Picornavirus Genome Replication

小核糖核酸病毒基因组复制

• 批准号: 10640512
• 负责人: CRAIG E. CAMERON
• 金额: $ 4.82万
• 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/10640512
• 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 这是一份申请书，要求更新研究小核糖核酸病毒基因组复制的资助。脊髓灰质炎病毒研究 (PV)继续建立所有正链RNA病毒的分子和细胞生物学范式 能够导致人类发病和/或死亡。PV复制其基因组与 膜。事实上，这种病毒用一种独特的脂质创造了自己的基因组复制细胞器（RO 在一些实施方案中，所述组合物包含丰富的磷酸肌醇（PIP）、磷脂酰肌醇-4-磷酸（PI 4P）。 在过去的五年中，许多实验室一直在寻找PI 4P 生物合成由各种小核糖核酸病毒（包括PV）诱导。总的来说，这些研究测试了 假设单个病毒蛋白质劫持单个细胞PI 4激酶（PI 4K），导致激酶重新定位 以及PI 4P的合成。由于肠道病毒3A（B）蛋白与 鸟嘌呤核苷酸交换因子，GBF 1，大多数早期研究集中在3A（B）上，并得出结论， 这种病毒蛋白通常通过间接机制负责劫持PI 4K。然而，这个曾经的 舆论现在又回到了不确定性。多年前，我们的实验室获得了 3CD在PV RO生物发生中可能发挥作用的证据。在上一个资助期内， 3CD和RO生物发生之间的明确联系，表明3CD是必要的和充分的 用于诱导细胞中的PI 4P生物合成。我们证明了正常细胞的GBF 1-Arf 1-PI 4 K轴是 就业。我们鉴定了两种在3C结构域中具有氨基酸取代的3CD衍生物（3CmD）或3D 结构域（3CDm），其在该途径中的离散步骤中对PI 4P生物合成的诱导有缺陷。无论是 在某些情况下，衍生物表现出对3CD的PIP结合活性的干扰。除了PI 4P，3CD还 诱导细胞中PI（4，5）P2（PIP 2）的生物合成。PIP 2诱导不是由3CD依赖性的 PI 4P增加，但似乎是一个不同的过程，基于观察到3CmD和3CDm 蛋白质保持对PIP 2诱导的能力。PV 3CD是PIP结合蛋白，是多种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为什么。在下一个供资期间， 具体目标如下：（1）明确PIP绑定的结构-功能关系 （2）阐明PI 4P的诱导机制 通过单独的3CD和在感染的情况下的生物合成;和（3）阐明诱导的机制， 通过单独的3CD和在感染的情况下的PIP 2生物合成。