Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less “pointed” than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.
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 Å resolution using synchrotron radiation and belonged to the trigonal space group P3 2 , with unit-cell parameters a = b = 251.0, c = 640.0 Å . 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.
Reovirus type 1 has been grown in green monkey kidney cells and harvested 24 hr after infection ("early" virus) and 96 hr after infection ("late" virus). A number of biological parameters have been determined on purified preparations of both "early" and "late" harvests of reovirus. There were no significant differences in values obtained for the molecular weight, RNA content, and buoyant density of virions prepared from early or late harvests. The size of the capsids and their morphology were also identical. Late harvests of reovirus were particularly rich in empty viral capsids (density, 1.28 in cesium chloride), and a significant number of empty inner capsid shells were routinely found. These shells could be prepared readily by controlled digestion of complete virus particles with trypsin. The inner shell appears to be composed of subunits packed with icosahedral symmetry to form a 45-m,u foundation on which the outer 92-subunit capsid is assembled. The inner shell is somewhat reminiscent in size and morphology of the capsid of papovaviruses. The fact that it can exist as a discrete entity has prompted us to propose some modifications to the current models for the reovirus capsid.
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