Adeno-associated virus (AAV) is a non-pathogenic virus that shows great promise as a delivery vehicle or vector for gene therapy. The focus of our group has been a structure-function analysis of the AAV capsid. We have combined structural analysis (X-ray crystallography and cryo-EM) with mutagenic and biochemical analysis toward identifying regions of the capsid that are essential for infection. This has led to important information that has allowed the development of new vector production strategies and the promise of targeted vectors. We have used X-ray crystallography to identify regions of the AAV capsid that undergo a structural change when the capsid is subjected to acidic pHs and used circular dichroism (CD) to show that unique region of the minor capsid viral protein VP1 (VP1u), which contains a phospholipase A2 (PLA2) function, becomes unfolded under similar conditions. These pHs (pH 4-6) have been shown to be essential for productive AAV infections and are comparable to those that the capsid encounters in endosomal compartments during cell entry and trafficking. Our studies led to two unexpected novel discoveries. The first is that the capsid has a previously unknown enzymatic activity: a pH sensitive protease that can catalyze autolytic cleavage of the capsid as well as external substrates. Both the mechanism of the protease activity and its function are unknown and appear to be unique compared to other virus encoded proteases. The second is that mutations in the pH sensitive region of the capsid have a profound effect on gene expression, even after the viral DNA is uncoated in the nucleus, suggesting that the capsid plays a role in gene expression after DNA uncoating in the nucleus. Furthermore, the CD studies suggested a mechanism for the externalization of the VP1u which is normally buried in the capsid interior but is extruded during trafficking through acidic endosomal compartments. In this proposal, we wish to explore these novel findings by (1) identifying the active site of the protease(s) as well as its cleavage targets; (2) determining the role of the pH sensitive capsid region in gene expression after nuclear uncoating; and (3) examining the effect of pH and cations on the other enzymatic activity in the capsid, the VP1u associated PLA2.

Public Health Relevance

It is now clear that AAV can be used successfully for a variety of applications in gene therapy of genetic and acquired diseases. Our research will provide important new information about the mechanism of AAV entry into cells, trafficking to the nucleus, and subsequent gene expression. What we learn in the course of these studies will lead to the development of more potent and more specific next generation AAV vectors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM109524-04
Application #
9341366
Study Section
Virology - A Study Section (VIRA)
Program Officer
Sakalian, Michael
Project Start
2014-09-15
Project End
2019-08-31
Budget Start
2017-09-01
Budget End
2019-08-31
Support Year
4
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Tan, Yong Zi; Aiyer, Sriram; Mietzsch, Mario et al. (2018) Sub-2?Å Ewald curvature corrected structure of an AAV2 capsid variant. Nat Commun 9:3628
Bennett, Antonette; Patel, Saajan; Mietzsch, Mario et al. (2017) Thermal Stability as a Determinant of AAV Serotype Identity. Mol Ther Methods Clin Dev 6:171-182
Mietzsch, Mario; Hering, Henrik; Hammer, Eva-Maria et al. (2017) OneBac 2.0: Sf9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA. Hum Gene Ther Methods 28:15-22
Callaway, Heather M; Feng, Kurtis H; Lee, Donald W et al. (2017) Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages. J Virol 91:
Drouin, Lauren M; Lins, Bridget; Janssen, Maria et al. (2016) Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging. J Virol 90:8542-51
Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna et al. (2016) Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids. J Virol 90:7196-7204
Ling, Chen; Wang, Yuan; Lu, Yuan et al. (2015) The Adeno-Associated Virus Genome Packaging Puzzle. J Mol Genet Med 9:
Kailasan, Shweta; Agbandje-McKenna, Mavis; Parrish, Colin R (2015) Parvovirus Family Conundrum: What Makes a Killer? Annu Rev Virol 2:425-50
Snijder, Joost; van de Waterbeemd, Michiel; Damoc, Eugen et al. (2014) Defining the stoichiometry and cargo load of viral and bacterial nanoparticles by Orbitrap mass spectrometry. J Am Chem Soc 136:7295-9