JOURNAL OF VIROLOGY, Feb. 2011, p. 1871–1874
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 4
Seeing the Portal in Herpes Simplex Virus Type 1 B Capsids?
R. H. Rochat,1,2X. Liu,2K. Murata,2K. Nagayama,3F. J. Rixon,4and W. Chiu1,2*
Structural Computational Biology and Molecular Biophysics Program, Baylor College of Medicine, Houston, Texas 770301;
National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Microbiology,
Baylor College of Medicine, Houston, Texas 770302; Okazaki Institute for Integrative Bioscience, 5-1 Higashiyama,
Myodaiji-cho, Okazaki, Aichi 444-8787, Japan3; and MRC—University of Glasgow Centre for Virus Research,
Church Street, Glasgow G11 5JR, United Kingdom4
Received 7 August 2010/Accepted 16 November 2010
Resolving the nonicosahedral components in large icosahedral viruses remains a technical challenge in
structural virology. We have used the emerging technique of Zernike phase-contrast electron cryomicroscopy
to enhance the image contrast of ice-embedded herpes simplex virus type 1 capsids. Image reconstruction
enabled us to retrieve the structure of the unique portal vertex in the context of the icosahedral capsid and, for
the first time, show the subunit organization of a portal in a virus infecting eukaryotes. Our map unequivocally
resolves the 12-subunit portal situated beneath one of the pentameric vertices, thus removing uncertainty over
the location and stoichiometry of the herpesvirus portal.
Herpes simplex virus type 1 (HSV-1) is an enveloped mam-
malian virus approximately 200 nm in diameter, consisting of a
125-nm icosahedral capsid surrounded by tegument and enve-
lope (17). The HSV-1 genome is packaged into the capsid
through a unique structure called the portal, which is present
at one of the 12 pentameric vertices (15, 19). The location of
the portal with respect to the capsid shell and its oligomeric
structure have not been unambiguously defined. Previous stud-
ies have suggested positions both outside and inside the capsid
(2, 3, 19) and that in vitro-assembled HSV-1 portals can form
as multimers of 11 to 14 subunits with roughly equal frequency
(19). To date, no study has definitively determined the location
and quaternary structure of the HSV-1 portal in its native
Single-particle virus reconstructions are typically generated
with icosahedral symmetry imposed, meaning that any features
that are not icosahedrally arranged will be lost. As the portal is
believed to be located at just one of the 12 5-fold vertices in the
capsid, it cannot be definitively resolved in icosahedral recon-
structions, necessitating the use of a nonicosahedral, symme-
try-free approach. Unfortunately, reconstructions of viruses
free of icosahedral enforcement, which are ideal for resolving
portal structures, are limited by difficulties in identifying the
orientation of the unique vertex (2, 3). In the case of the
HSV-1 capsid, there are no sentinel markers for the location of
the portal, such as those provided by the external tail structures
in some double-stranded DNA bacteriophages (1, 4, 9, 10, 20).
The lack of a nonicosahedrally arranged protein density that
protrudes from the surface of the HSV-1 capsid presents a
computational challenge in determining, from the noisy elec-
tron cryomicroscopy (cryo-EM) image, which of the 12 vertices
is the portal vertex. An additional complication is that the
portal is approximately the same size (?814 to 1,036 kDa in
total, assuming 11 to 14 copies of UL6 ), as the pentons
(745 kDa) that occupy the other 5-fold vertices and, conse-
quently, does not show up as an obvious excess or absence of
density in projection images of capsids. Even though the
HSV-1 capsid lacks a tail density, a feature that has proven to
be an efficient marker of the portal vertex in bacteriophages (4,
10, 13), we posited that recent advances in Zernike phase-
contrast EM (ZPC-EM), which produces dramatic low-resolu-
tion contrast enhancement (5, 6), would prove useful in help-
ing to identify this unique vertex. Furthermore, when used in
conjunction with an advanced reconstruction algorithm (12),
our ability to identify this vertex should be greatly enhanced.
Identification of this vertex would then make it possible to
reconstruct HSV-1 capsids to a moderate resolution using sin-
gle-particle cryo-EM without enforcing any symmetry (14).
