Three-dimensional structure of the human herpesvirus 8 capsid

Abstract

Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated herpesvirus, is a gammaherpesvirus implicated in all forms of Kaposi's sarcoma and certain lymphomas. HHV-8 has been extensively characterized, both biochemically and immunologically, since its first description in 1994. However, its three-dimensional (3D) structure remained heretofore undetermined largely due to difficulties in viral purification. We have used log-phase cultures of body cavity-based lymphoma 1 cells induced with 12-O-tetradecanoylphorbol-13-acetate to obtain HHV-8 capsids for electron cryomicroscopy and computer reconstruction. The 3D structure of the HHV-8 capsids revealed a capsid shell composed of 12 pentons, 150 hexons, and 320 triplexes arranged on a T=16 icosahedral lattice. This structure is similar to those of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV), which are prototypical members of alpha- and betaherpesviruses, respectively. The inner radius of the HHV-8 capsid is identical to that of the HSV-1 capsid but is smaller than that of the HCMV capsid, which is consistent with the relative sizes of the genomes they enclose. While the HHV-8 capsid exhibits many structural similarities to the HSV-1 capsid, our reconstruction shows two major differences: its hexons lack the "horn-shaped" VP26 densities bound to the HSV-1 hexon subunits, and the HHV-8 triplexes appear smaller and less elongated than those of HSV-1. These differences are in excellent agreement with our sequence comparisons of HHV-8 and HSV-1 capsid proteins. This gammaherpesvirus capsid structure complements previous structural studies on alpha- and betaherpesviruses in providing an account of structural similarities and differences among capsids representing all human herpesvirus subfamilies.

FIG. 1

cryoEM of HHV-8 capsids. (A) An area of a micrograph of the ice-embedded HHV-8 capsids showing predominantly intermediate capsids and one full capsid (arrow). The underfocus value of this image was estimated to be 1.6 μm based on incoherently averaged Fourier transforms (54). The enlarged view of an intermediate capsid (B) shows the capsomers (e.g., arrow) that form a characteristic hexagonal pattern with adjacent capsomers. The full-capsid (C) image reveals the characteristic “fingerprint” pattern.

FIG. 2

3D structure of the HHV-8 capsid at 24-Å resolution as viewed along the icosahedral twofold axis from the outside (A) and inside (B) of the capsid. The map was color coded according to the particle radius (see color bar at the bottom right), such that the upper domains of the pentons and hexons are in blue (between radii of 570 and 650 Å), the connecting triplexes are in green (between radii of 510 and 560 Å), the shell is in yellow (between radii of 460 and 510 Å), and the densities inside the capsid shell are in red (<460-Å radius). The capsid has a T=16 icosahedral symmetry (3 of the 12 fivefold axes are labeled 5, and 1 of the 20 triangular faces is outlined by a red dashed line in panel A), with the unique structural components in one asymmetric unit labeled, following the nomenclature established for HSV-1 (41, 57). These components include one-fifth of a penton (labeled 5), two and one-half hexons (one P, one C, and one-half of an E), and five and one-third triplexes (one each of the Ta, Tb, Tc, Td, and Te triplexes and one-third of the Tf triplex). The inside view in panel B is the same as that in panel A except that the upper half of the capsid was computationally removed to show the cutaway side views of some of the triplexes (dashed red arrows) and the inner floor of the HHV-8 capsids. Dot-dashed lines indicate icosahedral five-, three-, and twofold axes, which pass through a penton channel, a Tf triplex, and an E hexon channel, respectively. The densities inside the capsid shells (red) lack structural information because they are not icosahedrally disposed and thus have been removed computationally in the right half of panel B to show the internal surface of the capsid shell.

FIG. 3

Structural comparison of HHV-8 capsid and HSV-1 B capsid. The two capsid maps are radially colored as in Fig. 2 and are shown in a montage as viewed along the icosahedral threefold axis. The HSV-1 B capsid was reconstructed similarly to the same resolution (24 Å) from a subset of images selected from those used in a structure published previously (53). One penton (5), three types of hexon (P, E, and C), and six types of triplexes (Ta to Tf) are labeled.

FIG. 4

Radial density distributions of the HHV-8 capsid and HSV-1 B-capsid reconstructions. The density profiles were generated by spherically averaging the HHV-8 and HSV-1 capsid maps and were plotted as a function of particle radius. The HSV-1 and HHV-8 capsids have identical inner radii of about 460 Å. For the HSV-1 capsid, the locations of the four capsid proteins have been established (29, 34, 48, 55) and the density peaks corresponding to radial locations of the capsid floor, the triplexes, and the smallest capsid protein, VP26, are indicated accordingly. The HHV-8 capsid profile lacks the prominent peak corresponding to that attributed to VP26 in the HSV-1 capsid. The triplex peak is narrower and shifted to a lower radius in HHV-8 than in HSV-1. arb, arbitrary.

FIG. 5

Structural comparisons of the penton (A and C), hexon (B and D), and their subunits in HHV-8 capsid (A and B) and HSV-1 B capsid (C and D). The top views of the HHV-8 and HSV-1 pentons (A and C, respectively) and E hexons (B and D, respectively) reveal an axial channel approximately 25 Å in diameter in each penton and E hexon. The side views were generated by rotating the top view about the short edge of the figure by about 80° and show interactions between their adjacent subunits. In the side views of the HHV-8 penton and hexon subunits, the upper (u, blue), middle (m, green), lower (l, green to yellow), and floor (f, yellow) domains of a subunit are labeled, following the same designations used for HSV-1 (52, 57). Arrow in the HHV-8 hexon subunit (B), density protrusion that forms the constriction inside the hexon channel. The red dotted lines on the lower and floor domains of these subunits (A and B) illustrate the angles between the two domains. The two side views of the HHV-8 penton and hexon subunits (A and B, right) are related to each other by a roughly 90° rotation about the long edge of the Figure. (C and D, right) Side views of HSV-1 penton and hexon. The coiled lines drawn on the floor domain of penton side views (A and C) illustrate the location of the long α helix joining the adjacent major capsid proteins together at the floor domains, which was first visualized in the 8.5-Å structure of HSV-1 B capsid (53). (E and F) Superposition of penton (shown in semitransparent red) and hexon subunits (shown as wire frames using the same radial coloring scheme as in panels A to D) extracted from the HHV-8 capsid (E) and from the HSV-1 capsid (F). In the HSV-1 hexon subunit (D and F), a horn-shaped VP26 density attaches to the upper domain of each subunit (arrow) (48, 55). No horn-shaped or other extra density of similar size can be identified at the corresponding location on the HHV-8 hexon subunit (B and E). The pentons and hexons were displayed using a density threshold of 1.2 standard deviations (SD) above the mean, and their extracted subunits were shown at 1.5 SD above the mean density. Except for the penton subunits in panels E and F, the maps are colored according to the capsid radius as in Fig. 2 and 3 (see color bar in Fig. 2).