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

描述（由申请人提供）：腺病毒（Ad）是一种非包膜、二十面体、双链DNA病毒，在儿童和成人中造成约5%的上呼吸道感染。Ad是世界范围内肠胃炎的重要病原，也是免疫抑制和发展中国家重要的人类病原体。除了将Ad用于人类基因治疗之外，这还表明，为了开发治疗方式或更好地改进基因治疗应用，需要进一步了解Ad复制周期。感染后，Ad复制周期改变宿主细胞代谢，促进病毒基因组向高拷贝复制。随后，病毒基因组被包装到一个空衣壳中，病毒通过细胞裂解释放出来。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 IVa2蛋白在不同来源的Ad中高度保守，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 IVa2蛋白具有与ASCE ATPases的催化结构域相似/同源的特征氨基酸序列基序。氨基酸序列分析将Ad IVa2蛋白置于一个独特的位置，作为一种预测的atp酶，它类似于转运体atp酶的ABC超家族，但在任何其他病毒谱系中都没有表现出来。提出的研究目标是通过使用x射线晶体学确定其三维结构来推进我们对这种蛋白质功能的理解。确定ad2iva2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，就像噬菌体包装马达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一样。三维结构也将允许设计抗病毒药物。该蛋白将在Sf9细胞中大量表达并纯化至均匀性。通过将纯化蛋白单独或与ATP和/或DNA复合置于各种沉淀剂、缓冲液、温度等条件下，可以发现结晶条件。这些条件将被改进，直到适合高分辨率结构测定的高质量晶体出现。接下来，将通过单波长异常色散（SAD）或多重同构替换（MIR）技术解决相位问题来确定IVa2的晶体结构。

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.