Understanding the proviral role for TRAF6 interaction with the viral protease in flavivirus replication and pathogenesis

肿瘤坏死因子受体相关因子6 （TRAF6）是宿主免疫反应的核心，对病原体的保护性反应很重要。我们在这里报告TRAF6的非规范角色。蜱传黄病毒（tbfv）已经进化出一种独特的机制，利用TRAF6来促进感染。了解TRAF6的抗病毒和原病毒作用之间的平衡是如何倾向于TBFV复制的，可能会揭示黄病毒如何克服细胞抗病毒环境，最终导致疾病。我们发现TRAF6与TBFV蛋白酶NS3pro之间存在相互作用。TRAF6是一种E3泛素连接酶，在病毒感染的情况下通常与抗病毒功能相关。令人惊讶的是，tbfv利用TRAF6在细胞中进行复制。在这里，我们发现TBFV NS3pro蛋白在感染过程中与TRAF6相互作用支持TBFV复制。在蚊媒黄病毒中未发现TRAF6的原病毒作用，这与缺乏保守的TRAF6结合基序一致。通过定点诱变破坏TRAF6-NS3pro相互作用，导致成熟蛋白酶蛋白表达显著降低，体外病毒复制减弱。为了研究TRAF6的前病毒功能，我们评估了其泛素化状态，发现NS3pro在TRAF6存在下泛素化。本应用程序的总体目标是确定NS3-TRAF6相互作用如何影响TBFV感染小鼠模型中的蛋白酶结构和功能、病毒复制和发病机制。我们假设NS3pro通过TRAF6的直接相互作用和泛素化激活，最终促进病毒的高效复制。为了验证我们的假设，我们将使用两个特定的目标。1)为了确定TRAF6如何促进TBFV复制，我们将使用细胞生物学和生化方法来确定NS3pro如何被泛素化修饰以及泛素化如何影响NS3pro的结构和功能。2)为了确定TRAF6在原代细胞（如树突状细胞和神经元）和体内发病机制中的前病毒作用，我们将使用治疗和遗传方法来操纵TRAF6在原代细胞和TBFV发病机制小鼠模型中的表达。通过了解TRAF6如何使NS3pro受益及其在感染过程中的作用，我们将深入了解一种至关重要的病毒酶的调控。然后，通过使用TBFV感染小鼠模型，我们将确定NS3-TRAF6相互作用在多大程度上可能成为黄病毒药物开发的目标。

Tumor necrosis factor receptor associated factor 6 (TRAF6) is central to host immune responses and is important for protective responses to pathogens. We report here a non-canonical role for TRAF6. The tick- borne flaviviruses (TBFVs) have evolved a unique mechanism to use TRAF6 to benefit infection. Understanding how the balance between TRAF6’s antiviral and proviral effects is tipped in favor of TBFV replication may reveal how flaviviruses overcome the cellular antiviral environment, to ultimately cause disease. We identified an interaction between TRAF6 and the TBFV protease, NS3pro. TRAF6 is an E3 ubiquitin ligase that is normally associated with antiviral functions in the context of virus infections. Surprisingly, TBFVs use TRAF6 to replicate in cells. Here, we show that TBFV NS3pro protein interaction with TRAF6 during infection supports TBFV replication. The proviral role of TRAF6 was not seen with mosquito-borne flaviviruses, consistent with the lack of a conserved TRAF6 binding motif. Disruption of the TRAF6-NS3pro interaction by site-directed mutagenesis resulted in a significant reduction of mature protease protein expression, and attenuation of in vitro virus replication. To investigate the TRAF6 proviral function, we assessed the ubiquitination status and found that NS3pro is ubiquitinated in the presence of TRAF6. The overall objective of this application is to determine how NS3-TRAF6 interaction affects protease structure and function(s), virus replication, and pathogenesis in a mouse model of TBFV infection. We hypothesize that NS3pro is activated by TRAF6 through direct interaction and ubiquitination, ultimately to promote productive virus replication. To test our hypothesis, we will use two specific aims. 1) To determine how TRAF6 benefits TBFV replication we will use cell biology and biochemical approaches to determine how NS3pro is modified by ubiquitination and how ubiquitination affects the structure and function of NS3pro. 2) To determine the proviral role of TRAF6 in primary cells (e.g., dendritic cells and neurons) and in pathogenesis in vivo we will use therapeutic and genetic methods to manipulate TRAF6 expression in primary cells and in a mouse model of TBFV pathogenesis. By understanding how TRAF6 benefits NS3pro and its role during infection, we will gain insight into the regulation of a critically important viral enzyme. Then by using a mouse model of TBFV infection we will determine the extent to which the NS3-TRAF6 interaction may be targeted for flavivirus drug development.