Picornavirus Genome Replication

小核糖核酸病毒基因组复制

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要这是一份续签研究小核糖核酸病毒基因组复制拨款的申请。脊髓灰质炎病毒的研究（PV）继续建立所有正链RNA病毒的分子和细胞生物学范例能够导致人类发病和/或死亡。 PV 复制其基因组与膜。事实上，该病毒利用独特的脂质创建了自己的基因组复制细胞器（RO）成分，包括丰富的磷酸肌醇 (PIP)、4-磷酸磷脂酰肌醇 (PI4P)。在过去的五年里，许多实验室一直在寻找 PI4P 的作用机制。生物合成由多种小核糖核酸病毒（包括PV）诱导。总的来说，这些研究测试了假设单个病毒蛋白劫持单个细胞 PI4 激酶 (PI4K)，导致激酶重新定位以及PI4P的合成。由于肠道病毒 3A(B) 蛋白与鸟嘌呤核苷酸交换因子 GBF1，大多数早期研究集中在 3A(B) 并得出结论：这种病毒蛋白通常通过间接机制劫持 PI4K。然而，这个曾经举办过的共识现在又回到了不确定性。很多年前，我们的实验室获得了遗传 3CD 在 PV RO 生物发生中可能发挥作用的证据。在上一个资助期间，我们做了一个通过证明 3CD 是必要且充分的，得出 3CD 和 RO 生物发生之间的明确联系用于诱导细胞内 PI4P 生物合成。我们证明正常细胞 GBF1-Arf1-PI4K 轴是受雇。我们鉴定了 3C 结构域 (3CmD) 或 3D 中氨基酸取代的 3CD 的两种衍生物结构域 (3CDm)，该结构域在该途径的离散步骤中诱导 PI4P 生物合成有缺陷。在两者中例如，衍生物对 3CD 的 PIP 结合活性表现出干扰。除了PI4P之外，还有3CD 诱导细胞内 PI(4,5)P2 (PIP2) 生物合成。 PIP2 诱导并非由 3CD 依赖性引起 PI4P 增加，但根据 3CmD 和 3CDm 的观察，这似乎是一个不同的过程蛋白质仍然能够诱导 PIP2。 PV 3CD 是一种 PIP 结合蛋白，也是多种 PIP 的调节剂生物合成途径。我们提出的研究旨在解决如何以及为何。在下一个资助期内，我们将追求以下具体目标：（1）定义PIP结合的结构-功能关系单独的 3C 和 3D 领域以及 3CD 背景下的领域； (2)阐明PI4P的诱导机制单独通过 3CD 进行生物合成以及在感染情况下进行生物合成； (3) 阐明诱导机制单独通过 3CD 以及在感染情况下进行 PIP2 生物合成。