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Journal of Virology, October 2000, p. 9646-9654, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Three-Dimensional Structure of the Human Herpesvirus 8 Capsid
Lijun
Wu,1
Pierrette
Lo,2
Xuekui
Yu,2
James K.
Stoops,2
B.
Forghani,1 and
Z.
Hong
Zhou2,*
Viral and Rickettsial Disease Laboratory,
Division of Communicable Disease Control, California Department of
Health Services, Berkeley, California 94720,1
and Department of Pathology and Laboratory Medicine,
University of Texas-Houston Medical School, Houston, Texas
770302
Received 31 May 2000/Accepted 13 July 2000
 |
ABSTRACT |
Human herpesvirus 8 (HHV-8), or Kaposi's sarcoma-associated
herpesvirus, is a gammaherpesvirus implicated in all forms of Kaposi's
sarcoma and certain lymphomas. HHV-8 has been extensively characterized, both biochemically and immunologically, since its first
description in 1994. However, its three-dimensional (3D) structure
remained heretofore undetermined largely due to difficulties in viral
purification. We have used log-phase cultures of body cavity-based
lymphoma 1 cells induced with
12-O-tetradecanoylphorbol-13-acetate to obtain HHV-8
capsids for electron cryomicroscopy and computer reconstruction. The 3D
structure of the HHV-8 capsids revealed a capsid shell composed of 12 pentons, 150 hexons, and 320 triplexes arranged on a T=16 icosahedral
lattice. This structure is similar to those of herpes simplex virus
type 1 (HSV-1) and human cytomegalovirus (HCMV), which are prototypical
members of alpha- and betaherpesviruses, respectively. The inner radius
of the HHV-8 capsid is identical to that of the HSV-1 capsid but is
smaller than that of the HCMV capsid, which is consistent with the
relative sizes of the genomes they enclose. While the HHV-8 capsid
exhibits many structural similarities to the HSV-1 capsid, our
reconstruction shows two major differences: its hexons lack the
"horn-shaped" VP26 densities bound to the HSV-1 hexon subunits, and
the HHV-8 triplexes appear smaller and less elongated than those of
HSV-1. These differences are in excellent agreement with our sequence
comparisons of HHV-8 and HSV-1 capsid proteins. This gammaherpesvirus
capsid structure complements previous structural studies on alpha- and
betaherpesviruses in providing an account of structural similarities
and differences among capsids representing all human herpesvirus subfamilies.
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INTRODUCTION |
Human herpesvirus 8 (HHV-8), also
known as Kaposi's sarcoma (KS)-associated herpesvirus, has been
implicated in all forms of KS, in primary effusion lymphoma (PEL), and
in multicentric Castleman's disease, based on serological analyses and
epidemiological studies (8, 9, 24, 37, 40). HHV-8 was
initially identified from two novel herpesvirus-like DNA fragments
cloned from AIDS-associated KS (AIDS-KS), an angiogenic neoplasm
composed of endothelial and spindle cells (9). Subsequent
sequencing of an AIDS-KS genome library fragment hybridizing to
fragment KS330Bam demonstrated that HHV-8 belongs to the
Gammaherpesvirinae subfamily, which also includes two other
DNA tumor viruses: Epstein-Barr virus and Herpesvirus
saimiri (25). More recently, two new viruses with more
homology to HHV-8 than any other previously known herpesviruses have
been isolated from monkeys (15, 38). To date, the virus cannot be successfully propagated in vitro, although a susceptible cell
line with limited viral culturing capability has been reported (26). Instead, several tumor cell lines that appear to
harbor forms of HHV-8 DNA, such as body cavity-based lymphoma 1 (BCBL-1), BC-1, and BC-3, have been established. Cell-free virus can be generated by induction of lytic replication with tumor-promoting agents
such as 12-O-tetradecanoylphorbol-13-acetate (TPA)
(31).
Pulsed-field gel electrophoresis of DNA extracted from virions purified
from TPA-induced BCBL-1 cells revealed that the HHV-8 genome consists
of 165 to 170 kb (30, 50). This genome size is slightly
larger than the 153-kb genome of herpes simplex virus type 1 (HSV-1),
the prototypical herpesvirus and a member of the Alphaherpesvirinae subfamily (32), but
significantly smaller than the 230-kb genome of human cytomegalovirus
(HCMV), a member of the Betaherpesvirinae subfamily
(23). The HHV-8 genome contains at least 85 open reading
frames, of which 66 are homologous to those of other herpesviruses; the
others are unique to HHV-8 and are designated with the prefix K
(27, 33).
