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

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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.