This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff.
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 rapid access application is to obtain experimental beam time for X-ray diffraction data collection on the Adeno-associated virus serotypes 5, 8, and 9 (AAV5, AAV8 and AAV9) - being exploited for gene therapy applications, H-1 hamster parvovirus being exploited for cancer cell targeted gene delivery of cytokines, and the prototype strain of minute virus of mice (MVM) (a viral model for understanding virus host range adaptation) co-crystallized with carbohydrate components of its cell surface receptor. The AAV5 and MVMp data will be collected for capsids co-crystallized with sialic acid components of their cell surface receptors towards identifying the capsid site that facilitates infectious interaction. AAV5, AAV8, and MVM data will also be collected at pHs (6.0, 5.5 and 4.0) that mimic the endocytic pathway data in an effort to elucidate the nature of the capsid transistions associated with parvovirus trafficking to the nucleus. We are seasoned users of CHESS and have determined several structures from data collected at A1 and F1. 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. Our studies are focused on two aspects of parvovirology: (I) Understanding viral adaptation to new hosts and mechanisms of host pathogenicity, exploiting Minute virus of mice (MVM) and H-1 hamster parvovirus (H-1); Highly homologous MVM strains have disparate tissue tropism and infection pathogenicity, with one or two amino acids conferring a lethal outcome. Our work MVM has focused determining the structures of the homologous MVMp (prototype strain) and MVMi (the immunosuppressive strain) as well as virulent mutants of MVMp, alone and in complex with the sialic acid component of their infectious cell surface receptor. We have shown that the regions controlling their tissue tropism and pathogenicity phenotypes are on the capsid surface and configure a receptor attachment site1,-3. This observation suggests that host adaptation likely utilizes alteration of host receptor recognition, reminiscent of the host adaptation/jumping that has been reported for the influenza virus. The H-1 project is new to the lab and is aimed at structural characterization towards improving its use for cancer cell targeted gene delivery. (II) Structural characterization of host cellular entry, trafficking and transduction efficiency towards improved viral-based gene delivery, exploiting the Adeno-associated virus (HIV AAV) serotypes that hold significant promise for the correction of human diseases. The AAVs can be used to transfer non-host genes efficiently into primary cells in vivo, and in most cases, expression of the transgene appears to be long lived. Several serotypes are now in clinical trials of the treatment of animal and human diseases. 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 cell entry followed by uptake via endocytosis. The receptor binding regions are thought to be on capsid surfaces regions that differ between the serotypes. Our work is focused on characterizing the structures of the representative members of the AAV clades (AAV1-AAV9), their interactions with host receptor molecules, and entry. We are also working to identify the dominant antigenic regions of these capsids with the goal to engineering a second generation of gene delivery vectors with host immune evasion properties. This beam time application is for five projects;MVM, H-1, AAV5, AAV8, and AAV9. We have determined the structures of MVMi and MVMp1, plus those of virulent MVMp strains to 3.2 ? (in preparation) with data collected at CHESS, APS, BNL and at the Daresbury Laboratories (UK). Our current studies are aimed at continued characterization of the capsid regions used for receptor recognition and pH-mediated transitions associated with endosomal trafficking to the nucleus for replication. We have co-crystals of MVMp-sialic acids that specifically bind MVM capsids3 and crystals grown at pHs 6.0-4.0 that mimic the endocytic pathway. For H-1 we have crystals of empty capsids. For the AAVs, we have crystals of AAV5 and AAV8 at pHs that mimic the endocytic pathway and for AAV9, for which there is currently no structure information, we have small crystals for testing diffraction quality. We also have crystals of AAV5 co-crystallized with the sialic acid component of its receptor. We have already determined the crystal structures of wild type AAV5 and AAV8 at pH 7.54-6. F1 beam time allocation will enable us to collect X-ray diffraction data for our continued structure function studies on these viruses discussed above. The long-term goal of this research is to obtain a fundamental understanding of the structural determinants of the natural variations that exists in the Parvoviridae, along with information on their interactions with host cellular components and mutational data. Such information is important for understating the characteristics of emerging pathogens. We also aim to use this data towards the development of vaccines, antigen delivery systems, and gene therapy vectors for the treatment of animals and humans.
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