While extensive biochemical and immunological efforts have been
undertaken to characterize HHV-8 infection and pathogenesis since its
first description (9), structural studies of HHV-8 particles
have been limited. The morphology and structure of this human pathogen
are difficult to study because it remains refractory to cultivation in
either tissue culture systems or animals. In contrast to the detailed
descriptions of the three-dimensional (3D) structures of HSV-1 (5,
36, 53) and HCMV capsids (7, 10, 47), the only
available structural description of HHV-8 is a comparison of electron
microscopy images of HHV-8 and HCMV in virus-infected endothelial cells
(35). Based on negative-stain images of thin sections, it
was suggested that the morphological features of the HHV-8 and HCMV
nucleocapsids are similar (35). However, the HCMV virion is
generally larger (150 to 200 nm) than that of HHV-8 (120 to 150 nm),
and its tegument layer is denser than that of HHV-8 (35).
Here we report the 3D structure of the HHV-8 capsid, reconstructed from
electron cryomicroscopy (cryoEM) images of HHV-8 capsids purified from
TPA-induced BCBL-1 cells, and show its structural similarities and
differences with the HSV-1 capsid.
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MATERIALS AND METHODS |
Cell lines.
An HHV-8-positive BCBL-1 PEL cell line
(31) was obtained from the National Institutes of Health
AIDS Research and Reagent Program (Rockville, Md.). BCBL-1 cells were
propagated in T150 flasks containing RPMI 1640 medium (Sigma) with 10%
fetal bovine serum (HyClone), 100 U of penicillin/ml, 100 µg of
streptomycin/ml, and 50 µM 2-mercaptoethanol (Sigma) in 5%
CO2 at 37°C.
Purification of HHV-8 capsids.
A log-phase culture of BCBL-1
cells (1,000 ml) at a density of ~2 × 106 cells/ml
was induced with 20 ng of TPA (Sigma)/ml for 6 days to obtain the
maximum production of extracellular virus. The cells were pelleted by
centrifugation at 4,000 × g for 30 min, and the supernatant was collected. Polyethylene glycol 6000 (Sigma) was dissolved in the supernatant to a final concentration of 6% (wt/vol). After incubation overnight at 4°C, the precipitate was sedimented by
centrifugation at 4,000 × g for 30 min and the pellet
was resuspended in 10 ml of Tris-buffered saline (0.05 M Tris-0.15 M
NaCl, pH 7.4) containing 0.25% Nonidet P-40 (NP-40). The remaining
solution represented approximately a 100-fold concentration of the
original culture fluid.
Density gradient purification of HHV-8 capsids and virions was
performed as described previously for cytomegalovirus (17). Aliquots of the concentrated capsids (2 ml each) were loaded onto 10-ml
gradients of 20 to 60% sucrose with Tris-buffered saline and
centrifuged at 110,000 × g for 20 h at 4°C in a
Beckman SW 41Ti rotor. Two visible bands, corresponding to 35 and 50%
sucrose, respectively, were collected and separated into two pools.
Each pool was diluted with the original buffer and centrifuged at
75,000 × g for 60 min. The final pellet was
resuspended in 0.2 ml of Tris-HCl buffer (0.05 M, pH 7.4). Initial
negative-stain electron microscopy indicated that the upper band
contained virions and a significant amount of cellular debris while the
lower band contained predominantly capsids at approximately 5 × 107 particles/ml with less cellular debris. The
capsid-containing fraction was further concentrated using a Microcon
YM-100 centrifugal filter device (Millipore) and subsequently used for
cryoEM observation.
cryoEM.
cryoEM images of freeze-hydrated HHV-8 capsids were
recorded using standard procedures as described previously (42,
57). Briefly, 3 µl of a purified HHV-8 capsid sample was
applied to carbon-coated holey grids and quickly frozen in liquid
ethane so that the capsids were suspended in a thin layer of vitreous ice across the holes of the carbon support film. Most micrographs were
taken as focal pairs at ×30,000 magnification with a dosage of ~9
electrons/Å2 for each micrograph in a JEOL JEM1200
electron cryomicroscope operated at 100 kV with a cold stage at
167°C. Selected micrographs were digitized with a SCAI
microdensitometer (Carl Zeiss, Inc., Englewood, Colo.) using a step
size equivalent to 4.67 Å/pixel and subsequently averaged by combining
adjacent pixels to yield a step size of 9.34 Å/pixel on the specimen scale.
