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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）：丙型肝炎病毒影响约 2% 的美国人口，导致急性和慢性肝病、肝硬化、肝癌和肝功能衰竭。用于治疗 HCV 的现有疗法仅对一小部分患者耐受性差且有效。因此，我们重点关注开发治疗 HCV 感染的新疗法至关重要。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，因为参与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体 (RC) 的组装和功能，对这些酶的研究，特别是在其自然背景下的研究，受到了阻碍。我们缺乏关于 RC 的结构信息，尽管对分离的复制酶（例如非结构蛋白 NS3 和 NS5B）进行了大量研究，但越来越多的证据表明这些酶高度依赖于辅助因子，并且它们在 RC 机器内运行时功能不同。因此，为了开发生物学和药理学相关的检测方法并了解有关 HCV 复制酶的新信息，我们建议分离 HCV RC 并研究其作为完整复制全酶的结构和功能。这一挑战将通过使用两种不同的、互补的方法来应对： 1. 完整 RC 的体外翻译、处理和组装。 2.从肝细胞系中分离和纯化RC。 HCV 疗法发展的第二个限制是没有关于 HCV RC 结构的信息。我们不知道蛋白质如何组合在一起，RC 如何整合到膜中或它如何结合 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位点并建立 RC 复杂结构的三维模型。这项工作将为 RC 蛋白的协调活动提供新的见解，并将揭示可作为小分子抑制剂开发目标的蛋白质-蛋白质界面。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法相结合。 2. 遗传抑制分析以鉴定偶联氨基酸。 3.从HCV序列比对中检测功能耦合残基的系统发育方法。这项工作的成功将取决于项目领导团队中化学家、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.