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).