3D reconstruction and visualization.
Data processing and
visualization were carried out on SGI Octane dual-processor
workstations (Silicon Graphics, Inc.) using parallel programs for
refinement (52) and reconstruction (20), which
are based on Fourier common lines (12, 18) and
Fourier-Bessel synthesis (13). Individual HHV-8 capsid
particles were boxed out from digitized micrographs into separate image
files. A list of 28 initial orientation estimates was generated for
each particle image using a program based on self common lines. A
preliminary 3D model was then computed at 45 Å from 10 particles that
showed the best self-common-line phase residuals and that had been
refined by cross-common-line phase residual minimization among all 10 particles (52). The incorrect orientations were eliminated
from the list of initial orientation estimates of each particle by evaluating the cross-common-line phase residual between the particle and projection images of the preliminary model. The selected
orientations were then refined, first by a global refinement that
minimized the cross-common-line phase residuals across all selected
particles (52) and then by a projection-based refinement
that optimized the match between the image and the projections computed
from the preliminary 3D model. The new model reconstructed from these refinements was used as a template for the next round of particle selection and refinement, resulting in a further improved model. This
process was iterated for several cycles utilizing the entire data set
until no more particles with correct orientation parameters could be
obtained and no improvement was evident in the cross-common-line phase
residual between particle images and the computed projections. The
icosahedral (5-3-2-2) symmetry was imposed in the final reconstruction.
The final reconstruction was calculated by merging data up to 22-Å
resolution from 431 particle images with defocus values
ranging from
0.8 to 3.5 µm, which were determined from the incoherently
averaged
Fourier transforms of particle images in each micrograph
(
54). The contrast transfer function was corrected as
described
previously (
51) with an estimated temperature
factor (or B factor)
of 500 Å
2 and an amplitude contrast
of 7% (
39). The effective resolution
of the final map was
estimated to be 24 Å, where the cross-correlation
coefficient between
two independent reconstructions reaches 0.5.
For comparison, an HSV-1
B-capsid map was reconstructed similarly
at 24-Å resolution from 431 particle images; the particles were
a subset of those selected from
images used in the determination
of a previously published HSV-1 capsid
structure (
53). The magnification
difference between the two
electron cryomicroscopes used for imaging
HSV-1 and HHV-8 capsids was
estimated to be 2.0%, using the HCMV
particle as a calibration
standard, by matching the diameters
of HCMV reconstructions
independently determined from images taken
in both microscopes (not
shown) (
10).
The 3D visualization was carried out using Iris Explorer (NAG, Inc.,
Downers Grove, Ill.) with custom-designed modules (
16).
The
maps were displayed using a contour level of 1 standard deviation
above
the mean density of the map unless otherwise indicated.
The radial
density distribution profiles were obtained by spherically
averaging
the 3D density maps, and their relative densities were
scaled by
matching the height of the floor density
peaks.
Protein sequence analyses.
Pairs of HHV-8 and HSV-1 capsid
proteins were assigned based on homologies among their amino acid
sequences and/or the relative positioning of their genes among HHV-8
(33), HSV-1 (22), and other gammaherpesviruses
(1). The amino acid sequences for these capsid proteins were
downloaded from GenBank. Homologies were calculated using a variety of
programs, including the CLUSTALW (45) and LALIGN
(19) modules in Biology Workbench, version 3.0 (http://biology.ncsa.uiuc.edu) (11), the BESTFIT program of
the Genetics Computer Group package (Wisconsin package; Genetics Computer Group, Inc., Madison, Wis., 1998) in BioNavigator
(http://www.bionavigator.com), and the BLASTP program of the BLAST
2 tool (44) (http://www.ncbi.nlm.nih.gov/gorf/b12.html). A
preliminary comparison was made using the default parameters given by
Biology Workbench for both CLUSTALW and LALIGN. In order to improve the
alignments for the ORF62 and VP19C and ORF26 and VP23 pairs, LALIGN was
rerun using different gap penalties to get the optimum length of
aligned sequence. The open and extended gap penalties were 12 and 2, respectively, for ORF25 and VP5 and ORF17 and VP40 and 5 and 3, respectively, for ORF26 and VP23 and ORF62 and VP19c.
| |
RESULTS |
Purification and cryoEM of HHV-8 capsids.
