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: 8258391
• 负责人: Nicolas Nassar
• 金额: $ 7.16万
• 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/8258391
• 关键词: Structure; Function; Relationship; Adenovirus; Assembly

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（由申请方提供）：腺病毒（Ad）是一种无包膜、二十面体、双链DNA病毒，约5%的儿童和成人上呼吸道感染由其引起。Ad是世界范围内胃肠炎的重要病原体，并且在免疫抑制背景下和在发展中国家是重要的人类病原体。这，除了利用广告的人类基因治疗，主张进一步了解广告的复制周期，以开发治疗方式或更好的基因治疗应用的改进。感染后，Ad复制周期改变宿主细胞代谢，以促进病毒基因组复制到高拷贝。随后，将病毒基因组包装到空衣壳中，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，也是感染的必要步骤。Ad IVa 2蛋白在来自不同来源的Ad中是高度保守的，并且是病毒组装和病毒基因组包装的关键。它具有ATP酶的氨基酸序列特征，并在体外结合ATP。Ad IVa 2蛋白具有与ASCE ATP酶的催化结构域相似/同源的结构一致的特征氨基酸序列基序。氨基酸序列分析将Ad IVa 2蛋白置于一个独特的位置，作为一种预测的ATP酶，类似于ABC超家族的转运蛋白ATP酶，但不代表任何其他病毒谱系。这项研究的目标是通过使用X射线晶体学确定其三维结构来促进我们对这种蛋白质功能的理解。确定Ad 2 IVa 2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，类似于噬菌体包装马达ATP酶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。三维结构也将允许设计抗病毒剂。大量的这种蛋白质将在Sf 9细胞中表达并纯化至均一。结晶条件将通过将纯化的蛋白质单独或与ATP和/或DNA复合，以各种沉淀剂，缓冲液，温度等来确定。这些条件将被细化，直到适合于高分辨率结构测定的高质量晶体在手。接下来，IVa 2的晶体结构将通过单波长异常色散（SAD）或多个同晶置换（MIR）技术解决相位问题来确定。 公共卫生相关性：将腺病毒基因组包装到空衣壳中是病毒复制和感染的重要步骤。ATP结合蛋白IVa 2是空衣壳组装所必需的，并参与随后的病毒DNA包装。这项研究的目的是确定Ad 2 IVa 2的三维结构，以更好地了解其功能，并作为设计抗病毒剂的一种手段。