Electron tomography of nascent herpes simplex virus virions

Abstract

Cells infected with herpes simplex virus type 1 (HSV-1) were conventionally embedded or freeze substituted after high-pressure freezing and stained with uranyl acetate. Electron tomograms of capsids attached to or undergoing envelopment at the inner nuclear membrane (INM), capsids within cytoplasmic vesicles near the nuclear membrane, and extracellular virions revealed the following phenomena. (i) Nucleocapsids undergoing envelopment at the INM, or B capsids abutting the INM, were connected to thickened patches of the INM by fibers 8 to 19 nm in length and < or =5 nm in width. The fibers contacted both fivefold symmetrical vertices (pentons) and sixfold symmetrical faces (hexons) of the nucleocapsid, although relative to the respective frequencies of these subunits in the capsid, fibers engaged pentons more frequently than hexons. (ii) Fibers of similar dimensions bridged the virion envelope and surface of the nucleocapsid in perinuclear virions. (iii) The tegument of perinuclear virions was considerably less dense than that of extracellular virions; connecting fibers were observed in the former case but not in the latter. (iv) The prominent external spikes emanating from the envelope of extracellular virions were absent from perinuclear virions. (v) The virion envelope of perinuclear virions appeared denser and thicker than that of extracellular virions. (vi) Vesicles near, but apparently distinct from, the nuclear membrane in single sections were derived from extensions of the perinuclear space as seen in the electron tomograms. These observations suggest very different mechanisms of tegumentation and envelopment in extracellular compared with perinuclear virions and are consistent with application of the final tegument to unenveloped nucleocapsids in a compartment(s) distinct from the perinuclear space.

FIG. 1.

One-nanometer-thick slices from a tomographic reconstruction of a type B capsid attached to the INM. Cells infected with HSV-1 were high-pressure frozen and freeze substituted. The slice number of each image (out of a total of 180 slices) is indicated in the lower left of each panel. The cytoplasm is at the top of the image, and the nucleus is at the bottom. An arrow indicates a single bridging rod emanating obliquely from the capsid surface. Cyto, cytoplasm; Nuc, nucleus.

FIG. 2.

Models of the tomogram illustrated in Fig. 1. A. Model created by computationally fitting surfaces to densities in the tomogram, representing the capsid surface (purple) and the thickened region of the INM (green). Bridging rods (yellow) were segmented by manual tracing in sequential 1-nm sections. B. Similar to panel A, but the INM is made translucent to better illustrate the attachments of the bridging rods. C. Side view of a model created by manual tracing and surface rendering of the resulting contours. The inner and outer surfaces of the capsid (purple) and the INM (green) are shown, with the bridging rods (yellow) connecting the outer surface of the capsid with the inner surface of the thickened region of the INM. D. Same as panel A, but viewed from the cytoplasmic side to show the orientation of the bridging rods relative to the capsid surface. The inner protein sphere or scaffold does not appear in the model.

FIG. 3.

One-nanometer-thick slices from a tomographic reconstruction of a nucleocapsid completing envelopment at the INM. The sample was prepared as for Fig. 1. The slice number of each image (out of a total of 235 slices) is indicated in the lower left of each panel. The position of the INM is indicated with an arrow. Triangles in slice 120 indicate two adjacent angular vertices with bridging rods, whereas the relatively planar surface between them is largely devoid of bridging rods. Nuc, nucleus; Cyto, cytoplasm.

FIG. 4.

Models of the tomogram shown in Fig. 3. A. Thick slice through the center of the virion. The nucleocapsid shell (purple) and virion envelope (green) are modeled by computationally fitting surfaces to densities in the tomogram, while the ONM (blue), INM (green), and bridging rods (yellow) are modeled by manual tracing. The DNA within the core is not shown. B. Side view of the nucleocapsid surface (purple) and the attached bridging rods (yellow). The horizontal elongation of the rods is an artifact due to the limited tilt range of the tomogram. C and D. Cutaway views of the enveloping nucleocapsid shown as a surface-rendered model after manual tracing. The nucleoplasm is on the bottom and cytoplasm on the top. The near portion of the model has been cut away for illustration. The nucleocapsid (purple), bridging rods (yellow), virion envelope (green), INM (green), and ONM (blue) are shown. As only the surfaces were rendered, the nucleocapsid and nascent virion envelope appear as hollow shells in this cutaway view. Note that the virion envelope is more than twice as thick as the neighboring INM.

FIG. 5.

One-nanometer-thick slices from a tomographic reconstruction of an extracellular virion. The sample was prepared as described in the legend to Fig. 1. The number of the section (of a total of 180) is indicated at the bottom of each image.

FIG. 6.

Electron tomographic reconstruction of virion in the perinuclear space and putative transport vesicle. Cells were infected with HSV-1(F) and were embedded and stained conventionally. The number of the slice in the reconstruction (of 150 total) is indicated in each image. The membrane of the putative transport vesicle is indicated with a large arrow. The position of the ONM is indicated with a small arrow.