Our initial attempt
to purify HHV-8 capsids from BC3 cells (2) induced either by
TPA or by a combination of TPA and butyric acid yielded barely
detectable viral particles using a density gradient procedure, though a
higher percentage of cells was induced into lytic replication in BC3
cells (40 to 60%) than in BCBL-1 cells (30 to 40%), as confirmed by
an immunofluorescence assay using a mouse monoclonal antibody against
the late antigen glycoprotein K8.1. It appeared possible that, prior to
achieving maximum yield of virions in the medium, BC3 cells lysed into
debris, which could have interfered with the subsequent purification of
viral particles. Our best yield was obtained by inducing lytic
replication of HHV-8 in BCBL-1 cells with TPA. It is noted, however,
that the HHV-8 capsid preparation, including capsids from BCBL-1 cells,
was much less homogeneous and had a significantly lower yield than
preparations routinely obtained for HSV-1 and HCMV capsids, although
various techniques, including those used for HSV-1 and HCMV in previous studies, were tried to optimize the isolation procedure.
Our subsequent cryoEM effort was focused on imaging the lower band of
the HHV-8 preparation from BCBL-1 cells, which contained
HHV-8 capsids
with the least cellular debris. The cryoEM micrographs
of the
ice-embedded HHV-8 capsids (Fig.
1) show
that the capsids
have a polyhedral shape with characteristic capsomer
protrusions,
similar to those seen in HSV-1 (
5,
36), HCMV
(
7,
10,
47), and bovine herpesvirus (
3) capsids.
The HHV-8 capsids
are approximately 1,250 Å in diameter, similar to
HSV-1 capsids.
Two types of HHV-8 capsids can be seen, designated
intermediate
and full capsids based on their resemblance to cryoEM
images of
the intermediate and full HSV-1 capsids (
5,
36).
Most capsids
are of the intermediate type, which are less electron
opaque (Fig.
1A and B). It is possible to identify the hexagonally
arranged
capsomers and capsomer channels (Fig.
1B) from the images of
the
intermediate capsids but not the full capsids (Fig.
1C). The full
HHV-8 capsids (Fig.
1A) have a striated, fingerprint-like appearance
(Fig.
1C) similar to that caused by the presence of viral DNA
in HSV-1
(
5,
51) and HCMV (
4) C-capsids. Since the HSV-1
full and intermediate capsids were shown to have similar structures
(
5,
36,
57), we subsequently focused on the more prevalent
intermediate capsids for in-depth data analyses.
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FIG. 1.
cryoEM of HHV-8 capsids. (A) An area of a micrograph of
the ice-embedded HHV-8 capsids showing predominantly intermediate
capsids and one full capsid (arrow). The underfocus value of this image
was estimated to be 1.6 µm based on incoherently averaged Fourier
transforms (54). The enlarged view of an intermediate capsid
(B) shows the capsomers (e.g., arrow) that form a characteristic
hexagonal pattern with adjacent capsomers. The full-capsid (C) image
reveals the characteristic "fingerprint" pattern.
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Structural organization of the HHV-8 capsid.
We analyzed two
sets of cryoEM images recorded for two separate HHV-8 capsid
preparations, which were obtained through independent purifications.
The 3D reconstructions obtained independently from these two sets of
images show almost identical structural features except for slightly
different resolutions, which were most likely due to differences in the
number of capsid particles used for the two reconstructions. The final
reconstruction was computed from 431 particle images of the second
preparation and had an effective resolution of 24 Å based on a
cross-correlation coefficient evaluation.
Each capsid, as shown in the shaded surface view along an icosahedral
twofold axis (Fig.
2A),
contains 12 pentons (blue, denoted
by 5) and 150 hexons (blue, denoted by P, E, and C), which are
interconnected by 320 triplexes (green). These capsomers or structural
components are
arranged in a T=16 icosahedral lattice with 20
triangular faces, one of
which is outlined in Fig.
2A. Each asymmetric
unit (one-third of a
triangular face) of the capsid contains one-fifth
of a penton at the
vertex; one each of P, C, and one-half E hexons;
and one each of Ta,
Tb, Tc, Td, Te, and one-third Tf triplexes.
These structural components
were designated following the nomenclature
established for HSV-1
(
41,
57). Each penton and hexon contain
an axial channel
that connects the inside of the capsid to the
outside (Fig.
2B). The
penton and E hexon channels coincide with
the fivefold and twofold
axes, respectively, as indicated by Fig.
