PhiX174 genome-capsid interactions: Evidence for a scaffolding-like function for the genome during morphogenesis

Persistent Link:
http://hdl.handle.net/10150/280355
Title:
PhiX174 genome-capsid interactions: Evidence for a scaffolding-like function for the genome during morphogenesis
Author:
Hafenstein, Susan
Issue Date:
2003
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The assembly of viral proteins and nucleic acids into mature and biologically active virions involves a diverse spectrum of macromolecular interactions. After capsid formation, structural and packaging proteins must interact with viral nucleic acids. These interactions may confer packaging specificity, spatially organize the genome, enhance particle stability, or contribute directly to capsid quaternary structure. In the Microviridae, packaging and capsid proteins are tightly associated, tethering the genome to the inner surface and guiding it into the overall icosahedral symmetry of the particle. All of these factors may influence the final stages of maturation, which involves an inward collapse of coat proteins around the packaged genome. These packaging parameters were altered in three ways. (1) The DNA binding residues of the DNA binding protein were altered. Although the genome and protein are in the interior of the capsid, alterations were expressed on the capsid's outer surfaces. The results of second site genetic analyses illustrate how coat protein modifications can compensate for defective phenotypes. (2) Non-DNA binding amino acid residues believed to be of structural importance were mutated. The results of these analyses elucidate the function of these residues in optimizing DNA-protein interactions and organizing the DNA into the capsid's symmetry. Again, the results of second site genetic analyses demonstrate the inherent evolutionary plasticity of the system. (3) Packaged DNA was changed by altering base composition and folding parameters. The experimental results support a model in which the secondary structure of the packaged genome acts in a scaffold-like manner during the final stage of virion morphogenesis, affecting the biophysical and biological properties of the mature virion. Finally a chimeric particle was constructed by placing a wild type DNA binding protein in a wild type, but foreign environment. The biophysical characterization of these particles is consistent with the mentioned model. A structural analysis was performed, in collaboration with Dr. Rossmann's group (Purdue University), to provide a structural context in which to interpret the observed biophysical effects. While not all questions were answered or hypotheses verified, the results elucidate the limitations and interpretation of structural analyses, a current debate in the structural biology field.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Molecular.; Biology, Microbiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Veterinary Science and Microbiology
Degree Grantor:
University of Arizona
Advisor:
Fane, Bentley A.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titlePhiX174 genome-capsid interactions: Evidence for a scaffolding-like function for the genome during morphogenesisen_US
dc.creatorHafenstein, Susanen_US
dc.contributor.authorHafenstein, Susanen_US
dc.date.issued2003en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe assembly of viral proteins and nucleic acids into mature and biologically active virions involves a diverse spectrum of macromolecular interactions. After capsid formation, structural and packaging proteins must interact with viral nucleic acids. These interactions may confer packaging specificity, spatially organize the genome, enhance particle stability, or contribute directly to capsid quaternary structure. In the Microviridae, packaging and capsid proteins are tightly associated, tethering the genome to the inner surface and guiding it into the overall icosahedral symmetry of the particle. All of these factors may influence the final stages of maturation, which involves an inward collapse of coat proteins around the packaged genome. These packaging parameters were altered in three ways. (1) The DNA binding residues of the DNA binding protein were altered. Although the genome and protein are in the interior of the capsid, alterations were expressed on the capsid's outer surfaces. The results of second site genetic analyses illustrate how coat protein modifications can compensate for defective phenotypes. (2) Non-DNA binding amino acid residues believed to be of structural importance were mutated. The results of these analyses elucidate the function of these residues in optimizing DNA-protein interactions and organizing the DNA into the capsid's symmetry. Again, the results of second site genetic analyses demonstrate the inherent evolutionary plasticity of the system. (3) Packaged DNA was changed by altering base composition and folding parameters. The experimental results support a model in which the secondary structure of the packaged genome acts in a scaffold-like manner during the final stage of virion morphogenesis, affecting the biophysical and biological properties of the mature virion. Finally a chimeric particle was constructed by placing a wild type DNA binding protein in a wild type, but foreign environment. The biophysical characterization of these particles is consistent with the mentioned model. A structural analysis was performed, in collaboration with Dr. Rossmann's group (Purdue University), to provide a structural context in which to interpret the observed biophysical effects. While not all questions were answered or hypotheses verified, the results elucidate the limitations and interpretation of structural analyses, a current debate in the structural biology field.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Molecular.en_US
dc.subjectBiology, Microbiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineVeterinary Science and Microbiologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorFane, Bentley A.en_US
dc.identifier.proquest3106995en_US
dc.identifier.bibrecord.b44660467en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.