Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions

Abstract

Herpes simplex virus type 1 virions were examined by electron cryomicroscopy, allowing the three-dimensional structure of the infectious particle to be visualized for the first time. The capsid shell is identical to that of B-capsids purified from the host cell nucleus, with the exception of the penton channel, which is closed. The double-stranded DNA genome is organized as regularly spaced ( approximately 26 A) concentric layers inside the capsid. This pattern suggests a spool model for DNA packaging, similar to that for some bacteriophages. The bulk of the tegument is not icosahedrally ordered. However, a small portion appears as filamentous structures around the pentons, interacting extensively with the capsid. Their locations and interactions suggest possible roles for the tegument proteins in regulating DNA transport through the penton channel and binding to cellular transport proteins during viral infection.

FIG. 1

Electron cryomicrograph (a) and reconstruction (b) of HSV-1 virions. (a) The electron micrographs of ice-embedded HSV-1 virions were recorded at 400 kV in a JEOL 4000 electron cryomicroscope at ×30,000 magnification, using a dose of 6 electrons/Ų. The underfocus value of this image was determined to be 2.7 μm. Scale bar, 1,000 Å. (b) Shaded surface representation of the 3D map of the HSV-1 virion viewed along a threefold symmetry axis. The map is displayed at 0.7ς and colored radially using the color scheme shown at the bottom. In this scheme, all mass outside a radius of 650 Å is colored purple.

FIG. 2

Averaged density distribution of the virion and B-capsid reconstructions as a function of particle radius. For the virion, the radial dimensions of the tegument and membrane, capsid shell, and dsDNA are indicated. The B-capsid profile terminates at 700 Å, which is at the limit of the boxed-out area.

FIG. 3

Visualization of icosahedrally ordered tegument. (a) HSV-1 virion reconstruction displayed at 1.0ς. At this density threshold, the nonicosahedrally related densities in the tegument/membrane layers are not visible. (b) HSV-1 B-capsid reconstruction at the same resolution as the virion reconstruction (20 Å) displayed also at 1.0ς. (c) Difference map between the virion and B-capsid reconstructions. In this display, the densities inside the capsid shell are excluded for clarity. Consequently, differences occurring at all of the pentonal positions including those on the hemisphere facing toward (labeled 1, 2, and 3) and away from (labeled 4, 5, and 6) the observer can be seen. (d) Superposition of the difference map on the B-capsid reconstruction (gray). The tegument proteins are highlighted in color. Labeled are the unique structural components in one asymmetric unit (the unique building block of the entire icosahedron) of the T=16 capsid, which comprises 1 penton (5) subunit, 2½ hexons (P, C, and E), and 5 triplexes (T_a, T_b, T_c, T_d, T_e, and T_f).

FIG. 4

Capsid-tegument interactions. (Upper left) Close-up view of a computationally isolated portion of the superposition map (Fig. 3), including the penton (red), the five P hexons (blue), and the T_a and T_c triplexes (green). The additional density (yellow) that is not present in the B-capsid is attributed to tegument proteins, which clearly make contact with the penton, hexon, and triplexes. (Lower left panel and right column) Enlarged top and two side views of the tegument density interacting with its adjacent penton subunits and triplexes T_a and T_c. In these views, the P hexon, which also interacts with the tegument density, is removed for clarity.

FIG. 5

Closure of the penton channel in the virion. (a) Sectional view of the B-capsid penton. In the middle of the channel, densities from the VP5 subunits (arrows) protrude inward, resulting in a constriction. (b) Sectional view of the virion capsid penton. The arrow indicates the closure in the axial channel at the region that is constricted in the B-capsid channel. Also indicated are the densities attributed to tegument (T) and viral DNA.

FIG. 6

Central cross-section (100 Å thick) through the virion reconstruction as viewed along a twofold axis. The concentric shells of density inside the capsid are attributable to the viral DNA. The spacing between the layers is 26 Å. The map is colored radially, using the color scheme shown at the bottom.

FIG. 7

Organization of the dsDNA inside the HSV-1 virion. (a through d) Representative projection views of raw virion images, revealing characteristic patterns of DNA, including circular ring (a), dotted (b), striation (c), and dotted-striation (d) patterns. The orientations [(θ, φ), listed below each image] were determined by assuming icosahedral symmetry. Although the orientations are similar for panels a and b and for panels c and d, their DNA projections are strikingly different. (e through f) Computed diffraction patterns of panels a through d, respectively, showing distinctive patterns due to differences in the DNA genome projections. All the diffraction patterns show 1/26 Å⁻¹ spacing (arrows in panel e) with different distributions.