ZPC-EM relies on using a thin carbon film with a small hole
in the middle, suspended at the back focal plane of the micro-
scope’s objective lens, to retard the phase of scattered elec-
trons by ?/2 with respect to the unscattered beam (7). The
resulting shift in phase is manifested in dramatically enhanced
low-resolution image contrast compared to that of conven-
tional imaging methods.
HSV-1 B capsids, purified by sucrose gradient sedimentation
(16), were applied to thin-carbon-film-coated Quantifoil grids
(14) and plunge-frozen in liquid ethane using a Vitrobot (FEI,
Eindhoven). The grids were kept at 100K in a JEM2200FSC
field-emission electron microscope (JEOL, Tokyo, Japan) op-
erating at 200 kV, equipped with an in-column energy filter
and a Zernike-type phase plate. Images were recorded on a
Tietz 4,000- by 4,000-pixel SlowScan charge-coupled-device
* Corresponding author. Mailing address: Graduate Program in
Structural and Computational Biology and Molecular Biophysics, Na-
tional Center for Macromolecular Imaging, Verna and Marrs McLean
Department of Biochemistry and Molecular Biology, Baylor College of
Medicine, Houston, TX 77030. Phone: (713) 798-6985. Fax: (713)
798-8682. E-mail: email@example.com.
?Published ahead of print on 24 November 2010.
(CCD) camera (TVIPS, Germany) at ?56,000 detector mag-
nification, with a total dose of 20 e/Å2. The ZPC-EM image
was targeted at close to zero defocus in order to obtain max-
imum contrast enhancement. The contrast of the resulting
images is substantially higher than that of conventional
cryo-EM images (Fig. 1a and b). The white halo around each
particle in Fig. 1b is a consequence of the phase-contrast optics
and is characteristic of ZPC-EM images (6, 14).
The particle images were boxed with the e2boxer.py program
(18) and reconstructed using the Multi-Path Simulated An-
nealing algorithm (11–13). From 353 CCD frames, 6,033 sin-
gle-particle images of HSV-1 B capsids were collected. Under
ZPC-EM conditions, we were able to reconstruct an icosahe-
dral map (Fig. 1c) without the need for contrast transfer func-
tion correction (6, 14). The resolved icosahedral orientation of
each particle was subsequently used to initiate the process of
determining its asymmetric orientation (12). In effect, the al-
gorithm compares an iteratively refined de novo model of the
portal density to specific regions of the raw data in order to
determine which one of the 60 equivalent icosahedral orienta-
tions is most likely to be the “true” asymmetric orientation.
This procedure does not require a priori knowledge of the
portal structure or its location in the capsid (12). To enhance
the specificity of this selection process, we chose only those
particles where the mean score for one vertex was statistically
higher (P value ? 0.05) than the score for the other 11 vertices.
The asymmetric model was refined until no further improve-
ments were observed. The 2,308 particles whose asymmetric
orientations satisfied these statistical conditions were used for
reconstructing the final asymmetric map, estimated at ?25 Å
by the 0.5 Fourier Shell Correlation criterion (Fig. 2a). The
seemingly low resolution, in comparison to the total number of
particles used (14), is a reflection of the fact that the B capsid’s
portal vertex is not readily apparent in the raw images, and as
a result, the asymmetric orientations for some of the particle
images might be incorrectly assigned.
Both the icosahedral and the asymmetric reconstruction of
the HSV-1 B capsid (Fig. 1c and 2a) show the characteristic
FIG. 1. Zernike phase-contrast electron microscopy (ZPC-EM) of HSV-1 B capsids. (a) Image of ice-embedded HSV-1 B capsids collected
using conventional cryo-EM. Bar ? 50 nm. (b) Equivalent image collected using ZPC-EM. Bar ? 50 nm. (c) Radially colored icosahedral
reconstruction of the HSV-1 B capsid showing the characteristic T?16 capsid shell.
FIG. 2. Reconstruction of the HSV-1 B capsid without symmetry enforcement. (a) Radially colored asymmetric reconstruction of the HSV-1
B capsid showing a large density situated beneath one of the 12 5-fold vertices (pink). The internal scaffold density is shown in white. (b) Axial
view of a 5-fold vertex (red arrow in panel a) showing a typical penton density. (c) Axial view of the unique vertex (orange arrow in panel a) showing
the lack of obvious penton density (compare to panel b). (d) Axial slice of the asymmetric map (violet slicing plane in panel a) showing the 12-fold
symmetry of the portal complex (pink).
