Three-dimensional structure of Aleutian mink disease parvovirus: implications for disease pathogenicity

Abstract

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.

FIG. 1

Micrograph of unstained ADV_G-VP2 particles suspended in a layer of vitreous ice over holes in a carbon support film. (A) A low-magnification survey image of nine user-selected areas (black open squares) over holes in the carbon support film and one in the carbon film. (B) A portion of a 1K² image recorded with spot-scan procedures on a slow-scan CCD (43).

FIG. 2

Shaded-surface representations of the ADV_G-VP2 3D reconstruction viewed down a twofold icosahedral axis (in stereo) (A), down a threefold axis (B), and down a fivefold axis (C). The reconstruction was computed to 22 Å resolution from 87 different ADV_G-VP2 particle images.

FIG. 3

Sequence alignment of the VP2 capsid proteins of ADV-G, ADV-Utah 1, and CPV. Arrowheads demarcate residues that consistently differ between ADV-G and the pathogenic ADV strains. The ADV hypervariable region is indicated by asterisks (∗) under the arrowheads. Boldface lettering is used to highlight completely conserved residues. Colored lettering highlights residues that comprise the β-barrel structural motif (blue), insertions IN1-IN8 in the ADV capsid protein (red), and insertions in the CPV capsid protein relative to ADV (green). The sequence numberings are for ADV-G (top) and CPV (bottom). The peptides (pVP2a-i) used in a nonoverlapping peptide expression study (19) are indicated by bars with arrowheads at each end above the capsid sequence and are colored. Orange, pVP2b; green, pVP2c; dark blue, pVP2d; light blue, pVP2e; yellow, pVP2f; red, pVP2; and black, pVP2a, pVP2h, and pVP2i.

FIG. 4

Shaded-surface representations of ADV_G-VP2 (A), CPV (B), and B19 (C), all viewed down a twofold icosahedral axis of symmetry. Panels A and C show the 3D cryo-EM reconstruction of ADV_G-VP2 and B19 VP2 capsids (25) to 22 and 26 Å resolution, respectively, and panel B shows the atomic structure of CPV (63), rendered at 21 Å resolution. Locations of some two- (2), three- (3), and fivefold (5) icosahedral axes are indicated.

FIG. 5

Ribbon drawings of the ADV_G-VP2 pseudoatomic model. (A) VP2 monomer, viewed down an icosahedral twofold axis. (B) VP2 dimer, viewed as in panel A. (C) VP2 trimer, viewed down a threefold axis. The blue and red colors identify, respectively, the β-barrel motif and the insertions (IN1 to IN8) relative to the CPV capsid protein (Fig. 3). An icosahedral asymmetric unit (large open triangle) includes the region bounded by one fivefold (filled pentagon) and two threefold axes (filled triangles) separated by a twofold axes (filled oval) in panels A and B. The yellow circle in panel C highlights the clustering of insertion loops IN1, IN2, IN4, IN6, and IN7, which form the mounds near the threefold axes. The figure was generated with the program MOLSCRIPT (39).

FIG. 6

The pseudoatomic model of ADV-G VP2 fitted into the cryo-EM reconstruction (gray isodensity contour). (A) Stereo diagram of the mounds viewed parallel to an icosahedral twofold axis, showing the right-hand-side trimer with the reference VP2 in blue and the icosahedral threefold-related VP2s in red and green. Shown also is the viral asymmetric unit depicted as an open triangle. (B) Close-up view of panel A with inserted peptides IN1, IN2, IN4, IN6, and IN7 in ADV. (C) Close-up view of the ADV mound density with superimposed CPV atomic model, which has no equivalence structure to the inserted peptides in ADV. This figure was generated with the program MacInPlot (57).

FIG. 7

The locations of amino acids that differ between ADV-G and pathogenic ADV strains (strain-specific amino acids), projected onto a roadmap (23) representation of the residues on the capsid surface of the ADV_G-VP2 pseudoatomic model colored according to radial distance (in angstroms) from the viral center. The orientation is the same as in Fig. 5A and B. The asymmetric unit contains contributions from several symmetry-related VP2 subunits. A letter preceding the sequence number identifies a symmetry operation used to generate the residue from the unlettered, reference VP2 subunit. G denotes a clockwise fivefold operation with respect to the threefold axis at the bottom right of the diagram. The boundaries of the projected surface-exposed amino acids are labeled according to residue type and sequence number. Residues 84 and 236 differ only between ADV-G and ADV-Utah 1.

FIG. 8

The locations of nonoverlapping ADV-G peptides, pVP2b-pVP2g (Fig. 3), projected onto a roadmap (23) representation of the residues on the capsid surface of the ADV_G-VP2 pseudoatomic model. The peptides are colored in the same scheme as in Fig. 3. The view is the same as in Fig. 5A and B and 7. Residue labeling is as explained in the legend for Fig. 7. A denotes a twofold operation, G denotes a clockwise fivefold operation, and I denotes a clockwise threefold operation with respect to the threefold axis at the bottom right of the diagram.