Novel Antiviral Strategy to Combat Arenaviruses

对抗沙粒病毒的新型抗病毒策略

• 批准号: 6680989
• 负责人: Juan C. de la Torre
• 金额: $ 23.46万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680989
• 关键词: Arenaviridae; Lassa virus; bioterrorism /chemical warfare; genetic techniques; hemorrhagic fever; host organism interaction; intermolecular interaction; lymphocytic choriomeningitis virus; mass spectrometry; proline; protein protein interaction; protein structure; protein structure function; site directed mutagenesis; tissue /cell culture; vesicle /vacuole; virus cytopathogenic effect; virus genetics; virus protein; virus replication

DESCRIPTION (provided by applicant): The prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) is an excellent model to study the molecular and cellular biology of hemorrhagic fever arenaviruses, including Lassa fever virus (LFV). These viruses cause severe human disease, and they pose a real threat as agents of bioterrorism. We have developed a reverse genetic system for LCMV. We can now probe the function of viral genes in cell entry, virus replication, assembly and budding. We have obtained evidence that Arenavirus Z protein is the main driving force of virus budding. Arenavirus Z proteins contain proline-rich motifs PPXY and PT/SAP, which have been identified as essential for budding of several viruses including HIV-1 and Ebola. Evidence indicates that many enveloped viruses are capable of hijacking the cellular multivesicular body (MVB) machinery to escape the cell. We hypothesize that budding of LFV is mediated by the interaction of Z proline-rich domains with specific host factors, including components of the MVB pathway. Understanding these interactions will facilitate targeting of these virally reprogrammed host cellular processes as a new strategy to combat arenaviruses. We propose two specific aims. First, to determine the role of PPPY and PTAPP motifs in LFV Z mediated budding. Z proteins with mutations in PPPY or PTAPP, or both, motifs will be evaluated for their competence to promote budding of virus like particles (VLPs). Second, to identify host factors required for Arenavirus budding. We will test the hypothesis that TSG101 and Nedd4 proteins, both members of the MVB pathway, interact with LFV Z and that these interactions are relevant in LFV Z mediated budding. We will use the tandem affinity purification (TAP) method combined with mass spectrometry procedures to identify other Z-interacting cellular proteins. We will use biochemical and cellular assays to characterize Z/host factor interactions. The functional significance of these host factors in Arenavirus budding will be investigated by quantifying budding of VLPs in cells where expression of the host protein of interest has been disrupted by RNA interference.

描述（由申请人提供）：原型体育症病毒淋巴细胞脉络膜脑膜炎病毒（LCMV）是研究出血性田纳病毒（包括Lassa Fever Virus（LFV））的分子和细胞生物学的绝佳模型。 这些病毒引起了严重的人类疾病，并且作为生物恐怖主义的药物构成了真正的威胁。 我们已经为LCMV开发了反向遗传系统。 现在，我们可以探测病毒基因在细胞进入，病毒复制，组装和萌芽中的功能。 我们已经获得了竞技病毒Z蛋白是病毒萌芽的主要驱动力的证据。 体育症病毒Z蛋白包含富含脯氨酸的序列PPXY和PT/SAP，这些基序被确定为包括HIV-1和埃博拉病毒在内的几种病毒的萌芽必不可少的。 证据表明，许多包围病毒能够劫持细胞多囊体（MVB）机械以逃脱细胞。 我们假设LFV的萌芽是由富含Z脯氨酸域与特定宿主因子（包括MVB途径的组件）的相互作用介导的。 理解这些相互作用将有助于靶向这些病毒重编程的宿主细胞过程，以此作为对抗体ar虫病毒的新策略。 我们提出了两个具体目标。 首先，确定PPPY和PTAPP基序在LFV Z介导的芽中的作用。 Z蛋白具有PPPY或PTAPP中突变的Z蛋白，或两者兼有基序，以促进颗粒（VLP）等病毒的萌芽能力评估。 第二，确定体育症病毒芽所需的宿主因素。 我们将检验以下假设：MVB途径的两个成员TSG101和NEDD4蛋白与LFV Z相互作用，并且这些相互作用与LFV Z介导的芽伴相关。 我们将使用结合质谱程序的串联亲和力纯化（TAP）方法来识别其他Z相互作用的细胞蛋白。 我们将使用生化和细胞测定法来表征Z/宿主因子相互作用。 这些宿主因子在体育症病毒萌芽中的功能意义将通过量化VLP在RNA干扰中破坏宿主蛋白的表达中的细胞中的萌芽来研究。