Natural Phenotypic Diversity of HCV NS3/4A Protease

描述（由申请人提供）：在 HCV 感染患者的一生中，宿主的先天免疫和病毒进化之间会发生相互作用。先天反应在控制发病机制和疾病严重程度方面发挥着重要作用。病毒复制或蛋白质产物与特定细胞病原体识别受体（例如 Toll 样受体 1 (TLR1) 和 RIG-I）的结合会触发先天抗病毒反应。 HCV NS3/4A 丝氨酸蛋白酶通过关键接头分子（例如 TRIF 和 IPS1）的蛋白水解裂解来破坏先天免疫信号传导途径。这些途径对于 I 型 IFN 诱导至关重要，并提供了 HCV 破坏先天免疫并建立持续感染的机制。在我们对人类自然感染过程中 HCV 遗传进化的研究中，我们分离出了与体内轻度和重度疾病表型相关的新颖且有趣的 NS3/4A 突变体。我们现在建议描述这种自然发生的 NS3/4A 变体的生物化学和分子生物学特征，因为它们随着时间的推移在不同的临床结果中在人类中进化。第一个目标将对来自 5 名轻度和 5 名重度疾病患者的 NS3/4A 基因盒进行扩增和测序，这些病毒血症标本是先前从我们的 1,200 名阿拉斯加原住民和美洲印第安人队列 (AN/AI) 中获得的存档病毒血症样本，这是 NIH 连续资助的第 12 年，用于描述未经治疗的长期自然发生的慢性丙型肝炎期间的体内病毒宿主动态。我们将克隆和表达感兴趣的病毒酶复合物，用于功能研究，视具体情况而定。 Aim 1 还将与我们的机构同事、人类先天免疫和 HCV NS3/4A 生物化学方面的专家 Michael Gale Jr. 博士合作，将天然变体 NS3/4A 复合物的蛋白酶生物化学与原型复合物进行比较，包括稳定性、底物识别（包括病毒和宿主靶点）和酶动力学。目标 2 将使用反式救援方法研究蛋白酶针对先天免疫的生物学功能。因此，我们将描述未经治疗的人类中 NS3/4A 基因和酶功能随时间的自然进化，比较感染后早期和晚期的活性。我们假设，与来自轻症病例的蛋白酶变体相比，从重症患者中分离出的 NS3/4A 基因变体将表现出不同的体内进化动力学，病毒多蛋​​白加工也有所不同，并且将对宿主先天免疫途径发挥更广泛和更有效的控制作用。该提案意义重大，因为它描述并测试了轻度和重度疾病进展期间 HCV 基因组中自然发生的突变的潜在致病意义。

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.