Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 9

Abstract

Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P3(2), with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress.

Figure 1

Purification and crystallization of AAV9 viral capsids. (a) SDS–PAGE gel of the purified AAV9 sample, showing the presence of viral proteins VP1, VP2 and VP3. Note that on the SDS–PAGE gel the molecular weights of AAV9 VP1, VP2 and VP3 run slightly higher than the calculated molecular weights of 81, 66 and 60 kDa, respectively. (b) Negatively stained electron micrograph of AAV9 capsids. (c) Optical photograph of a rod-shaped AAV9 crystal of approximately 0.2 × 0.05 × 0.03 mm in size.

Figure 2

X-ray diffraction image of an AAV9 crystal. (a) Image of a typical 0.3° oscillation photograph. The circles indicate resolution ranges of 40, 20, 8.0, 4.0 and 2.8 Å. (b) Close-up of the boxed region in (a) showing observed reflections to greater than 2.8 Å resolution.

Figure 3

Stereographic projections showing self-rotation function search results for the X-ray diffraction data. The three viral capsids in the unit cell have the same orientation and the crystallographic threefold axis is parallel to the icosahedral threefold axis; thus, the self-rotation peaks from all three capsids overlap with each other. (a) The rotation function for κ = 72°, showing a search for fivefold icosahedral symmetry elements, using the observed data set in the 10–5.0 Å resolution range with a radius of integration of 120 Å. (b) The rotation function for κ = 120°, showing a search for threefold symmetry elements. (c) The rotation function for κ = 180°, showing a search for twofold symmetry elements.