Characterization and disruption of host protein interations required for budding

出芽所需的宿主蛋白质相互作用的表征和破坏

• 批准号: 7670071
• 负责人: ANTHONY P SCHMITT
• 金额: $ 27.04万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-03 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7670071
• 关键词: Affinity Chromatography; Animal Diseases; Antiviral Agents; Binding; Binding Proteins; Development; Drug Delivery Systems; Drug Design; Filovirus; Henipavirus; Henipavirus Infections; Human; Integration Host Factors; Maps; Nipah Virus; Paramyxovirus; Play; Probability; Process; Production; Protein Binding; Proteins; Recruitment Activity; Retroviridae; Rhabdoviridae; Role; Screening procedure; System; Testing; Viral; Viral Proteins; Virus; Virus-like particle; Yeasts; biodefense; drug development; parainfluenza virus; particle; research study; yeast two hybrid system

Enveloped viruses are formed by a budding process that often requires participation of host proteins. Retroviruses, rhabdoviruses, and filoviruses all use similar late domain sequences within the viral proteins to recruit host factors for budding. Although paramyxoviruses generally lack the same late domain sequences used by these viruses, we have obtained evidence that paramyxoviruses, similar to other enveloped viruses, likely recruit host factors for budding. The viral protein:host protein binding interfaces used during virus budding have the potential to be effective as targets for antiviral drug design. Hendra and Nipah viruses (Henipaviruses) are recently emerged, zoonotic paramyxoviruses that are deadly to humans. Although progress has been made in characterizing the entry mechanisms for these viruses, very little is known about mechanisms of Henipavirus budding. We propose to take advantage of our expertise in the field of paramyxovirus budding by conducting experiments with the following aims: 1. Define requirements for Henipavirus particle production. We have established virus-like particle (VLP) assembly systems for Hendra and Nipah virus. We will define which Henipavirus proteins are important for VLP production, and hence which proteins may play roles in the recruitment of host factors for budding. 2. Identify host factors involved in Henipavirus budding. Two independent strategies will be employed: co-affinity purification of host proteins from Henipavirus VLPs, and yeast two-hybrid screening. An siRNAbased secondary screen will determine which candidate binding proteins are actually important for budding. 3. Define host and viral targets for antiviral drug development. Binding interfaces between viral and host proteins will be mapped. Minimal binding fragments of host proteins will be tested for the ability to block virus budding, similar to the effect analogous fragments have on retrovirus and parainfluenza virus 5 (PIV5) budding.

有包膜病毒是通过一个出芽过程形成的，该过程通常需要宿主蛋白的参与。 逆转录病毒、弹状病毒和丝状病毒都在病毒蛋白内使用类似的晚期结构域序列， 为萌芽招募宿主因子。虽然副粘病毒一般缺乏相同的晚期结构域序列 我们已经获得了副粘病毒，类似于其他包膜病毒， 可能会招募宿主因子来发芽。病毒蛋白：病毒感染过程中使用的宿主蛋白结合界面 出芽具有作为抗病毒药物设计的有效靶点的潜力。 亨德拉病毒和尼帕病毒（亨尼帕病毒）是最近出现的致命的人畜共患副粘病毒 对人类虽然在描述这些病毒的进入机制方面取得了进展， 对亨尼帕病毒出芽的机制知之甚少。我们建议利用我们的 通过进行具有以下目的的实验，利用副粘病毒出芽领域的专业知识： 1.定义亨尼帕病毒颗粒生产的要求。我们已经建立了病毒样颗粒 (VLP)亨德拉和尼帕病毒的组装系统。我们将定义哪些亨尼帕病毒蛋白是 重要的VLP生产，因此，蛋白质可能发挥作用，在招募宿主因子， 发芽 2.鉴定参与亨尼帕病毒出芽的宿主因子。将采用两种独立的策略： 来自亨尼帕病毒VLP的宿主蛋白的共亲和纯化，以及酵母双杂交筛选。基于siRNA的 第二次筛选将确定哪些候选结合蛋白对出芽实际上是重要的。 3.确定抗病毒药物开发的宿主和病毒靶点。病毒和 将绘制宿主蛋白质。将测试宿主蛋白质的最小结合片段的阻断能力。 病毒出芽，类似于类似片段对逆转录病毒和副流感病毒5（PIV5）的作用 发芽