T?16 capsid shell. The asymmetric reconstruction shows a
large density underneath one of the 12 vertices (Fig. 2a). In
addition, the capsid shell at this vertex is markedly different
from the other 11 vertices and lacks the typical penton density
(Fig. 2b and c) seen there. This finding is consistent with the
tomographic analysis of chemically treated HSV-1 B capsids,
which concluded that the outer surface of the capsid at this
position is formed by the top of the portal (3). The relatively
weak capsid shell density seen at this vertex may arise due to
difficulty in accurately assigning the asymmetric orientations,
which would result in the inclusion of some penton density at
this vertex. Even though the portal sits beneath the proteins
that comprise the capsid shell, a portion of it is externally
exposed and would be accessible to other viral or cellular
proteins, as well as to antibodies specific to the portal pro-
tein UL6 (2, 15).
Figure 2d shows a cross-section of the capsid passing
through the region of density beneath the unique vertex. The
cross-section clearly shows that this density (Fig. 3a) has an
apparent 12-fold symmetry, as confirmed by rotational corre-
lation analysis (Fig. 3b). It is important to note that no sym-
metry was imposed during the generation of this map and that
both the 5-fold nature of the surrounding capsid shell and the
12-fold symmetry of the internal density arise directly from the
data. The simultaneous visualization of these two symmetry-
mismatched features is only possible if they are not free to
adopt different rotational positions but are fixed relative to
each other. As the B capsids used in this study contained
cleaved scaffolding proteins, we cannot eliminate the possibil-
ity that these proteins contribute to the density we see. How-
ever, the presence of this structure at only one vertex and its
12-fold nature, which is consistent with previously described
portal structures for tailed bacteriophages (4, 10, 13), gives us
confidence that we are detecting the HSV-1 portal. Although a
12-subunit structure was one of several forms found for iso-
lated in vitro-assembled HSV-1 portals (19), this is the first
definitive demonstration that the portal in a virus of eukaryotes
shares the same stoichiometry as that of bacteriophages.
To assess the general structural features and arrangement of
the HSV-1 portal subunits, we applied c12 symmetry to our
asymmetric map and extracted the 12-fold-averaged portal
density (Fig. 3c). The organization and overall dimensions of
our portal structure resemble those previously published for
the 12-subunit form of the in vitro-assembled HSV-1 portal.
However, comparison with the published figures appears to
show subtle differences, for example, in the dimensions and
features of the peripheral flanges (19). It is not clear whether
this reflects genuine variation between the two structures in
biochemically isolated and capsid-resident states or differences
in the methods used for the two reconstructions.
Previous studies, which have reported two incompatible po-
sitions for the portal complex, one inside (3, 8) and the other
outside the floor of the capsid shell (2), cast uncertainty over its
true location. These studies, which used cryoelectron tomog-
raphy to examine the nonicosahedral features of the capsid,
were unable to resolve the subunit organization and provide
unambiguous identification of the portal, leading to the con-
fusion over its true location. The description of the portal in
HSV-1 B capsids presented here resolves this uncertainty. The
12-fold symmetry of the internal density (Fig. 3) convincingly
identifies it as the portal and establishes the location inside the
capsid shell as being correct.
Protein structure accession number. The asymmetric capsid
reconstruction and 12-fold-averaged portal of HSV-1 have
been deposited in EMDB (accession number EMD-5255).
FIG. 3. 12-Fold symmetry of the HSV-1 B capsid portal. (a) External view of the portal density extracted from the c1 map (Fig. 2a) showing
12 radially arranged densities. (b) Rotational correlation of an annulus of data from the c1 reconstruction shown in panel a confirms the 12-fold
arrangement of the portal. (c) The c12 average of the portal density shown in panel a sitting within the HSV-1 B capsid shell. Side, external, and
oblique views are shown.
VOL. 85, 2011 NOTES 1873
We thank David McNab of the MRC Virology Unit for excellent
technical assistance and Michael F. Schmid at the NCMI for his nu-
merous helpful discussions.
