Picornavirus Genome Replication

项目摘要 这是一项研究微小核糖核酸病毒基因组复制的拨款续期申请。脊髓灰质炎病毒的研究 (Pv)继续为所有正链RNA病毒的分子和细胞生物学建立范例 能够导致人类发病和/或死亡的。光伏病毒复制其基因组与 膜。事实上，这种病毒创造了自己的基因组复制细胞器(RO)，具有一种独特的脂质 组成，包括丰富的磷脂酰肌醇(PIP)，磷脂酰肌醇-4-磷酸(PI4P)。 在过去的五年里，许多实验室一直在寻找PI4P 包括细小核糖核酸病毒在内的各种微小核糖核酸病毒都能诱导生物合成。总的来说，这些研究测试了 一种假设，即单一病毒蛋白劫持单一细胞PI4K(PI4K)，导致激酶重新定位 和PI4P的合成。因为肠道病毒3A(B)蛋白和 鸟嘌呤核苷酸交换因子GBF1，早期的研究大多集中在3A(B)上，并得出结论 这种病毒蛋白通常通过间接机制劫持PI4K。然而，这一曾经举行的 共识意见现在又回到了不确定的状态。许多年前，我们实验室获得了基因 3CD在PV RO生物发生中可能作用的证据。在上一个资助期内，我们作出了 3CD和RO生物发生之间的明确联系--证明3CD既是必要的也是充分的 用于在细胞中诱导PI4P的生物合成。我们证明了正常细胞的GBF1-Arf1-PI4K轴是 受雇的。我们鉴定了两个在3C结构域(3CmD)或3D具有氨基酸取代的3CD的衍生物 结构域(3CDm)，在此途径的不连续步骤中，对于诱导PI4P的生物合成是缺陷的。在这两个地方 实例中，衍生物表现出对3CD的PIP结合活性的扰动。除了PI4P，3CD还 诱导细胞内PI(4，5)P2(PIP2)的生物合成。PIP2的诱导不是由3CD依赖引起的 PI4P的增加，但根据3CmD和3CDM的观察，似乎是一个不同的过程 蛋白质仍然具有诱导PIP2的能力。PV 3CD是一种PIP结合蛋白，是多个PIP的调节因子 生物合成途径。我们提议的研究旨在解决如何以及为什么。在下一个资助期内， 我们将追求以下具体目标：(1)确定PIP结合的结构-功能关系 (2)阐明了PI4P的诱导机制 3CD单独和在感染环境下的生物合成；以及(3)阐明了诱导的机制 PIP2的生物合成由3CD单独和在感染的背景下进行。

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.