2B. Triplex Tf lies on the
icosahedral threefold axis, while the
other triplexes are located at
the local threefold positions (Fig.
2B). The triplexes are triangularly
pyramidal in shape and are
positioned around small holes (~10 Å diameter) penetrating the
capsid floor (Fig.
2B). They appear to be
connected to the capsid
floor by small legs (Fig.
2B) and interact with
adjacent pentons
and hexons with "head" and "tail" domains
(Fig.
2A), in a manner
similar to triplexes of HSV-1 (
34,
53,
57) and HCMV (
7,
10,
47) capsids. The inside of the
capsid contains some discontinuous
densities (Fig.
2B, left, red), but
these are not icosahedrally
ordered as judged by their lack of
reproducibility among independent
reconstructions. This lack of
icosahedral symmetry inside the
capsid is similar to what was observed
in HSV-1 capsids (
56).
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FIG. 2.
3D structure of the HHV-8 capsid at 24-Å
resolution as viewed along the icosahedral twofold axis from the
outside (A) and inside (B) of the capsid. The map was color coded
according to the particle radius (see color bar at the bottom right),
such that the upper domains of the pentons and hexons are in blue
(between radii of 570 and 650 Å), the connecting triplexes are in
green (between radii of 510 and 560 Å), the shell is in yellow
(between radii of 460 and 510 Å), and the densities inside the capsid
shell are in red (<460-Å radius). The capsid has a T=16 icosahedral
symmetry (3 of the 12 fivefold axes are labeled 5, and 1 of the 20 triangular faces is outlined by a red dashed line in panel A), with the
unique structural components in one asymmetric unit labeled, following
the nomenclature established for HSV-1 (41, 57). These
components include one-fifth of a penton (labeled 5), two and one-half
hexons (one P, one C, and one-half of an E), and five and one-third
triplexes (one each of the Ta, Tb, Tc, Td, and Te triplexes and
one-third of the Tf triplex). The inside view in panel B is the same as
that in panel A except that the upper half of the capsid was
computationally removed to show the cutaway side views of some of the
triplexes (dashed red arrows) and the inner floor of the HHV-8 capsids.
Dot-dashed lines indicate icosahedral five-, three-, and twofold axes,
which pass through a penton channel, a Tf triplex, and an E hexon
channel, respectively. The densities inside the capsid shells (red)
lack structural information because they are not icosahedrally disposed
and thus have been removed computationally in the right half of panel B
to show the internal surface of the capsid shell.
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The montage of the HHV-8 capsid reconstruction with that of an HSV-1 B
capsid at the same resolution (Fig.
3)
clearly shows
that they have identical sizes and capsomer
organizations. However,
some notable differences are seen on closer
inspection. The HHV-8
capsid appears slightly more spherical than the
HSV-1 capsid,
which exhibits a somewhat angular, polyhedral shape (Fig.
3).
When viewed from the top (e.g., the C hexons surrounding Tf at
the
center of Fig.
3), the hexons in the HHV-8 capsid appear flower
shaped,
whereas those of HSV-1 have slightly tilted subunits and
as a result
appear more gear shaped (see below). Also, the HHV-8
triplexes are
slightly smaller and deviate less from threefold
symmetry than the
much-elongated triplexes in the HSV-1 capsid.
The color differences in
the upper domains of HSV-1 and HHV-8
triplexes indicate that the HSV-1
triplexes are slightly taller
(Fig.
3).
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FIG. 3.
Structural comparison of HHV-8 capsid and HSV-1 B
capsid. The two capsid maps are radially colored as in Fig. 2 and are
shown in a montage as viewed along the icosahedral threefold axis. The
HSV-1 B capsid was reconstructed similarly to the same resolution (24 Å) from a subset of images selected from those used in a structure
published previously (53). One penton (5), three types of
hexon (P, E, and C), and six types of triplexes (Ta to Tf) are
labeled.
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Radial distribution of capsid components.
The 3D maps of the
HHV-8 and HSV-1 capsids were spherically averaged to generate their
radial density distribution profiles as a function of particle radius
(Fig. 4). These profiles show that the
HHV-8 and HSV-1 capsids have identical inner radii of 460 Å. Because
both viruses also have similar genome sizes (30), their
identical inner radii suggest that their DNA packing densities inside
the capsids are similar. In contrast, betaherpesvirus capsids, such as
those of HCMV, have a somewhat larger internal volume than HSV-1 or
HHV-8 capsids (7, 10, 47). However, the increase in volume
is disproportionate to the large increase in the size of the HCMV
genome over the HSV-1 and HHV-8 genomes. This implies that the viral
DNA is more densely packed into HCMV virions (4) than into
HSV-1 or HHV-8 virions.
