JOURNAL OF VIROLOGY, Mar. 1994, P. 1935-1941
Copyright © 1994, American Society for Microbiology
Structure of Intracellular Mature Vaccinia Virus Observed
by Cryoelectron Microscopy
JACQUES DUBOCHET,1* MARC ADRIAN,1 KARSTEN RICHTER,'t
JAVIER GARCES,2t AND RICCARDO WITEK2
Laboratoire dAnalyse Ultrastructurale,' and Institut de Biologie Animale,2 Bdtiment de Biologie,
Universite de Lausanne, CH-1015 Lausanne, Switzerland
Received 29 September 1993/Accepted 3 December 1993
Intracellular mature vaccinia virus, also called intracellular naked virus, and its core envelope have been
observed in their native, unfixed, unstained, hydrated states by cryoelectron microscopy of vitrified samples.
The virion appears as a smooth rounded rectangle of ca. 350 by 270 nm. The core seems homogeneous and is
surrounded by a 30-nm-thick surface domain delimited by membranes. We show that surface tubules and most
likely also the characteristic dumbbell-shaped core with the lateral bodies which are generally observed in
negatively stained or conventionally embedded samples are preparation artifacts.
Vaccinia virus, the prototype orthopoxvirus, is associated
with several important discoveries in the history ofvirology (9).
The large size of the virions allowed their visualization with
light microscopy by Buist as early as 1886, and these descrip-
tions of small granules founded the concept of the particulate
nature of viruses (3a). Vaccinia virus was also the first animal
virus to be purified to an extent which allowed accurate
chemical analysis. Finally, vaccinia virus also gave its name to
vaccination and was closely associated with the development of
the science of immunology.
Vaccinia virus was also among the first viruses to be exam-
ined by electron microscopy. Our current knowledge of the
structure of the virion (Fig. 1 and 2) is based on the work of
many scientists and has been reviewed recently (9). Briefly, two
forms of virions can be distinguished. Intracellular naked
virions (INV), which have recently also been called intracellu-
lar mature virions (IMV) (20), remain cell associated and may
be recovered after experimental cell lysis. Virions which are
naturally released from the cell are surrounded by an envelope
derived from an intracellular cisterna and have been desig-
nated extracellular enveloped virions (EEV) (18). Both IMV
(or INV) and EEV are infectious.
For details of the structure, we refer here to the scheme of
Fenner et al. (8), reproduced in Fig. 1, and to typical views
shown in Fig. 2. The IMV particles are said to be brick-shaped,
measuring ca. 300 by 230 nm. As seen by negative staining (Fig.
2a), the outer membrane ofIMV particles consists ofrandomly
arranged tubular elements (surface tubules). The inside of the
virion is best visualized in thin sections of infected cells (Fig. 2b
and c). They reveal the presence of the viral core which
contains the viral genome and which assumes, in the virion, a
dumbbell shape owing to the presence of two lateral bodies of
unknown function. The
associated with a regularly spaced palisade.
Cores released from the virions by treatment of IMV
particles with nonionic detergents and reducing agents adopt a
core envelope frequently appears
*Corresponding author. Mailing address: Laboratoire d'Analyse
Ultrastructurale, Batiment de Biologie, Universite de Lausanne, CH-
1015 Lausanne, Switzerland. Phone: 21 692 2475. Fax: 21 692 2540.
Electronic mail address: JDUBOCHE@ULYS.UNIL.CH.
t Present address: Veilchenweg 20, D-68775 Ketch, Germany.
t Present address: Laboratorios Clinicos dePuebla,Diaz-Ordaz808,
Puebla, Pue 72530, Mexico.
more rectangular shape. The isolated core envelope is com-
posed of a smooth membrane, with regularly arranged spikes
about 10 nm long and 5 nm in diameter.
