Production, purification, crystallization and structure determination of H-1 Parvovirus

Abstract

Crystals of H-1 Parvovirus (H-1PV), an antitumor gene-delivery vector, were obtained for DNA-containing capsids and diffracted X-rays to 2.7 Å resolution using synchrotron radiation. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a=255.4, b=350.4, c=271.6 Å, β=90.34°. The unit cell contained two capsids, with one capsid per crystallographic asymmetric unit. The H-1PV structure has been determined by molecular replacement and is currently being refined.

Figure 1

Purification and crystallization of H-1PV full and empty capsids. (a) A 15% SDS–PAGE gel of the purified H-1PV capsids showing the relative ratios and positions of VP1, VP2 and VP3 (molecular weights of 81, 65 and 63 kDa, respectively). The positions of low-molecular-weight standards (labeled in kDa; Bio-Rad, Hercules, California, USA) are indicated on the right-hand side. (b, c) Negatively stained electron micrographs of H-1PV full capsids viewed at 100 000× magnification (b) and H-­1PV empty capsids viewed at 60 000× magnification (c). (d) Optical photograph of H-1PV full capsid crystals in a rod-shaped habit.

Figure 2

X-ray diffraction image for a crystal of H-1PV full capsids. The image is a typical 0.3° oscillation photograph. The concentric rings indicate the 10.0, 5.0 and 3.0 Å resolution shells. (b) The inset shows a close-up of the boxed region in the upper right-hand corner.

Figure 3

Stereographic projections of the self-rotation function search for the H-1PV full capsid X-ray diffraction data. (a) κ = 72°, (b) κ = 120° and (c) κ = 180°, searching for fivefold, threefold and twofold icosahedral symmetry elements, respectively. The peaks representing fivefold positions are delineated by the dashed pentagons in (a). The peaks belonging to each of the two viral capsids in the unit cell are circled in red and blue, respectively. The peaks interpreted as Klug peaks (5f1, 5f2 and 3f1–4) are circled in green and labeled in (a) and (b). The a*, b* and c* axes are labeled.