Characterization of AAV2 Genome Packaging

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Material Information

Title:
Characterization of AAV2 Genome Packaging
Physical Description:
1 online resource (143 p.)
Language:
english
Creator:
Drouin, Lauren M
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences, Biochemistry and Molecular Biology (IDP)
Committee Chair:
AGBANDJE-MCKENNA,MAVIS
Committee Co-Chair:
MUZYCZKA,NICHOLAS
Committee Members:
SRIVASTAVA,ARUN
FLANEGAN,JAMES B
CONDIT,RICHARD C

Subjects

Subjects / Keywords:
aav -- adeno-associated -- capsid -- complexes -- gene -- packaging -- rep -- rep-capsid -- therapy -- virus
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
Genre:
Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
The Adeno-associated viruses (AAVs) are single-stranded DNA (ssDNA)-containing parvoviruses under development for therapeutic gene delivery applications. However, a need to improve vector production requires a better understanding of their capsid assembly and genome packaging mechanisms. The parvoviruses are reported to assemble their empty capsids in the nucleus prior to genome packaging by the Rep proteins. However, it still remains unclear exactly how these processes occur. The Rep proteins encoded by the genome have been shown to interact with the AAV capsid proteins and the ssDNA genome, but Rep-capsid protein complexes have not previously been visualized and the region on the capsid surface at which Rep interacts has not been structurally mapped. We have applied biochemical, molecular, and structural approaches to attempt identification of the Rep binding site on the surface of the AAV capsid using wt AAV2 and an AAV2 packaging deficient mutant, AAV2-R432A, shown to have an increased affinity for Rep proteins. A FLAG-tagged AAV2-R432A capsid was constructed to enrich the yield of the Rep-capsid complexes, and putative packaging complexes were observed by negative stain electron microscopy. However, immunolabeling experiments of these Rep-capsid complexes with gold particles were not conclusive in pinpointing the globular attachment as Rep. Cryo-electron microscopy and image reconstruction of wild-type and AAV2-R432A capsids at low resolution have indicated that a closure of the 5-fold pore may be responsible for the inability of AAV-R432A to package viral genome. Further structural rearrangements observed in the AAV2-R432A capsid may lead to disruption of intermonomer interactions, and ultimately may serve to weaken the capsid, as was confirmed by DSC studies. These findings provide better insight into the mechanism of AAV genome packaging.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Lauren M Drouin.
Thesis:
Thesis (Ph.D.)--University of Florida, 2013.
Local:
Adviser: AGBANDJE-MCKENNA,MAVIS.
Local:
Co-adviser: MUZYCZKA,NICHOLAS.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-12-31

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0046190:00001