E3 Ubiquitin Ligase Functions in Enveloped Virus Budding

描述（由申请人提供）：病毒出芽（膜变形远离宿主细胞）是包膜病毒（如HIV和埃博拉病毒（EBOV））复制的最后必要步骤。为了出芽，HIV和EBOV通过多泡体（MVB）途径劫持通常参与细胞表面受体下调的细胞运输蛋白，包括nedd4家族泛素连接酶和运输所需的内体分选复合物（escrt）。通过选择泛素连接酶和escrt，病毒感染可以干扰内源性蛋白的分选，如表皮生长因子受体和CXCR4趋化因子受体，并可能影响其他受体的分选，如δ阿片受体和β 2-肾上腺素能受体，具有广泛的影响。例如，病毒感染破坏阿片类受体运输可能对t细胞活化和艾滋病毒复制产生影响，特别是在阿片类药物滥用的情况下。泛素连接酶和escrt在葡萄球菌中高度保守，使其成为理解这些相互作用的良好模型系统。在MVB分选中，泛素连接酶具有酶（泛素化）和非酶（接头）两种作用，但目前尚不清楚哪种连接酶活性是病毒出芽所必需的。我们将利用酵母系统系统地研究泛素连接酶相互作用在病毒结构蛋白HIV Gag和EBOV VP40出芽中的作用。我们将首先绘制Gag与泛素连接酶Rsp5 （Nedd4同源物）的相互作用，然后研究连接酶结合和泛素化缺陷对Gag和VP40病毒样颗粒（vlp）出芽的功能影响。然后，我们将研究病毒蛋白对内源性MVB货物运输的影响，在Gag和VP40表达的情况下，使用脉冲追踪动力学分析货物运输。最后，我们将开始使用一种合理的设计方法来开发病毒出芽抑制剂，该方法结合了硅抑制剂筛选和基于实验室的实验测试和验证。这项研究的目的是了解HIV和埃博拉病毒出芽的分子机制，这是复制的最后一步。这项工作将有助于开发治疗病毒感染的新疗法。此外，这些知识可以帮助我们了解病毒感染的潜在广泛后果，包括肿瘤发生、心肌病、成瘾和神经退行性变。本研究成果将广泛应用于其他包膜病毒的研究，包括Epstein- Barr病毒。人类t细胞嗜淋巴病毒。流感病毒和其他病毒。

DESCRIPTION (provided by applicant): Viral budding (membrane deformation away from the host cell) is the final required step in the replication of enveloped viruses such as HIV and Ebola virus (EBOV). To bud, HIV and EBOV hijack cellular trafficking proteins normally involved in cell surface receptor downregulation through the multivesicular body (MVB) pathway, including Nedd4-family ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs). By co-opting ubiquitin ligases and ESCRTs, viral infections can interfere with the sorting of endogenous proteins, such as epidermal growth factor receptor and the CXCR4 chemokine receptor and may impact the sorting of other receptors, such as the delta opioid receptor and beta2- adrenergic receptors, with wide ranging ramifications. For instance, disruption of delta opioid receptor trafficking by viral infection could have implications for T-cell activation and HIV replication, particularly in a setting of opioid abuse. Ubiquitin ligases and the ESCRTs are highly conserved in S. cerevisiae, making it a good model system for understanding these interactions. In MVB sorting ubiquitin ligases have both enzymatic (ubiquitination) and non-enzymatic (adaptor) roles, but it is less clear which ligase activities are required for viral budding. We will systematically address the role of ubiquitin ligase interactions on the budding ofthe viral structural proteins HIV Gag and EBOV VP40, using the yeast system. We will begin by mapping the interactions of Gag with the ubiquitin ligase Rsp5 (a Nedd4 homolog) and then examine the functional impact of defects in ligase binding and ubiquitination on the budding of Gag and VP40 viral-like particles (VLPs). Then we will investigate the impact of viral proteins on the trafficking of endogenous MVB cargos using pulse-chase kinetic analysis of cargo trafficking in the setting of Gag and VP40 expression. Finally, we will begin the development of viral budding inhibitors using a rational design approach that combines In silico inhibitor screening with laboratory based experimental testing and validation. The goal of this research is to understand the molecular mechanisms of HIV and Ebola virus budding, the final step in replication. This work will contribute to the development of novel therapeutics to treat viral infections. Additionally, this knowledge may help us to understand potentially wide-ranging consequences of viral infection, including oncogenesis, cadiomyopathy, addiction, and neurodegeneration. The insights gained from this research will have broad application to the study of other enveloped viruses, including Epstein- Barr virus. Human T-cell Lymophotropic virus. Influenza virus, and others.