Structure and function of the HCV replication complex

HCV复制复合体的结构和功能

基本信息

批准号：
8685099
负责人：
Brett D. Lindenbach
金额：
$ 59.17万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8685099
关键词：
Acute Address Affinity Amino Acids Antiviral Therapy Architecture Binding Proteins Biochemical Bioinformatics Biological Assay Cell Extracts Cell Line Cells Chemicals Cirrhosis Complex Coupled Development Drug Design Environment Enzymes Gene Expression Genetic Genetic Structures Genetic Suppression Genome Goals HCV Vaccine Hepatitis C Hepatitis C virus Hepatocyte Holoenzymes Human In Vitro Individual Leadership Learning Light Literature Liver Failure Malignant neoplasm of liver Maps Mass Spectrum Analysis Membrane Methods Modeling Molecular Molecular Target Nonstructural Protein Patients Phylogenetic Analysis Polyproteins Population Process Proteins Proteomics RNA Binding RNA replication Replicon Reporting Research Personnel Resolution Role Sequence Alignment Site Structure System Testing Therapeutic Translating Translation Process Translations Viral Viral Nonstructural Proteins Viral Proteins Virus Virus Replication Work chronic liver disease cofactor crosslink hepatoma cell improved inhibitor/antagonist insight intermolecular interaction overexpression protein complex protein protein interaction replicase small molecule success three-dimensional modeling

项目摘要

DESCRIPTION (provided by applicant): Hepatitis C virus afflicts ~2% of the U.S. population, causing acute and chronic liver disease, cirrhosis, liver cancer, and liver failure. Available therapies for treating HCV are poorly tolerated and effective in only a fraction of patients. It is therefore vital that we focus on the development of new therapies to treat HCV infection. The success of this effort hinges on developing an understanding of the molecular mechanisms underlying HCV gene expression and replication, as the enzymes involved these processes are the molecular targets for drug design. The study of these enzymes, particularly in their natural context, has been hampered by the lack of robust in vitro systems to study the assembly and function of the intact replication complex (RC). We lack structural information on the RC and, despite numerous studies on the isolated replicative enzymes (such as the nonstructural proteins NS3 and NS5B) there is growing evidence that these enzymes are highly dependent on cofactors and that they function differently when operating within the RC machine. Therefore, to develop biologically and pharmacologically relevant assays and to learn new information about the HCV replicative enzymes, we propose to isolate the HCV RC and to study its structure and function as an intact replicative holoenzyme. This challenge will be confronted by using two different, complementary approaches: 1. In vitro translation, processing and assembly of the intact RC. 2. Isolation and purification of the RC from liver cell lines. A second limitation to the development of HCV therapeutics is that there is no information on the architecture of the HCV RC. We do not know how the proteins fit together, how the RC integrates into membranes or how it binds the RNA genome. To address these issues, a second goal of our project is to determine sites of intermolecular interaction within the RC and to build a three-dimensional model of the RC complex structure. This effort will provide new insights into the coordinated activities of the RC proteins and it will reveal protein-protein interfaces that can serve as targets for the development of small molecule inhibitors. Three different approaches are being used to build the interaction map for the HCV RC: 1. Biochemical methods combined with high-resolution mass spectrometry. 2. Genetic suppression analysis to identify coupled amino acids. 3. Phylogenetic methods to detect functionally coupled residues from HCV sequence alignments. The success of this effort will be contingent on the coordinated work of chemists, biochemists and viral geneticists that are represented in the project leadership team.

描述（由申请人提供）：丙型肝炎病毒受苦约2％的美国人群，导致急性和慢性肝病，肝硬化，肝癌和肝衰竭。用于治疗HCV的可用疗法仅在一小部分患者中耐受性且有效。因此，至关重要的是，我们将重点放在治疗HCV感染的新疗法的开发上。这项工作的成功取决于对HCV基因表达和复制的分子机制的理解，因为涉及这些过程的酶是药物设计的分子靶标。由于缺乏强大的体外系统来研究完整复制复合物（RC）的组装和功能，对这些酶的研究，特别是在自然环境中。我们缺乏有关RC的结构信息，尽管进行了许多关于分离的复制酶（例如非结构蛋白NS3和NS5B）的研究，但越来越多的证据表明，这些酶高度依赖于辅助因子，并且在RC机器内运行时它们的功能有所不同。因此，为了开发生物学和药理相关的测定法，并学习有关HCV复制酶的新信息，我们建议隔离HCV RC并研究其结构和功能作为完整的复制性全酶。使用两种不同的互补方法：1。完整RC的体外翻译，处理和组装。 2。从肝细胞系的RC分离和纯化。 HCV疗法发展的第二个局限性是没有有关HCV RC架构的信息。我们不知道蛋白质如何结合在一起，RC如何整合到膜中或如何结合RNA基因组。为了解决这些问题，我们项目的第二个目标是确定RC内分子间相互作用的地点，并构建RC复合体结构的三维模型。这项工作将为RC蛋白的协调活性提供新的见解，它将揭示蛋白质 - 蛋白质界面，这些蛋白质界面可以作为开发小分子抑制剂的靶标。正在使用三种不同的方法来构建HCV RC的相互作用图：1。生化方法与高分辨率质谱。 2。遗传抑制分析以鉴定耦合的氨基酸。 3。从HCV序列比对检测功能偶联残基的系统发育方法。这项努力的成功将取决于项目领导团队中代表的化学家，生物化学家和病毒遗传学家的协调工作。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Molecular mechanics of RNA translocases.

DOI：
10.1016/b978-0-12-396546-2.00006-1
发表时间：
2012
期刊：
METHODS IN ENZYMOLOGY
影响因子：
0
作者：
Ding, Steve C.;Pyle, Anna Marie
通讯作者：
Pyle, Anna Marie

Structural insights into RNA recognition by RIG-I.

DOI：
10.1016/j.cell.2011.09.023
发表时间：
2011-10-14
期刊：
Cell
影响因子：
64.5
作者：
Luo D;Ding SC;Vela A;Kohlway A;Lindenbach BD;Pyle AM
通讯作者：
Pyle AM

A conserved NS3 surface patch orchestrates NS2 protease stimulation, NS5A hyperphosphorylation and HCV genome replication.

DOI：
10.1371/journal.ppat.1004736
发表时间：
2015-03
期刊：
PLoS pathogens
影响因子：
6.7
作者：
Isken O;Langerwisch U;Jirasko V;Rehders D;Redecke L;Ramanathan H;Lindenbach BD;Bartenschlager R;Tautz N
通讯作者：
Tautz N

Parts, assembly and operation of the RIG-I family of motors.