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（TRAF 6）是宿主免疫应答的中心，并且对于针对病原体的保护性应答是重要的。我们在这里报告一个非典型的作用TRAF 6。蜱传黄病毒（tick-borne flaviviruses，TBFV）已经进化出一种独特的机制来利用TRAF 6来有益于感染。了解TRAF 6的抗病毒和前病毒作用之间的平衡如何有利于TBFV复制，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。我们确定了TRAF 6和TBFV蛋白酶NS 3 pro之间的相互作用。TRAF 6是一种E3泛素连接酶，通常与病毒感染背景下的抗病毒功能相关。令人惊讶的是，TBFV使用TRAF 6在细胞中复制。在这里，我们表明TBFV NS 3 pro蛋白在感染过程中与TRAF 6的相互作用支持TBFV复制。在蚊媒黄病毒中未观察到TRAF 6的前病毒作用，这与缺乏保守的TRAF 6结合基序一致。通过定点诱变破坏TRAF 6-NS 3 pro相互作用导致成熟蛋白酶蛋白表达的显著降低，以及体外病毒复制的减弱。为了研究TRAF 6前病毒的功能，我们评估了泛素化状态，发现NS 3 pro在TRAF 6存在下被泛素化。本申请的总体目标是确定NS 3-TRAF 6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NS 3 pro通过TRAF 6直接相互作用和泛素化被激活，最终促进生产性病毒复制。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TRAF 6如何有利于TBFV复制，我们将使用细胞生物学和生物化学方法来确定NS 3 pro如何被泛素化修饰以及泛素化如何影响NS 3 pro的结构和功能。2)为了确定TRAF 6在原代细胞中的前病毒作用（例如，树突状细胞和神经元）和体内发病机制中，我们将使用治疗和遗传方法来操纵原代细胞和TBFV发病机制的小鼠模型中的TRAF 6表达。通过了解TRAF 6如何使NS 3 pro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感染的小鼠模型，我们将确定NS 3-TRAF 6相互作用可用于黄病毒药物开发的程度。

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.