Another typical form of the virion is frequently observed in
negatively stained preparations. These particles have a smooth
outer surface and are delimited by a characteristic 25-nm-thick
zone of less-electron-dense material. The core appears homo-
geneous. This aspect is depicted for example in Fig. 2 and 8 of
Muller and Williamson (16). This form of the virion is gener-
ally believed to be the consequence of stain penetration and to
be less representative of the true structure than the views
shown in Fig. 2.
The structure of the virions described above was derived
mainly from micrographs obtained by conventional electron
microscopy. This procedure involves full dehydration of the
material, which can be a source of artifacts (11). Artifacts are
particularly severe with membranous structures and enveloped
particles whose size and material distribution can be severely
affected by the preparation procedure.
Most preparation artifacts are avoided in cryoelectron mi-
croscopy of vitrified samples (4). In this technique, the speci-
mens are neither chemically fixed nor strained and they are
observed in their fully hydrated environment. The size of
virions is precisely conserved, and the structure is well pre-
served (1, 2, 14, 17). In the present work, we have observed
vaccinia virus IMV particles and isolated cores by cryoelectron
microscopy of vitrified samples and we have found that the
structure of the virion differs markedly from that which is
generally accepted to be the true structure.
MATERIALS AND METHODS
Viral strains. The virus strains used were WR, the Western
Reserve strain of vaccinia virus that most likely does not
express the intact P4C protein (18a), and A392, the Western
Reserve strain of vaccinia virus expressing the entire P4C
Virus preparations. IMV were purified by the method of
Joklik (10), with minor modifications. After centrifuging the
crude virus suspension through 36% sucrose, the virus was
resuspended by mild sonication in 10 mM Tris-hydrochloride
(pH 9), layered onto a 15 to 40% (wt/vol) sucrose gradient, and
centrifuged for 15 min at 13,000 rpm (Kontron, TST 55 rotor).
The virus band was extracted, and the virus was collected after
Vol. 68, No. 3
DUBOCHET ET AL.
1. Schematic representation of the structure of vaccinia virus,
section of enveloped virion; left-hand side, surface structure of non-
enveloped particle revealed by negative staining. T, surface tubules; B,
lateral bodies; E, envelope; OM, outer membrane; C, core; CE, core
envelope with palisade. Magnification,
produced by permission from Fenner et
x 200,000. Bar,
100 nm. Re-
al. (8; Fig. 2.1, p. 76), with
a threefold dilution in 10 mM Tris-hydrochloride (pH 9) by
centrifugation at 50,000 x g for 30 min. After a second round
resuspended in 10 mM Tris-hydrochloride (pH 9) at a concen-
tration of at least 10 mg/ml and stored at 40C. Before electron
Isolation of viral
cores were prepared
scribed by Easterbrook (7), with minor modifications. Purified
virus were diluted with 50 mM Tris-hydrochloride (pH 7.5) to
2 mg/ml, andvolume of 1% Nonidet P-40-2% 2-mercapto-
ethanol-SO mM Tris-hydrochloride (pH 7.5) was added. After
for 30 min
Electron microscopic view of IMV. (a) Particle negatively
stained in 2% uranyl acetate. The general brick shape and the surface
in Lowicryl. The
palisade (P), and the lateral bodies (B)
x 100,000. Bar, 100 nm. Panels b and c are used by the courtesy of G.
core envelope (CE),
are marked. Magnification,
centrifuged through 36% sucrose for 30 min at 20,000 x g
(TST 55 rotor). The cores were gently resuspendedin 50 mM
Tris-hydrochioride (pH 7.5) and stored at 40C.
Preparation of viral core envelopes. MgCl2was added to the
core preparation at a final concentration of 10 mM. After the
addition of 10 U of DNase (Promega) per0.1 mg of cores, the
suspensionwas incubated for 1 h at 370C. Core envelopeswere
purified by centrifugation through 36% sucrose at 20,000 rpm
(TST 55 rotor) for 1 h. The pellet fraction was gently resus-
pended in 10 mM Tris-hydrochioride (pH 7.5)-l mM EDTA
and stored at -20'C.
