。 在过去的五年中，许多实验室一直在寻找PI4P的机制 生物合成是由包括PV在内的各种PICORNAVIRES诱导的。通常，这些研究测试了 假设单个病毒蛋白劫持单个细胞PI4激酶（PI4K），导致激酶重新定位 和PI4P的合成。由于肠病毒3a（b）蛋白之间的长期联系 鸟嘌呤核苷酸交换因子GBF1，大多数早期研究都集中在3a（b）上，并得出结论认为 这种病毒蛋白通常是通过间接机制劫持PI4K的原因。但是，这曾经有过 共识意见现在已经恢复了不确定性。许多年前，我们的实验室获得了遗传 3CD在PV RO生物发生中可能作用的证据。在上一个资金期间，我们做了一个 通过证明3CD既需要且足够 用于诱导细胞中的PI4P生物合成。我们证明了正常的细胞GBF1-ARF1-PI4K轴为 雇用。我们在3C域（3CMD）或3D中鉴定了3CD的两个衍生物 在该途径中离散步骤中PI4P生物合成诱导的域（3CDM）。在这两个中 实例，衍生物对3CD的PIP结合活性表现出扰动。除了PI4P，3CD也 在细胞中诱导PI（4,5）P2（PIP2）生物合成。 PIP2诱导不是由3CD依赖性引起的 基于3CMD和3CDM的观察结果，PI4P的增加，但似乎是一个独特的过程 蛋白质仍然具有PIP2诱导能力。 PV 3CD是一种结合蛋白，是多个PIP的调节剂 生物合成途径。我们提出的研究旨在解决如何和原因。在下一个资金期间， 我们将追求以下特定目的：（1）定义管道结合的结构功能关系 仅3C和3D的域，在3CD的背景下； （2）阐明PI4P诱导的机理 仅3CD和感染的背景下，生物合成； （3）阐明了诱导的机制 PIP2生物合成单独和感染的背景下。