The role of the host ubiquitin system in Zika virus replication

在目前的寨卡病毒(ZIKV)暴发期间，感染ZIKV的关联增加 小头症和其他先天性神经疾病促使人们广泛努力开发抗病毒药物 然而，到目前为止，还没有一种药物被批准用于人类。宿主细胞的鉴定 病毒复制所需的因素可能导致新的治疗策略。主机泛素(Ub)系统是 一种保守的细胞通路，在许多功能中都很重要，包括先天免疫信号和病毒 复制。这项建议的目标是定义ZIKV复制的分子机制，涉及 泛素系统，并确定可作为治疗干预靶点的宿主前病毒因子。 黄病毒的包膜(E)蛋白对病毒复制是必不可少的，因为它介导与 宿主细胞受体，从而促进病毒内化。此外，E蛋白还可能赋予特定的 通过识别由特定细胞类型表达的宿主因子来实现细胞趋向性。尽管密切相关的E蛋白 相关的黄病毒，如登革病毒(DENV)，特征很好，对ZIKV-E的功能知之甚少， 以及宿主细胞因素是否与ZIKV期间观察到的特征性神经疾病有关 感染是未知的。关于ZIKV如何建立生产力，知识存在很大差距 以及它是否使用泛素系统进行复制。因为它的关键作用是 病毒内化，E蛋白已被认为是治疗干预的靶点。因此， 通过调节E蛋白功能识别病毒复制所需的宿主细胞因子可能提供 新的抗病毒靶点。 我们的初步数据表明，ZIKV-E蛋白被E3-Ub成员TRIM7泛素化 连接酶三部分基序(TRIM)家族。敲除TRIM7导致E泛素化减少，这是 与胎盘来源的细胞系中ZIKV复制减少相关，表明TRIM7具有前病毒 TRIM7在胎盘和生殖组织中高度表达，这是两个重要的 ZIKV复制站点。因此，我们的假设是ZIKV的包膜蛋白是由 TRIM7在宿主的特定站点内促进ZIKV复制。通过使用体外生化 方法、重组ZIKV突变病毒和活体小鼠模型，我们将确定 E的泛素化对ZIKV的趋向性很重要，并揭示了TRIM7是如何促进ZIKV复制的。我们 提出以下具体目标：1)确定ZIKV囊膜蛋白泛素化在 病毒复制和2)确定TRIM7调控ZIKV的分子机制 复制。这些研究的结果意义重大，因为它将提供关于 ZIKV的复制机制，这可能适用于黄病毒科的其他成员。这部作品 还确定了一种宿主细胞因子TRIM7，它可能被作为一种新的治疗策略。

During the current outbreak of Zika virus (ZIKV), there has been an increased association of ZIKV infections with microcephaly and other congenital neurological disorders prompting extensive efforts to develop antiviral strategies; however, none have thus far been approved for use in humans. The identification of host cellular factors required for virus replication may lead to novel therapeutic strategies. The host ubiquitin (Ub) system is a conserved cellular pathway important in many functions, including innate immune signaling and virus replication. The goal of this proposal is to define the molecular mechanisms of ZIKV replication involving the ubiquitin system, and identify host proviral factors that can be targeted for therapeutic intervention. The envelope (E) protein of flaviviruses is essential for virus replication because it mediates attachment to host cell receptors, thereby promoting virus internalization. Further, the E protein may also confer specific cellular tropism by recognizing host factors expressed by specific cell types. Although the E proteins of closely related flaviviruses, like dengue virus (DENV), are well characterized, little is known about ZIKV-E function, and whether host cellular factors are linked to the characteristic neurological disorders observed during ZIKV infections is unknown. There is a major gap in knowledge regarding how ZIKV establishes productive infections in its host and whether it uses the ubiquitin system for replication. Because of its critical function in virus internalization, the E protein has been proposed as a target for therapeutic intervention. Therefore, the identification of host cellular factors required for virus replication by regulating E protein function may provide novel antiviral targets. Our preliminary data indicate that the ZIKV-E protein is ubiquitinated by TRIM7, a member of the E3-Ub ligase tripartite motif (TRIM) family. Knockdown of TRIM7 resulted in reduced ubiquitination of E, which correlated with reduced ZIKV replication in placenta-derived cell lines, suggesting that TRIM7 has a proviral function by ubiquitinating E. TRIM7 is highly expressed in placenta and reproductive tissues, two important sites of ZIKV replication. Therefore, our hypothesis is that the envelope protein of ZIKV is ubiquitinated by TRIM7 to promote ZIKV replication within specific sites of the host. By using in vitro biochemical approaches, recombinant ZIKV mutant viruses, and an in vivo mouse model, we will determine whether ubiquitination of E is important for ZIKV tropism and reveal how TRIM7 contributes to ZIKV replication. We propose the following specific aims: 1) Determine the role of ubiquitination of the ZIKV envelope protein in virus replication and 2) Determine the molecular mechanism by which TRIM7 regulates ZIKV replication. The outcome of these studies is significant because it will provide fundamental knowledge about the mechanism of ZIKV replication, which may apply to other members of the Flaviviridae family. This work also identifies a host cellular factor, TRIM7, that can potentially be targeted as a novel therapeutic strategy.