Natural Phenotypic Diversity of HCV NS3/4A Protease

描述（由申请方提供）：在HCV感染患者的一生中，宿主的先天免疫和病毒进化之间发生相互作用。先天性反应在控制发病机制和疾病严重程度中起重要作用。病毒复制或蛋白产物与特定细胞病原体识别受体（例如Toll样受体1（TLR 1）和RIG-I）的结合触发先天性抗病毒应答。HCV NS 3/4A丝氨酸蛋白酶通过蛋白水解切割关键衔接分子如TRIF和IPS 1来破坏先天免疫信号传导途径。这些途径是I型IFN诱导所必需的，并提供了HCV破坏先天免疫并建立持续感染的机制。在我们的研究丙型肝炎病毒的遗传进化过程中，在自然的人类感染，我们已经分离出新的和有趣的NS 3/4A突变体与轻度和严重的疾病表型在体内。我们现在建议表征这种天然存在的NS 3/4A变体的生物化学和分子生物学，因为它们在不同的临床结果中随着时间的推移在人类中进化。第一个目标将从5名轻度和5名重度疾病患者中扩增和测序NS 3/4A基因盒，这些患者是先前从我们的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标本，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力学。我们将克隆和表达感兴趣的病毒酶复合物的功能研究，在个案的基础上。与我们的同事Michael Gale Jr.博士合作，作为人类先天免疫和HCV NS 3/4A生物化学方面的专家，Aim 1还将比较天然变体NS 3/4A复合物与原型复合物的蛋白酶生物化学，包括稳定性、底物识别（包括病毒和宿主靶标）和酶动力学。目标2将使用反式拯救方法来研究蛋白酶对先天免疫的生物学功能。因此，我们将描述NS 3/4A基因和酶功能在未经治疗的人类中随时间的自然演变，比较感染后早期和晚期的活性。我们假设，NS 3/4A的遗传变异体，从严重疾病的患者中分离，将显示不同的体内进化动力学，将不同的病毒多蛋白加工，并将发挥更广泛和更有效的控制宿主先天免疫途径相比，蛋白酶变异体从轻度疾病的情况。这一提议意义重大，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.