Conventional electron microscopy. Negative staining of
specimensand embedding inEpon and in Lowycrylwere done
by the standard methods (3, 20).
Cryoelectron microscopy. Thin layers of vitrified suspension
were prepared by the bare grid method (1, 4). A 3-pAl drop of
suspension containing ca. 50 mg ofparticles perml in 10 mM
Tris-hydrochioride (pH 9)wasputon aperforatedcarbon grid
mounted on a 200-mesh grid. Most of the drop was removed
with blotting paper,and the thin residual film was immediately
vitrified by projecting
nitrogen. The thinliquidfilm exists for about 0.1 s before being
vitrified in ca. 10-4 S.
For thin sections of vitrified material, a low-density pelletof
virions in 8% sucrose-7% short DNAfragmentswas obtained
by centrifugation. A small amount of this material (ca. 10 nl)
was mounted on a metal pin and immediately projected in
liquid ethane. The solution of sucrose-DNA gives an adequate
viscosity for manipulation and sufficient cryoprotective effect
to allow vitrification of a surface layer of usable thickness.
Deeper in the sample, water crystallizes into ice, producing
solute precipitation and dehydration of the virion. The frozen
sample was cut into thin cryosections in a Reichert FC4E
cryochamber of an Ultracut ultramicrotome (Leica, Vienna,
Austria), and the sections were flattened on a carbon film on a
200-mesh grid. During the entire procedure, the sample and
the sections were kept at or below -160'C.
The cold samples were mounted in a Gatan 626 cryospeci-
men holder (Gatan, Varrendale, Pa.) and observed at -170'C
in a Philips CM 12 cryoelectron microscope equipped with a
whether vitreous or crystalline, was tested by electron diffrac-
tion. Observations and image recording were made under
low-dose conditions. Kodak S0163 films developed for 12 mmn
in D 19, full-strength (speed, ca. 2 VLm2 per electron x optical
density unit) were used for recording micrographs at a typical
magnification of X 35,000.
Magnificationwas calibrated with across-grating replicaand
varied by less than 2% of the nominal value. Size measure-
ments were made directly on the negative or on prints with a
ruler or with an optical diffractometer for periodic structures.
it in liquid ethane cooled in liquid
state of the specimen,
General aspects. The general aspectof the freshly prepared
IMV particle as observed by cryoelectron microscopy of a
vitrified thin layer ofsuspension is shown in Fig. 3. The virion
appears as a smooth, rounded rectangle. One or two adjacent
corners are frequently more flattened than the others. At first
sight, the virus seems rather homogeneous, with a zone of
higher mass in the central region. A 30-nm-thick surface
domain (S)is visible in mostparticles.This domain is delimited
by two membranes (Fig. 3, arrows) which, however, are not
always visible. On closer examination, the virion has a fine
homogeneous grainy texture and
is also finely
VACCINIA VIRUS STRUCTURE REVISITED
FIG. 4. Aging solution of IMV prepared as described in the legend
to Fig. 3. S, surface domain; P, palisade. Arrows point toward a clearly
marked unit membrane. Arrowheads indicate discontinuities or rup-
tures in the outer membrane. Magnification, x 112,000. Bar, 100 nm.
FIG. 3. Vaccinia virus IMV in a thin layer of vitrified suspension, as
observed by cryoelectron microscopy. The 30-nm-thick surface domain
is marked S. The arrows point toward the two membranes limiting this
region. The thickness of the vitrified layer is ca. 300 nm. Magnification,
x 112,000. Bar, 100 nm.
irregular. There is no sign of a structure resembling surface
tubules or of a characteristic dumbbell-shaped core or lateral
Table 1 gives the sizes of isolated IMV particles, as deter-
mined in negatively stained and in vitrified preparations. The
standard deviations of the measurements, especially for the
length, are smaller in vitreous than in negatively stained
preparations. No differences could be observed in the size and
the aspect of WR and A392 strains.