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FIG. 4.
Radial density distributions of the HHV-8 capsid and
HSV-1 B-capsid reconstructions. The density profiles were generated by
spherically averaging the HHV-8 and HSV-1 capsid maps and were plotted
as a function of particle radius. The HSV-1 and HHV-8 capsids have
identical inner radii of about 460 Å. For the HSV-1 capsid, the
locations of the four capsid proteins have been established (29,
34, 48, 55) and the density peaks corresponding to radial
locations of the capsid floor, the triplexes, and the smallest capsid
protein, VP26, are indicated accordingly. The HHV-8 capsid profile
lacks the prominent peak corresponding to that attributed to VP26 in
the HSV-1 capsid. The triplex peak is narrower and shifted to a lower
radius in HHV-8 than in HSV-1. arb, arbitrary.
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The profiles also show peaks corresponding to the radial positions and
heights of the three major components of the capsid:
the floor (460- to
510-Å radius), the triplexes (510- to 540-Å
radius), and the upper
domains of the pentons and hexons (beyond
540-Å radius) (Fig.
4). The
floor peaks of HHV-8 and HSV-1 capsids
have similar shapes and radii,
but the triplex peaks are quite
different. The HHV-8 triplex peak is
narrower and at a smaller
radius than that of HSV-1, which coincides
with the observation
from Fig.
3 that the HHV-8 triplexes are shorter
than the HSV-1
triplexes. Finally, the peak attributed to the VP26
protein, which
makes the HSV-1 hexon gear shaped by attaching to the
upper domain
of the HSV-1 hexon subunit (see below), is not present in
the
HHV-8 profile (Fig.
4).
Comparison of the pentons and hexons of HHV-8 and HSV-1.
A
penton and an E hexon were computationally extracted from the capsid
structures for direct structural comparison. In HHV-8, both the penton
(Fig. 5A) and hexon (Fig. 5B) have a
cylindrical shape (140-Å diameter, 160-Å height) with a central,
axial channel approximately 25 Å in diameter. The penton and hexon
subunits both have an elongated shape with multiple domains, including upper, middle, lower, and floor domains (subunit views in Fig. 5A and
B). The middle domains of the subunits interact with the triplexes,
while the lower domains connect the subunits to each other and form the
axial channels. While the upper domains of adjacent hexon subunits
interact with one another, adjacent penton subunits are disconnected at
their upper domains, resulting in the V-shaped side view of the penton
(Fig. 5A). Another major difference between the penton and hexon
concerns their floor domains. These domains play an essential role in
maintaining capsid stability, as suggested by the higher-resolution
structural studies of the HSV-1 capsid (53), where a long
helix inserts into the floor domain of the adjacent subunit (penton
side views in Fig. 5A and C). The relative angle between the floor and
lower domains is about 110° in the penton subunit and becomes less
than 90° in the hexon subunit (Fig. 5A and B, subunit views). In
addition, unlike what is found for the penton channel, a ring-like
constriction exists in the middle of the hexon channel (Fig. 2B; Fig.
5B, top view); the constriction is formed by the association of a
density (Fig. 5B, subunit) protruding from the lower domain of each
subunit toward the center of the channel.
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FIG. 5.
