• Institute of Molecular Biophysics
  • Kasha Laboratory of Biophysics
  • Florida State University
  • Tallahassee, FL 32306-4380
  • phone: (850) 644-4104
  • fax: (850) 644-7244
  • odonnell@chem.fsu.edu

 

Single Particle Analysis

The Adeno-associated virus serotype 2 (AAV-2) has inherent qualities suitable for an ideal gene therapy vector. It is non-pathogenic, illicits a low immune response, and is widely tropic infecting both dividing and non-dividing cells. To this end, recombinantly engineered AAV vectors are in use for the treatment of a variety of diseases. However, AAV’s wide cell tropism is also a disadvantage and works against efficient delivery of genetic material. AAV-2’s infection process is initiated by binding to a primary (attachment) receptor, Heparan Sulfate Proteoglycan (HSPG) after which it binds to a co-receptor. Structural information regarding how it interacts with its receptors could greatly facilitate the achievement of tailored cell-specific vectors.

Cryo-EM single particle analysis (SPA)is becoming an increasingly popular technique for solving macromolecular structures. For those with a high order of symmetry such as icosahedral viruses, cryo-em lends the opportunity to resolve strucrual features at near atomic resolution. I performed the technique of SPA on vitrified images of AAV-2 in its native state and as a complex with a 17kDa fragment of its primary cellular receptor, heparin. Above in garnet and gold are stereo views of native(top row) and heparin-AAV-2(bottom row) at 7.8A and 8.3A respectively. The arrow points to extra density located near the shoulder of each three-fold related peak. A heparin-AAV-2 minus AAV-2 difference map (blue, below) reveals this extra density to be located directly over a cluster of amino acids previously implicated in binding by mutagenic studies.

A) Wild-type AAV-2 (garnet and gold) with a superimposed positive difference map (blue) contoured at two thresholds. The lower of two thresholds is shown with semi-transparency. B) Magnified stereo view of the white square in A in the same orientation. Colored differently are chains of the crystal structure after symmetry expansion (RSCB entry 1LP3). Shown as spheres are amino acids previously implicated in primary receptor binding. A gold arrow denotes the 3-fold axis of symmetry. C) Same as in A with 3 heparin 9-mers (gray, ball & stick) built by connecting rigidly fit monosaccharide units from RSCB entry 1SR5 into a difference map (white, semitransparent). D) Same as in C with the difference map removed.

PUBLICATIONS

  • Mitchell DA., O’Donnell J., Hare J.T, Chapman M.S. Serotype-specific detection of adeno-associated virus during laboratory preparation. J. Virol. Methods 136(1-2):277-82 (2006).
  • Jason O’Donnell, Kenneth A. Taylor & Michael S. Chapman. Adeno-Associated Virus-2 and its primary cellular receptor cryo-EM structure of a heparin complex. Virology 385 434-443 (2009).
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