The smooth surface of the virion shown in Fig. 3 is charac-
teristic of fresh samples prepared without harsh treatment.
Samples kept at 4°C for longer periods show a larger variety of
structures (degradation). An example is presented in Fig. 4. In
this case, the majority of the particles still have the general
TABLE 1. Sizes of IMV particles and core envelope in negatively
stained or vitrified preparations
363 ± 51
349 ± 18
288 ± 20
IMV, negative stained
Core membrane, vitreous
313 ± 11
267 ± 9
204 ± 13
shape of the intact virion, but the surface domain (S) is more
clearly delineated and is frequently marked by a ca. 4.8-nm
striation of the palisade (P). The outer membrane is frequently
seen as a typical unit membrane with ca. 4 nm of spacing
between the leaflets (Fig. 4, arrows). It frequently seems to be
broken, creating ridges with a more or less regular spacing of
25 nm (arrowheads). A number of particles appear to be more
seriously damaged (Fig. 4, *): they are more irregular in size or
more rounded, the outer unit membrane is more visible, and
the core frequently seems to separate from the surface domain.
In some cases, it resembles the typical dumbbell shape de-
scribed previously (not shown).
Surface tubules. In order to remove any doubts whether the
aspect of the virion seen in vitrified preparation (Fig. 3) is
more representative of the native state than when the sample
is prepared by negative staining (Fig. 2a), we have done the
following control experiment. A vitrified sample was prepared
similarly to the one shown in Fig. 3 except that a continuous
supporting film mounted on a finder grid was used. (A finder
grid is such that the observed region can easily be relocated in
a subsequent observation). It was verified in the cryoelectron
microscope that the virions had the usual aspect, and one
region of the grid was irradiated with an electron dose of ca.
3,000 electrons per nm2. This dose
considerable chemical transformations, though it is too small
to produce bubbling or a significant mass loss and to cause
visible structural damage at a dimension larger than about 5
nm. The sample was then slowly allowed to warm up until it
reached room temperature after ca. 2 h. Freeze-drying was
is sufficient to induce
VOL. 68, 1994
1938DUBOCHET ET AL.
FIG. 5. (a) Negatively stained preparation of IMV freeze-dried in
the electron microscope after fixation by electron irradiation (see text
for details). (b) Same sample as in panel a but recorded from a region
that was not irradiated previously. Magnification, x 60,000. Bar, 100
essentially finished before the temperature of - 100°C was
reached, and it was observed that the shape and the morphol-
ogy of the particle did not change significantly during freeze-
drying. The specimen was then withdrawn from the electron
microscope, immediately stained negatively with 2% uranyl
acetate, and observed again under normal room temperature
conditions. An obvious difference was noticeable between the
region previously irradiated at low temperature and that which
was not. In preirradiated regions, illustrated in Fig. 5a, most of
the virus particles resemble those observed in the vitreous state
though the surface domain
minority of the particles have the usual aspect of negatively
stained virions with surface tubules (*). In nonpreirradiated
regions, nearly all the particles have the usual aspect, with
surface tubules of negatively stained particles (Fig. 5b).
This experiment allows us to monitor the transformation of
the virion during the different steps of the preparation proce-
dure. It demonstrates that the structure shown in Fig. 5a
resembles that observed in previous preparation steps and is
therefore likely to be a faithful representation of the native
virion. On the contrary, the characteristic structure with sur-
face tubules seen in Fig. 5b appears only after staining. The
difference between panels a and b must therefore be attributed
to a staining artifact in panel b.
This experiment demonstrates further that electron irradia-
tion can prevent the artifactual formation of surface tubules
upon negative staining. This can be the consequence of a
porosity induced by electron beam damage in the surface
domain, making it insensitive to osmotic effect. It also suggests
that the surface domain is normally sensitive to osmotic stress,
either because it is delimited by two nonpermeable membranes
or because it has the structure of an osmotically active gel.