Structural comparisons of the penton (A and C), hexon (B
and D), and their subunits in HHV-8 capsid (A and B) and HSV-1 B capsid
(C and D). The top views of the HHV-8 and HSV-1 pentons (A and C,
respectively) and E hexons (B and D, respectively) reveal an axial
channel approximately 25 Å in diameter in each penton and E hexon. The
side views were generated by rotating the top view about the short edge
of the figure by about 80° and show interactions between their
adjacent subunits. In the side views of the HHV-8 penton and hexon
subunits, the upper (u, blue), middle (m, green),
lower (l, green to yellow), and floor (f, yellow)
domains of a subunit are labeled, following the same designations used
for HSV-1 (52, 57). Arrow in the HHV-8 hexon subunit (B),
density protrusion that forms the constriction inside the hexon
channel. The red dotted lines on the lower and floor domains of these
subunits (A and B) illustrate the angles between the two domains. The
two side views of the HHV-8 penton and hexon subunits (A and B, right)
are related to each other by a roughly 90° rotation about the long
edge of the Figure. (C and D, right) Side views of HSV-1 penton and
hexon. The coiled lines drawn on the floor domain of penton side views
(A and C) illustrate the location of the long helix joining the
adjacent major capsid proteins together at the floor domains, which was
first visualized in the 8.5-Å structure of HSV-1 B capsid
(53). (E and F) Superposition of penton (shown in
semitransparent red) and hexon subunits (shown as wire frames using the
same radial coloring scheme as in panels A to D) extracted from the
HHV-8 capsid (E) and from the HSV-1 capsid (F). In the HSV-1 hexon
subunit (D and F), a horn-shaped VP26 density attaches to the upper
domain of each subunit (arrow) (48, 55). No horn-shaped or
other extra density of similar size can be identified at the
corresponding location on the HHV-8 hexon subunit (B and E). The
pentons and hexons were displayed using a density threshold of 1.2 standard deviations (SD) above the mean, and their extracted subunits
were shown at 1.5 SD above the mean density. Except for the
penton subunits in panels E and F, the maps are colored according to
the capsid radius as in Fig. 2 and 3 (see color bar in Fig. 2).
|
|
The HSV-1 penton (Fig.
5C) and hexon (Fig.
5D) subunits have the same
basic shape as the HHV-8 subunits. Each consists of
upper, middle,
lower, and floor domains (Fig.
5C and D, subunit
views). However, the
upper domains of the HSV-1 penton subunits
point inward toward the
channel, whereas those of the HHV-8 penton
subunits point outward. The
upper domain of the HHV-8 subunit
has a rectangular shape (subunit view
in Fig.
5A), while that
of the HSV-1 penton subunit appears as a
triangle (subunit views
in Fig.
5C). The most striking difference is
that the HSV-1 hexon
subunits contain an extra horn-shaped density
(Fig.
5D), the VP26
protein, which is not found in either the HSV-1
penton (Fig.
5C)
(
48,
55,
57) or the HHV-8 hexon. This extra
density binds
to the top of each HSV-1 hexon subunit, forming a ring
around
the hexon at a radius of approximately 600 Å. This accounts for
the tilted or gear-like appearance of the HSV-1 hexon top view
(Fig.
5D). The superposition of HHV-8 penton and hexon subunits
(Fig.
5E)
shows that the domain features of HHV-8 penton and hexon
subunits are
roughly similar (except for the floor domain, as
described above),
whereas superposition of the HSV-1 penton and
hexon subunits clearly
shows an extra density bound to the upper
domain of the hexon subunit
(Fig.
5F).
Sequence homology between HHV-8 and HSV-1 capsid proteins.
HHV-8 capsid proteins were previously assigned to their HSV-1
counterparts by positional homology. Based on primary sequence analyses
using CLUSTALW, the major capsid proteins (HHV-8 ORF25 protein and
HSV-1 VP5) are almost identical in size and show significant identity
(25%) and similarity (60%) (Table 1).
In contrast, the smallest capsid proteins (HHV-8 ORF65 protein and
HSV-1 VP26) differ substantially in size and show almost no identity
(5%). LALIGN failed to align the ORF65 protein and VP26 pair even
after the gap and deletion penalties were relaxed.
| |
DISCUSSION |
Structural proteins of the HHV-8 capsid and their homologs in other
herpesviruses.
At least five major proteins are likely to be
involved in the assembly of the HHV-8 capsid, including a protease
(encoded by ORF17), the major capsid protein (encoded by ORF25), and
three other smaller capsid proteins (encoded by ORF62, ORF26, and
ORF65) (33) (Table 1). The protease, although not associated
with the capsid shell, plays an essential role in capsid assembly and is the most functionally conserved among members of the
Herpesviridae (49). Although our structure does
not directly indicate which proteins make up the capsomers, this can be
inferred from the positional and sequence homology between the HHV-8
and HSV-1 capsid proteins and also by comparing the structures of both
capsids. Therefore, our results provide structural evidence that the
HHV-8 pentons and hexons are composed of five and six copies,
respectively, of the ORF25 protein. The penton and hexon subunits
closely resemble those of HSV-1 in shape and size, and the ORF25
protein is homologous to HSV-1 major capsid protein VP5, which makes up
the pentons and hexons.
