Figure 1.
Visualization of a distinctive tegument structure associated with the portal vertex in HSV-1 virions.
(A) Projection image through a tomogram of HSV-1 virions embedded in vitreous ice (Video S2). (B–C) The symmetry-free virion averages generated from 213 subtomograms using MSA-guided classification and melon ball alignment methods, respectively. The maps are shown radially colored, with non-capsid densities trimmed to reveal the underlying capsid. Views are shown looking down a 2-fold axis of symmetry with the portal vertex at the bottom.
Figure 2.
Comparison of portal and pentonal vertices.
(A) Central section through an MSA-guided virion average. The left-hand panel shows a grey-scale density map. In the right-hand panel, the capsid shell is radially colored, with internal DNA density shown in cream, and portal density in purple. (B–F) Individual panels of the capsid shell cut away orthogonal to the axis running through the portal and opposing vertex, at the radii indicated by arrows in (A). Views are from the outside of the portal vertex (bottom) and its opposing pentonal vertex (top).
Figure 3.
Tegument-capsid interactions at the portal and pentonal vertices.
Close up views of portal (A–C) and pentonal (D–F) vertices. (A, D) Symmetry-free B-capsid reconstruction [6] low-pass filtered to 35 Å. (B, E) MSA-guided virion average (Figure 1B) shown in the same views as (A) and (D). (C, F) Difference maps (colored) between (A) and (B), and (D) and (E) respectively, superimposed on the B-capsid shell (grey), and shown tilted. The weak penton density seen in (A) is most likely due to occasional incorrect assignment of the asymmetric orientations [6].
Figure 4.
Influence of the PVAT on capsid-envelope eccentricity.
(A) Distribution of HSV-1 virion diameters for 112 of the virions used in the final reconstruction. (B) Graph showing the separation of the envelope from the capsid shell (defined as 625 Å from the center of the capsid) measured at the portal vertex (red line) and at the opposing pentonal vertex (blue line), and plotted as a function of virion diameter. The difference in slope between the two lines is a clear indication that the capsid is not randomly positioned within the envelope. Note that the vertex closest to the envelope is not always the portal vertex. (C) Schematic illustrating the spatial relationship between the HSV-1 capsid and the envelope for small, intermediate, and large sized virions, as predicted from (B).
Figure 5.
Workflow for MSA-guided classification.
(A) Projected slices from a representative tomogram of HSV-1 virions. (B) 12 MSA-derived class averages, top, side and lower views. The class average representing a putative portal vertex is shown in yellow. (C) The putative portal vertex (top), the average of all other vertices (middle) and the difference map calculated from them (bottom), with the positive difference densities in blue, and negative difference density in red. (D) Final MSA-guided average, radially colored. (E) Flow chart of the procedure (described in detail within the Materials and Methods).
Figure 6.
Workflow for melon ball alignment.
(A) Projected slices from a representative tomogram of HSV-1 virions. (B) 12 spherical masks (magenta), in an icosahedral arrangement, superimposed on a map of the capsid (grey), showing the relationship of the masks to the capsid surface. (C) Density extracted by the masking procedure in (B). After generating an initial model, the model may be updated iteratively by realigning the particles (red lines). (D) Final melon ball aligned average, radially colored. (E) Flow chart of the procedure (described in detail within the Materials and Methods).