Structure-Function Relationship of the Adenovirus Assembly and DNA packaging prot

腺病毒组装与DNA包装蛋白的结构-功能关系

• 批准号: 7976203
• 负责人: Nicolas Nassar
• 金额: $ 0.48万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2010-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7976203
• 关键词: ATP phosphohydrolase; Adenovirus Infections; Adenoviruses; Adult; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Amino Acid Sequence; Antiviral Agents; Bacteriophages; Binding; Binding Proteins; Biological; Buffers; Capsid; Catalytic Domain; Cell Culture Techniques; Cells; Child; Complex; Comprehension; Crystallization; Cytolysis; DNA; DNA Binding; DNA Packaging; DNA Viruses; Data; Double Stranded DNA Virus; Gastroenteritis; Genome; Goals; Hand; Human; Human Adenoviruses; Immunosuppression; In Vitro; Infection; Insecta; Metabolism; Modality; Motor; Phase; Positioning Attribute; Protein Sequence Analysis; Proteins; Research; Resolution; Structure; Structure-Activity Relationship; Techniques; Temperature; Testing; Therapeutic; Upper Respiratory Infections; Viral; Viral Genome; Virus; Virus Assembly; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; design; gene therapy; insight; pathogen; protein function; public health relevance; research study; three dimensional structure; viral DNA

DESCRIPTION (provided by applicant): Adenoviruses (Ad) are non-enveloped, icosahedral, double-stranded DNA viruses that are responsible for ~5% of upper respiratory infections in children and adults. Ad is a significant etiological agent of gastroenteritis worldwide and an important human pathogen in the context of immunosuppression and in the developing world. This, in addition to the utilization of Ad for human gene therapy, argues for further understanding of the Ad replication cycle in order to develop therapeutic modalities or better refinements for gene therapy applications. Following infection, the Ad replication cycle alters host cell metabolism to promote the replication of the viral genome to high copy. Subsequently, the viral genome is packaged into an empty capsid and the virus is released by cell lysis. DNA packaging is thus an important biological step in viral replication and an essential step for infection. The Ad IVa2 protein is highly conserved among Ads from diverse origins and key for virus assembly and viral genome packaging. It has the amino acid sequence hallmarks of an ATPase and binds ATP in vitro. The Ad IVa2 protein has signature amino acid sequence motifs that are consistent with a structure similar/homologous to the catalytic domain of ASCE ATPases. Amino acid sequence analysis places the Ad IVa2 protein in a unique position as a predicted ATPase that resembles the ABC superfamily of transporter ATPases but that is not represented in any other viral lineage. The goal of the proposed research is to advance our understanding of the function of this protein by determining its three-dimensional structure using X-ray crystallography. Determining the structure of the Ad2 IVa2 protein will certainly advance our comprehension of the packaging of Ad and of complex eukaryotic DNA viruses in a similar way the structures of bacteriophage packaging motor ATPases revealed important insights into their function and allowed predictions to be made and tested about their function. The three-dimensional structure will also allow for design of antiviral agents. Large quantities of this protein will be expressed in Sf9 cells and purified to homogeneity. Crystallization conditions will be found by subjecting the purified protein alone, or in complex with ATP and/or DNA, to a variety of precipitants, buffers, temperatures, etc. These conditions will be refined until high-quality crystals suitable for high-resolution structure determination are at hand. Next, the crystal structure of IVa2 will be determined by solving the phase problem by the single-wavelength anomalous dispersion (SAD) or multiple isomorphous replacement (MIR) techniques. PUBLIC HEALTH RELEVANCE: Packaging of the Adenovirus genome into an empty capsid is an essential step in viral replication and infection. The ATP-binding protein IVa2 is required for the assembly of empty capsids and is implicated in the subsequent packaging of viral DNA. The goal of the proposed research is to determine the three- dimensional structure of Ad2 IVa2 to better our understanding of its function and as a means toward the design of antiviral agents.

描述(申请人提供)：腺病毒(Ad)是一种无包膜、二十面体、双链DNA病毒，约占儿童和成人上呼吸道感染的5%。AD是世界范围内胃肠炎的重要病原体，也是免疫抑制背景下和发展中国家的重要人类病原体。这除了将Ad用于人类基因治疗外，还需要进一步了解Ad复制周期，以便开发治疗模式或更好地改进基因治疗应用。在感染后，Ad复制周期改变宿主细胞的代谢，促进病毒基因组的复制到高拷贝。随后，病毒基因组被包装成一个空的衣壳，病毒通过细胞裂解释放。因此，DNA包装是病毒复制的重要生物学步骤，也是感染的关键步骤。Ad IVa2蛋白在来自不同来源的广告中高度保守，是病毒组装和病毒基因组包装的关键。它具有ATPase的氨基酸序列特征，并在体外与ATP结合。Ad IVa2蛋白具有特征性的氨基酸序列基序，其结构与ASCE ATPase的催化结构域相似/同源。氨基酸序列分析将Ad IVa2蛋白定位在一个预测的ATPase的独特位置，该ATPase类似于ABC转运体ATPase超家族，但在任何其他病毒谱系中都不存在。这项拟议的研究的目标是通过使用X射线结晶学确定其三维结构来促进我们对该蛋白质功能的理解。确定AD2 IVa2蛋白的结构肯定会促进我们对Ad和复杂真核DNA病毒包装的理解，类似地，噬菌体包装运动ATPase的结构揭示了它们的功能，并允许对它们的功能进行预测和测试。这种三维结构还可以用于抗病毒药物的设计。大量该蛋白将在Sf9细胞中表达并纯化至均一。通过将纯化的蛋白质单独或与ATP和/或DNA复合，在各种沉淀剂、缓冲液、温度等条件下寻找结晶条件。这些条件将被改进，直到获得适合高分辨率结构测定的高质量晶体。接下来，将通过单波长反常色散(SAD)或多重同晶置换(MIR)技术解决位相问题来确定IVa2的晶体结构。 与公共卫生相关：将腺病毒基因组打包到空衣壳中是病毒复制和感染的关键步骤。ATP结合蛋白IVa2是组装空衣壳所必需的，并与随后病毒DNA的包装有关。这项研究的目的是确定AD2 IVa2的三维结构，以更好地了解其功能，并作为抗病毒药物设计的一种手段。