Natural Phenotypic Diversity of HCV NS3/4A Protease

HCV NS3/4A 蛋白酶的自然表型多样性

基本信息

批准号：
8047145
负责人：
David Richard Gretch
金额：
$ 22.3万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8047145
关键词：
Alaska Native American Indians Amino Acid Sequence Antiviral Agents Antiviral Response Archives Binding Biochemical Biochemistry Biological Biological Assay Biological Process Cells Chronic Hepatitis C Clinical Collaborations Complex Data Disease Disease Outcome Disease Progression Encephalomyocarditis virus Enzymatic Biochemistry Enzyme Gene Enzyme Kinetics Enzymes Evolution Funding Genes Genetic Genetic Variation Genome Grant Growth Hepatitis C Hepatitis C virus Human Immune Immune response In Vitro Indigenous Individual Infection Interferons Interleukin-1 Liver diseases Molecular Biology Multienzyme Complexes Mutation Natural Immunity Nature Outcome Pathogenesis Pathway interactions Patients Peptide Hydrolases Play Polyproteins Process Proteins Research Role Serine Protease Severity of illness Signal Pathway Signal Transduction Specificity Specimen Substrate Specificity Testing Time Toll-Like Receptor 1 Tretinoin United States National Institutes of Health Variant Viral Viral Proteins Virus case-by-case basis cohort disease phenotype genetic evolution genetic variant human TLR3 protein human disease in vivo interest mutant novel pathogen pressure promoter prototype receptor research study response

项目摘要

DESCRIPTION (provided by applicant): During the lifetime of an HCV infected patient, interplay occurs between innate immunity of the host and virus evolution. The innate responses play an important role in controlling pathogenesis and disease severity. The binding of either viral replicates or protein products to specific cellular pathogen recognition receptors, such as Toll-like receptor 1 (TLR1) and RIG-I, triggers the innate antiviral response. The HCV NS3/4A serine protease disrupts innate immunity signaling pathways via proteolytic cleavage of critical adaptor molecules such as TRIF and IPS1. These pathways are essential for Type I IFN induction, and provide a mechanism by which HCV subverts innate immunity and establishes persistent infection. During our study of HCV genetic evolution during natural human infection, we have isolated novel and interesting NS3/4A mutants associated with mild and severe disease phenotype in vivo. We now propose to characterize the biochemistry and molecular biology of such naturally occurring NS3/4A variants, as they evolve in humans over time during diverse clinical outcomes. The first Aim will amplify and sequence NS3/4A genetic cassettes from, 5 mild and 5 severe disease patients, archived viremic specimens previously obtained from our 1,200 Alaskan Natives and American Indian cohort (AN/AI), which is in it's 12th year of contiguous NIH funding to describe in vivo viral- host dynamics during untreated, long term naturally occurring chronic hepatitis C. We will clone and express viral enzyme complexes of interest for functional studies, on a case-by case basis. In collaboration with our institutional colleague, Dr. Michael Gale Jr., an expert on human innate immunity and the biochemistry of HCV NS3/4A, Aim 1 will also compare protease biochemistry of natural variant NS3/4A complexes with prototype complexes, including stability, substrate recognition (including both viral and host targets) and enzyme kinetics. Aim 2 will look at protease biological function against innate immunity using the trans-rescue approach. We will thus describe natural evolution of NS3/4A genes and enzyme function in untreated humans over time, comparing activities at early and late times post infection. We hypothesize that genetic variants of NS3/4A, isolated from patients with severe disease, will display different in vivo evolutionary dynamics, will differ in viral polyprotein processing, and will exert broader and more efficient control of host innate immune pathways compared to protease variants from mild disease cases. This proposal is significant because it describes and tests the potential pathogenic significance of naturally occurring mutations in HCV genomes during mild and severe disease progression. PUBLIC HEALTH RELEVANCE: The study will gather pilot information on the potential relationship between HCV enzyme functional evolution, and liver disease activity, and is therefore novel. The experiments will bridge in vivo pathogenesis research on viral quasispecies dynamics during human disease in the Alaskan Natives and Indigenous Peoples cohort, currently funded by an NIH R01, and in vitro mechanistic research on an important regulator of host innate immunity, the HCV NS3/4A protease. Studies proposed in this grant will take advantage of well-characterized research specimens previously obtained from infected patients over time.

描述（由申请人提供）：在HCV感染患者的寿命中，相互作用发生在宿主的先天免疫和病毒演化之间。先天反应在控制发病机理和疾病严重程度中起着重要作用。病毒性复制或蛋白质产物与特定细胞病原体识别受体的结合，例如Toll样受体1（TLR1）和RIG-I，触发了先天抗病毒反应。 HCV NS3/4A丝氨酸蛋白酶通过临界适配器分子（例如TRIF和IPS1）的蛋白水解裂解破坏了先天的免疫信号通路。这些途径对于I型IFN诱导至关重要，并提供了HCV颠覆先天免疫并建立持续感染的机制。在天然人类感染期间HCV遗传进化的研究中，我们在体内与轻度和重度疾病表型相关的新型且有趣的NS3/4A突变体。现在，我们建议表征这种天然发生的NS3/4A变体的生物化学和分子生物学，因为它们在各种临床结果中随着时间的流逝而在人类中演变。第一个目标将在5名轻度和5例严重疾病患者中放大和序列NS3/4A遗传盒，先前从我们的1200名阿拉斯加本地人和美洲印第安人同类（AN/AI）获得的存档病毒血管（AN/AI），该标本是在连续的NIH基金的第12年，在vivo Viral-her-He thr-trim thr-hepation them them thrictim thep and contranting heptime compationd tyment thep thep thep thep contranting comptime C.以情况为基础，表达功能研究的感兴趣的病毒酶复合物。在与我们的机构同事的合作中，迈克尔·盖尔（Michael Gale Jr. AIM 2将使用反式逆向方法来研究蛋白酶的生物学功能，以防止先天免疫。因此，随着时间的流逝，我们将描述NS3/4A基因和酶功能的自然演化，并比较感染后早期和晚期的活动。我们假设从患有严重疾病的患者中分离出的NS3/4A的遗传变异将显示出不同的体内进化动力学，在病毒多蛋白加工方面会有所不同，并且与与蛋白酶的蛋白酶相比，对宿主先天免疫途径的更广泛，更有效的控制将更广泛，更有效地控制。该提案很重要，因为它描述并测试了轻度和重度疾病进展过程中HCV基因组中天然突变的潜在致病意义。公共卫生相关性：该研究将收集有关HCV酶功能进化与肝病活性之间潜在关系的试点信息，因此是新颖的。该实验将桥接有关阿拉斯加当地人和本地人群人类疾病期间病毒准确性动力学动力学的体内发病机制研究，目前由NIH R01资助，以及对宿主先天免疫的重要调节剂的体外机械研究，HCV NSS3/4A蛋白酶。该赠款中提出的研究将利用先前从感染患者获得的特征良好的研究标本。