Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1

Abstract

By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry. The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 A, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site. Its architecture resembles those of bacteriophage portal/connector proteins.

FIG. 1.

Negative staining EM of the HSV-1 UL6 portal protein. (a) Field of UL6 molecules, prepared as described previously (16), and negatively stained with uranyl acetate. Bar, 200 Å. Round particles likely to represent axial views (a few examples are marked with asterisks) were analyzed with the symmetry detection algorithm, Rotastat (12), which detected 11-fold, 12-fold, 13-fold, and less strongly, 14-fold molecules (see Results). When the images were classified in terms of their strongest harmonics, they were estimated to be 23% 11-mers, 27% 12-mers, 22% 13-mers, and 16% 14-mers. Correlation-averaged images of molecules presenting the first three symmetries are shown in panels b, c, and d. Their resolutions were 28 Å for panel b and 26 Å for both panels c and d. These images were further enhanced by rotational symmetrization in panels e, f, and g. The 14-mers were not only fewer but appeared less regular, to judge by the symmetry detection analysis and markedly lower resolution achieved on averaging (data not shown). Bar, 100 Å.

FIG. 2.

Cryo-EM of the HSV-1 UL6 portal protein. (a) A field of ice-embedded molecules (protein is dark). Bar, 200 Å. (b to i) Various views of a 3D reconstruction of the 12-mer at 16-Å resolution. Bar, 50 Å. A movie clip of the reconstruction may be seen at http://www.niams.nih.gov/rcn/labbranch/lsbr/UL6_movie.html.

FIG. 3.

Robustness of the 3D reconstruction of the UL6 dodecamer with respect to the choice of starting model. (a) A resolution-limited, appropriately swollen, rendition of the bacteriophage φ29 portal led to (c), a model at 29-Å resolution, calculated from images of negatively stained molecules, which in turn led to (d) a model at 16-Å resolution calculated from cryoelectron micrographs. Starting the analysis with an entirely different, computer-generated, model (b) led to essentially the same result.

FIG. 4.

Fitting of the UL6 portal protein into a vacant vertex in the HSV-1 capsid. (a) Interior view of a capsid model at 18-Å resolution (4) from which a penton of VP5, the major capsid protein, was computationally excised and replaced by the portal (right-hand side at 3 o'clock). Panel b shows a section through a vertex site occupied by a VP5 pentamer and is compared in panels c and d with the same site with an implanted portal in both orientations. In one orientation (c), the portal fits snugly into this cavity. In the other orientation (d), the portal also fits but makes only tenuous contact with the surrounding VP5 molecules, leaving gaps between the molecules and the capsid (arrows), suggesting a less stable interaction. Bars, 100 Å.