This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.
The aim of our projects on the ssDNA viruses is to identify structural determinants of receptor attachment, tissue tropism, in vivo pathogenicity, and transduction efficiencies between highly homologous Parvoviridae strains and serotypes. Our structural studies so far indicate that slight capsid surface alterations, resulting from amino acid differences, are associated with pronounced differences in biological properties during the viral life cycle of parvoviruses. The long-term goal of our studies are to utilize the structural information obtained for the design of viral vaccines, foreign antigenic delivery systems, viral gene therapy vectors for the treatment of animal and human diseases. This application is to obtain experimental beam time for data collection on the adeno-associated virus serotypes 5 and 7 (AAV5 and AAV7) being actively developed for gene therapy applications, and on the prototype strain of minute virus of mice (MVMp) on studies aimed at structural elucidation of virulence determinants. The current application is to obtain beam time for data collection on crystals of wt AAV7 for which there is no structure data and on crystals of AAV5 at pHs encountered in the endosome. Both sets of crystals have been shown to diffract X-rays to at least 3.0 & resolution at either A1 or F1. We will also collect data on full (DNA containing) MVMp crystals, with the goal to mapping its genomic interaction site, and also on virulent MVMp mutant crystals soaked with sialylated oligosaccharide components that regular cellular infection and virulence determination. The Parvoviridae are spherical, non-enveloped, T=1 icosahedral viruses, with a wide range of natural hosts which includes humans, monkeys, pigs, dogs, cats, mink and mice. They are the cause of a number of serious diseases, especially in the young of the species that they infect, due to a requirement for cellular factors, produced during active cell division. Parvoviral capsids are ~260 & in diameter and encapsidate a ssDNA genome of ~5000 bases. Our correlation of available structural information on parvovirus capsids with molecular and biochemical data indicates that capsid surface variations modulate biological differences, such as receptor attachment, host range, pathogenicity and antigenicity, among members of the parvoviruses and between strains/serotypes of the same virus. More recently, our research efforts have been focused on the study of Adeno-associated virus (AAV) serotypes that hold significant promise for the correction of human diseases when used as gene therapy vectors. AAV can be used to transfer genes efficiently into primary cells in vivo, and in most cases, expression of the transgene appears to be long lived. Our work has focused on the AAV1, 2, 4, 5, 7 and 8 serotypess that show different tissue tropism. The cellular tropism of the different AAV serotypes is determined by the ability of the virus capsid to bind host cell receptors with different terminal carbohydrates that mediate the cell uptake of the virus via endocytosis. The receptor binding regions are thought to be on the capsid surfaces of the viruses in regions that differ between the serotypes. We have determined the structure of minute virus of mice (both MVMi and MVMp, the immunosuppressive and prototype strains, respectively, and mutants of MVMp) to 3.2 & with data collected at CHESS and at the Daresbury Laboratories (UK). Our current studies are aimed at identifying (I) regions on the capsid that interact with genomic DNA (for packaging), (II) regions that determine virulence, and (III) regions that are utilized for receptor attachment. Towards this goal we have obtained data (at CHESS) on crystals for MVMp and its virulent mutants soaked with sialic acid components shown to specifically recognize the MVM capsid in a glycan array. Work is in progress to identify the sialic acid binding site on the capsids of these viruses. With regards to our studies of the AAV serotypes, we have determined the wt structures of AAV4, AAV5, and AAV8 from X-ray diffraction data collected at CHESS to ~3.0 & resolution. In addition, in an effort to map potential structural re-arrangements encountered during cell infection and entry, we determined the structure of AAV8 at four different pHs, 7.5, 6.0. 5.5, and 4.0. Analyses of these structures are in progress. This beam time application is enable X-ray diffraction data collection on three projects; full (DNA containing) MVMp, AAV5 and AAV7. F1 beam time allocation will enable us to collect X-ray diffraction data on the full MVMp crystals grown for three different populations of Sfull infectious virions with different ratios of their capsid protein that represent different maturation stages. This will enable our continued mapping of the protein-DNA interactions inside parvovirus capsids and their contribution to icosahedra ordering. A1 beam time allocation will enable us to collect X-ray data on AAV5 (at pH 6.0, 5.5, and 4.0) and on wt AAV7 (for which there is no structural data). These studies are aimed at obtaining capsid surface information that will enable recombinant manipulations for vector re-targeting for specific gene delivery. The long-term goal of this research is to use the fundamental understanding of the natural variations that exists in the Parvoviridae, together with information on their interactions with host cellular components and mutational data, which can be deemed from structural analysis, to develop vaccines, antigen delivery systems and gene therapy vectors for the treatment of animals and humans.
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