Natural Phenotypic Diversity of HCV NS3/4A Protease

描述(由申请人提供)：在丙型肝炎病毒感染者的一生中，宿主的先天免疫和病毒进化之间发生相互作用。先天反应在控制发病机制和疾病严重程度方面起着重要作用。病毒复制或蛋白产物与特定的细胞病原体识别受体结合，如Toll样受体1(TLR1)和RIG-I，触发先天的抗病毒反应。丙型肝炎病毒NS3/4A丝氨酸蛋白酶通过蛋白水解性裂解TRIF和IPS1等关键适配子分子，扰乱先天免疫信号通路。这些途径对于诱导I型干扰素是必不可少的，并提供了一种机制，使丙型肝炎病毒颠覆天然免疫并建立持续感染。在我们对自然人类感染过程中丙型肝炎病毒基因进化的研究中，我们在体内分离到了与轻度和严重疾病表型相关的新的和有趣的NS3/4A突变体。我们现在建议表征这些自然产生的NS3/4A变体的生物化学和分子生物学，因为它们在人类体内随着时间的推移在不同的临床结果中进化。第一个目标是扩增和测序NS3/4A基因盒，来自5名轻度和5名重症患者，以前从我们的1,200名阿拉斯加原住民和美国印第安人队列(AN/AI)获得的存档病毒样本，这是NIH连续资助的第12个年头，以描述未经治疗的、长期自然发生的慢性丙型肝炎期间体内病毒宿主动态。我们将克隆和表达感兴趣的病毒酶复合体，用于逐个病例的功能研究。在我们的机构同事、人类先天免疫和丙型肝炎病毒NS3/4A生化专家小迈克尔·盖尔博士的合作下，AIM 1还将比较自然变异的NS3/4A复合体和原型复合体的蛋白酶生物化学，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将使用反式救援方法研究蛋白酶对天然免疫的生物学功能。因此，我们将描述未经治疗的人类随着时间的推移NS3/4A基因和酶功能的自然进化，比较感染后早期和晚期的活性。我们假设，从严重疾病患者中分离的NS3/4A基因变体将在体内显示不同的进化动力学，在病毒多蛋白加工方面将不同，并且与来自轻度疾病病例的蛋白酶变体相比，将对宿主天然免疫途径施加更广泛和更有效的控制。这一建议意义重大，因为它描述并测试了在轻度和严重疾病进展期间，丙型肝炎病毒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.