Previous structural studies have shown that the HSV-1 triplex is a
monomer of VP19c and a dimer of VP23 (
29,
34,
57)
and that
the HCMV triplex is similarly composed of a monomer and
a dimer
(
10). By analogy, the HHV-8 triplexes are likely also
composed of a monomer of the ORF62 protein and a dimer of the
ORF26
protein, which are the respective homologs of VP19c and
VP23. The
molecular masses of the ORF62 and ORF26 proteins are
similar, in
contrast to the significant molecular mass difference
between VP19c and
VP23 (Table
1). These differences accord well
with our observation that
the HHV-8 triplex is smaller and less
deviated from threefold symmetry
than the HSV-1
triplex.
The ORF65 protein is absent from the HHV-8 capsid
reconstruction.
Due to difficulties in purifying homogeneous HHV-8
virions and capsids (see Materials and Methods), it remains technically prohibitive to verify, through biochemical means such as sodium dodecyl
sulfate-polyacrylamide gel electrophoresis or Western blotting, whether
the ORF65 protein is associated with HHV-8 capsids. Difference imaging
of HSV-1 recombinant capsids has unambiguously shown that the extra
densities attached to the upper domains of the hexons can be attributed
to monomers of the VP26 protein. VP26 binds only to VP5 in hexons and
not to VP5 in pentons (48, 55), demonstrating the
quasiequivalent structures of identical VP5 proteins in different
locations. The HHV-8 hexons do not have this extra density (Fig. 5E),
which implies that the VP26 positional homolog, the ORF65 protein (also
referred to as small viral capsid antigen or sVCA) (21), is
not attached to the hexons in our reconstruction.
At present, the exact role of VP26 in HSV-1 capsid assembly is still
unclear. It has been shown that VP26 is not essential
for capsid
assembly in insect cells infected with recombinant
baculoviruses
expressing HSV-1 capsid proteins, although B-capsid
assembly is more
efficient when VP26 is present (
43,
46).
Moreover, VP26 is
not required for HSV-1 infection (
14). VP26
might serve to
connect the capsid proteins with the tegument layer,
given that VP26 is
located on the outermost surface of the hexons
(
51).
We are not able to offer a definitive explanation for the absence of
the ORF65 protein from our structure. It may have been
lost during the
purification procedure if it is not as tightly
bound to the hexons as
VP26 in HSV-1. In this regard, 2 M guanidine
chloride treatment
resulted in the dissociation of VP26 from HSV-1
capsids
(
28). Thus, NP-40 or Triton X-100 may have dissociated
the
ORF65 protein from the HHV-8 capsids in our preparation. If
this is the
case, the interaction between the ORF65 protein and
HHV-8 major capsid
protein must be weaker than that between its
counterpart in HSV-1 or
HCMV and the major capsid protein, since
its homologs in HCMV and
simian cytomegalovirus remain bound when
treated with 0.5% NP-40
(
7,
47) and since VP26 remains bound
when HSV-1 is treated
with 0.25% NP-40 or 1% Triton X-100 (
5,
28). It is also
possible that the mature HHV-8 capsid lacks
a binding site for the
ORF65 protein and, consequently, that the
ORF65 protein is not involved
in capsid assembly. This situation
is similar to that for the channel
catfish virus, a related virus
that lacks an ORF65 protein or VP26
positional homolog and showed
no horn-shaped densities on its hexon
subunits (
6).
In any event, this first step in overcoming the technical problem of
virus isolation has permitted the first view of this
gammaherpesvirus
capsid and the structural comparisons with other
herpesvirus capsids.
Our observations should encourage further
investigation of the HHV-8
structures at a higher resolution in
order to reveal in greater detail
structural differences that
are important to the understanding of their
pathogenesis in different
diseases.
| |
ACKNOWLEDGMENTS |
This research was supported in part by Public Health Service
grants (AI 46420 to Z.H.Z. and HL42886 to J.K.S.) and the March of
Dimes Birth Defect Foundation (5-FY99-852 to Z.H.Z.). Z.H.Z. is a Pew
Scholar in the Biomedical Sciences.
We thank L. Oshiro and H. Zhang for initial electron microscopy
assessment of sample conditions, John Stewart for preliminary data
processing, W. Chiu and F. Rixon for the use of the HSV-1 capsid
structure, and T. Dunnebacke-Dixon for comments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology and Laboratory Medicine, University of Texas-Houston Medical School, Houston, TX 77030. Phone: (713) 500-5358. Fax: (713) 500-0730. E-mail: Z.H.Zhou{at}uth.tmc.edu.
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Journal of Virology, October 2000, p. 9646-9654, Vol. 74, No. 20
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
