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: 8068317
• 负责人: Nicolas Nassar
• 金额: $ 7.57万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 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 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8068317
• 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（由申请人提供）：腺病毒（AD）是非发育的，二十面体，双链的DNA病毒，造成约5％的儿童和成人上呼吸道感染。 AD是全世界胃肠炎的重要病因，并且在免疫抑制和发展中国家中是重要的人类病原体。除了将AD用于人类基因疗法外，这还主张进一步理解AD复制周期，以开发治疗方式或更好的基因治疗应用细化。感染后，AD复制周期改变了宿主细胞代谢，以促进病毒基因组的复制到高拷贝。随后，将病毒基因组包装到空的衣壳中，并通过细胞裂解释放病毒。因此，DNA包装是病毒复制的重要生物学步骤，也是感染的重要步骤。 AD IVA2蛋白在不同起源的AD和病毒组装和病毒基因组包装的关键中高度保守。它具有ATPase的氨基酸序列标志，并在体外结合ATP。 ADIVA2蛋白具有标志性的氨基酸序列基序，与与ASCE ATPases的催化结构域相似/同源的结构一致。氨基酸序列分析将ADIVA2蛋白作为一种与Transporter ATPases的ABC超家族的预测ATPase的独特位置，但在任何其他病毒谱系中都不表示。拟议的研究的目的是通过使用X射线晶体学确定其三维结构来提高我们对该蛋白质功能的理解。确定AD2 IVA2蛋白的结构肯定会推进我们对AD包装和复杂真核DNA病毒包装的理解，以类似的方式，噬菌体包装运动ATPases ATPases的结构揭示了对其功能的重要见解，并允许对其功能进行预测和测试。三维结构还将允许设计抗病毒剂。大量该蛋白质将在SF9细胞中表达，并纯化为均匀性。通过单独对纯化的蛋白质进行纯化的蛋白质，或与ATP和/或DNA复杂化，可以找到结晶条件，以与各种沉淀剂，缓冲液，温度等。这些条件将被完善，直到适合高分辨率结构确定的高质量晶体为止。接下来，IVA2的晶体结构将通过单波长异常分散（SAD）或多种同构置换（MIR）技术来确定。 公共卫生相关性：将腺病毒基因组包装成空的衣壳是病毒复制和感染的重要一步。 ATP结合蛋白IVA2是空capsids组装所必需的，并与随后的病毒DNA包装有关。拟议研究的目的是确定AD2 IVA2的三维结构，以更好地理解其功能，并作为抗病毒药剂设计的手段。