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. 2012 Jul;86(13):7326-33.
doi: 10.1128/JVI.00448-12. Epub 2012 Apr 18.

Identification of the galactose binding domain of the adeno-associated virus serotype 9 capsid

Christie L Bell  1 , Brittney L Gurda, Kim Van Vliet, Mavis Agbandje-McKenna, James M Wilson
Affiliations

Identification of the galactose binding domain of the adeno-associated virus serotype 9 capsid

Christie L Bell et al. J Virol. 2012 Jul.

Abstract

Adeno-associated virus serotype 9 (AAV9) vectors show promise for gene therapy of a variety of diseases due to their ability to transduce multiple tissues, including heart, skeletal muscle, and the alveolar epithelium of the lung. In addition, AAV9 is unique compared to other AAV serotypes in that it is capable of surpassing the blood-brain barrier and transducing neurons in the brain and spinal cord. It has recently been shown that AAV9 uses galactose as a receptor to transduce many different cell types in vitro, as well as cells of the mouse airway in vivo. In this study, we sought to identify the specific amino acids of the AAV9 capsid necessary for binding to galactose. By site-directed mutagenesis and cell binding assays, plus computational ligand docking studies, we discovered five amino acids, including N470, D271, N272, Y446, and W503, which are required for galactose binding that form a pocket at the base of the protrusions around the icosahedral 3-fold axes of symmetry. The importance of these amino acids for tissue tropism was also confirmed by in vivo studies in the mouse lung. Identifying the interactions necessary for AAV9 binding to galactose may lead to advances in vector engineering.

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Figures

Fig 1
Fig 1
AAV9 capsid amino acids required for galactose binding. (A) After comparing the capsid amino acid sequence of AAV9 to that of other serotypes, specific amino acids unique to AAV9 that contain charged or polar side chains were mutated to alanine to identify those responsible for galactose binding. Fourteen mutant vectors were constructed and were named according to the native amino acid and its specific position followed by the new amino acid: alanine. The mutants were tested for binding to Pro-5, Lec-2, and Lec-8 cells, compared to AAV9. Vector was added (5 × 109 GC) to each cell line and incubated at 4°C for 1 h. After washing, total DNA was isolated to determine bound vector GCs by quantitative PCR. N470 was determined to be necessary for AAV9 galactose binding. (B) Subsequently, 19 amino acids located in close proximity to N470 were mutated to alanine to examine the effect on AAV9 galactose binding. In addition, four other mutants were produced containing mutations of the nonpolar amino acids A472 or V473 to either serine or aspartic acid. The binding of these mutant vectors was then assessed as described above. D271, N272, Y446, and W503, as well as A472 and V473, were found to be important for AAV9 galactose binding. The data are shown as means + the standard deviation (SD).
Fig 2
Fig 2
In vitro transduction efficiency of mutant vectors. The five mutants that lost the ability to bind to galactose (N470A, D271A, N272A, Y446A, and W503A), as well as two mutants that retained galactose binding (S469A and E500A) and an AAV9 control vector, all expressing ffLuc, were added to Lec-2 (A) or Pro-5 (B) cells at 109 GC/well (MOI = 104) and ffLuc expression was measured 48 h later. The data are shown as means + the SD. RLU, relative light units.
Fig 3
Fig 3
Mutant vector transduction of conducting airway epithelium. C57BL/6 mice were either given an intranasal instillation of 100 mU NA (+NA) or left untreated (−NA), 1 h prior to administration of AAV9 or mutant vectors expressing nLacZ (1011 GC). After 21 days, the lungs were inflated, removed, and stained for β-Gal expression. (A) Representative images of lung expression showing conducting airway and alveolar epithelium transduction. Images were taken at ×100 magnification. (B) Quantification of nLacZ-positive conducting airway epithelial cells per ×200 field of view. The data are shown as means + SD.
Fig 4
Fig 4
Structure of the AAV9 galactose binding pocket. Galactose was docked onto an AAV9 trimer using the molecular docking webserver PatchDock (36). (A) AAV9 trimer surface showing the location of the galactose binding pockets. Galactose (light green) is shown docked at one of the binding sites. (B) Side view of an AAV9 trimer depicting the galactose binding pocket. (C) Close-up view from the side of the AAV9 galactose binding pocket. (D) Structure of the galactose binding pocket indicating the distances between the amino acid side chains and galactose. N470, yellow; D271, red; N272, white; Y446, blue; W503, orange; A472, teal; V473, gray; and galactose, light green. Images were generated using the PyMOL Molecular Graphics System, version 1.3 (Schrödinger, LLC).
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References

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