The role of the host ubiquitin system in Zika virus replication

在目前寨卡病毒（ZIKV）爆发期间，ZIKV感染的相关性增加， 小头畸形和其他先天性神经系统疾病促使人们广泛努力开发抗病毒药物， 然而，迄今为止还没有一种被批准用于人类。宿主细胞的鉴定 病毒复制所需的因子可能导致新的治疗策略。宿主泛素（Ub）系统是 一种在许多功能中重要的保守细胞通路，包括先天免疫信号传导和病毒 复制的该提案的目标是定义涉及ZIKV复制的分子机制。 泛素系统，并确定宿主前病毒因子，可以针对治疗干预。 黄病毒的包膜（E）蛋白对于病毒复制是必需的，因为它介导与病毒的粘附。 宿主细胞受体，从而促进病毒内化。此外，E蛋白还可以赋予特异性的 通过识别由特定细胞类型表达的宿主因子来识别细胞向性。虽然E蛋白与 相关的黄病毒，如登革病毒（DENV），被充分表征，关于ZIKV-E功能知之甚少， 以及宿主细胞因子是否与ZIKV期间观察到的特征性神经系统疾病有关。 感染是未知的。关于ZIKV如何建立生产力的知识存在重大差距 感染，以及它是否使用泛素系统进行复制。由于其在以下方面的关键作用， 病毒内化，E蛋白已被提议作为治疗干预的靶点。因此 鉴定通过调节E蛋白功能进行病毒复制所需的宿主细胞因子可以提供 新的抗病毒靶点。 我们的初步数据表明ZIKV-E蛋白被TRIM 7泛素化，TRIM 7是E3-Ub的成员， 连接酶三联体基序（TRIM）家族。TRIM 7的敲低导致E的泛素化减少， 与胎盘来源的细胞系中减少的ZIKV复制相关，表明TRIM 7具有前病毒介导的转录因子。 通过泛素化E. TRIM 7在胎盘和生殖组织中高度表达，这是两个重要的 ZIKV复制的位点。因此，我们的假设是ZIKV的包膜蛋白被泛素化， TRIM 7促进宿主的特定位点内的ZIKV复制。通过使用体外生化 方法，重组ZIKV突变病毒和体内小鼠模型，我们将确定是否 E的泛素化对于ZIKV向性是重要的，并且揭示了TRIM 7如何有助于ZIKV复制。我们 1）确定ZIKV包膜蛋白的泛素化在ZIKV中的作用。 2）确定TRIM 7调节ZIKV的分子机制 复制的这些研究的结果是重要的，因为它将提供有关以下方面的基本知识： ZIKV复制的机制，这可能适用于黄病毒科的其他成员。这项工作 还鉴定了一种宿主细胞因子TRIM 7，其可以作为一种新的治疗策略潜在地靶向。

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.