This work was supported by the NIH (grants R01AI0175208 and
P41RR002250), the Robert Welch Foundation (grant Q1242), and
funding from the Core Research for Evolutional Science and Tech-
nology (CREST) of Japan Science and Technology Corporation to
K.N. R.H.R. is supported by NIH training grants (grant GM07330
through the MSTP and grant T15LM007093 through the Gulf Coast
Consortia). F.J.R. is funded by the United Kingdom MRC.
1. Agirrezabala, X., et al. 2005. Maturation of phage T7 involves structural
modification of both shell and inner core components. EMBO J. 24:3820–
2. Cardone, G., et al. 2007. Visualization of the herpes simplex virus portal in
situ by cryo-electron tomography. Virology 361:426–434.
3. Chang, J., M. Schmid, F. Rixon, and W. Chiu. 2007. Electron cryotomogra-
phy reveals the portal in the herpesvirus capsid. J. Virol. 81:2065–2068.
4. Chang, J., P. Weigele, J. King, W. Chiu, and W. Jiang. 2006. Cryo-EM
asymmetric reconstruction of bacteriophage P22 reveals organization of its
DNA packaging and infecting machinery. Structure 14:1073–1082.
5. Danev, R., R. Glaeser, and K. Nagayama. 2009. Practical factors affecting the
performance of a thin-film phase plate for transmission electron microscopy.
6. Danev, R., and K. Nagayama. 2008. Single particle analysis based on Zernike
phase contrast transmission electron microscopy. J. Struct. Biol. 161:211–
7. Danev, R., and K. Nagayama. 2001. Transmission electron microscopy with
Zernike phase plate. Ultramicroscopy 88:243–252.
8. Deng, B., C. M. O’Connor, D. H. Kedes, and Z. H. Zhou. 2007. Direct
visualization of the putative portal in the Kaposi’s sarcoma-associated her-
pesvirus capsid by cryoelectron tomography. J. Virol. 81:3640–3644.
9. Fokine, A., et al. 2004. Molecular architecture of the prolate head of bacte-
riophage T4. Proc. Natl. Acad. Sci. U. S. A. 101:6003–6008.
10. Jiang, W., et al. 2006. Structure of epsilon15 bacteriophage reveals genome
organization and DNA packaging/injection apparatus. Nature 439:612–616.
11. Liu, X., W. Jiang, J. Jakana, and W. Chiu. 2007. Averaging tens to hundreds
of icosahedral particle images to resolve protein secondary structure ele-
ments using a Multi-Path Simulated Annealing optimization algorithm. J.
Struct. Biol. 160:11–27.
12. Liu, X., R. H. Rochat, and W. Chiu. 15 June 2010. Reconstructing cyano-
bacteriophage P-SSP7 structure without imposing symmetry. Nat. Protoc.
13. Liu, X., et al. 2010. Structural changes in a marine podovirus associated with
release of its genome into Prochlorococcus. Nat. Struct. Mol. Biol. 17:830–
14. Murata, K., et al. 2010. Zernike phase contrast cryo-electron microscopy and
tomography for structure determination at nanometer and subnanometer
resolutions. Structure 18:903–912.
15. Newcomb, W. W., et al. 2001. The UL6 gene product forms the portal for
entry of DNA into the herpes simplex virus capsid. J. Virol. 75:10923–10932.
16. Roberts, A., et al. 2009. Differing roles of inner tegument proteins pUL36
and pUL37 during entry of herpes simplex virus type 1. J. Virol. 83:105–116.
17. Subak-Sharpe, J. H., and D. J. Dargan. 1998. HSV molecular biology:
general aspects of herpes simplex virus molecular biology. Virus Genes
18. Tang, G., et al. 2007. EMAN2: an extensible image processing suite for
electron microscopy. J. Struct. Biol. 157:38–46.
19. Trus, B., et al. 2004. Structure and polymorphism of the UL6 portal protein
of herpes simplex virus type 1. J. Virol. 78:12668–12671.
20. Xiang, Y., et al. 2006. Structural changes of bacteriophage phi29 upon DNA
packaging and release. EMBO J. 25:5229–5239.
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