Dumbbell-shaped core. As for the dumbbell shape of the
core presented in Fig. 2b and c, some of the above observations
suggest that it is also a preparation artifact. Supporting this
view is the fact that dumbbell-shaped cores are rarely observed
in vitrified samples of freshly prepared virions, whereas they
are more numerous in older preparations or after chemical
However, the possibility remains that in the thin vitrified
is more visible. Only a small
FIG. 6. Unstained and chemically unfixed cryosection of a concen-
trated solution of IMV. In order to make vitrification possible, 8%
sucrose-7% DNA was added to the solution. The specimen was
prepared as described in Materials and Methods. (a) Micrograph of a
region where the sample is vitrified. The virion appears with a dense,
nearly homogeneous body. (b) Micrograph of the same section deeper
in the sample, where the water is crystallized in hexagonal ice. Most
particles are nonhomogeneous, and many of them have a central
low-density dumbbell-shaped region. The quality of the specimen is
degraded by cutting-induced compression along the cutting direction
(marked by a circled double arrow). Crevices and deformation lines
are other visible cutting artifacts. The segregation pattern formed by
material excluded from the growing ice crystals and the Bragg reflec-
tions (circled) indicate that water is crvstallized in this region. The
section thickness is ca. 100 nm. Magnification, x 100,000. Bar, 100 nm.
layer, the particles are all oriented in such a way that the
dumbbell-shaped core could be visualized only in extreme
lateral views which are not feasible in the electron microscope.
In order to exclude this possibility, we have observed thin
cryosections of a vitified concentrated solution of isolated
virions. In such specimens, the orientation of the particles is
random and all projections are equally probable. The result,
presented in Fig. 6, shows, first of all, that vaccinia virus
particles are not well suited for cryosections and do not
compare favorably to other samples such as liver tissue (12),
muscle (13), DNA crystals (19), or apple leaves (15) prepared
by the same method. In particular, they suffer from severe
cutting damage due to compression along the cutting direction
(double arrows) and most of the fine details of virus structure
are lost. As for the aspect of the virus, two cases can be
distinguished. When the section is truly vitrified, as is the case
in Fig. 6a, most of the virus particles have a homogeneous mass
distribution without recognizable internal structure. In con-
trast, the characteristic dumbbell-shaped core is visible in many
particles in regions where ice is crystallized. This is the case for
the region shown in Fig. 6b, in which the crystalline state of the
ice is apparent from the segregation pattern of the solute
visible in the background and from the dark spots (circled) due
to Bragg reflections in the hexagonal ice crystals. In addition,
VACCINIA VIRUS STRUCTURE REVISITED
FIG. 8. Vitrified thin layer of isolated core envelopes. The particle
shown has lost many of its spikes during preparation. They are still in
the vicinity of the particle but are oriented in respect to the thin film
surface. Magnification, x 112,000. Bar, 100 nm.
FIG. 7. Thin layer of vitrified isolated core envelopes. The mem-
brane marked with an asterisk has a characteristic shape, whereas the
one marked by an arrowhead is torn off. Ordered spikes are visible
around the particle or, in some places, at the surface (circled area).
The thickness of the film is ca. 200 nm. Magnification, x 70,000. Bar,
the crystalline versus vitreous state of the water was tested
directly by electron diffraction. Since the two views presented
in Fig. 6 are from the same section, the difference must be
attributed to freezing damage, probably induced by a dehydra-
Core envelope. Isolated cores are rarely intact. They lose
variable amounts of their content and are heterogeneous in
shape and dimension. Removal of the residual core material by
digestion with DNase results in apparently empty core enve-
lopes which can be precisely characterized. Such a preparation
is shown in Fig. 7. The envelope seems to be formed by a
membrane with more or less regularly arranged spikes pointing
outward. The envelopes have roughly the same general shape
as IMV particles, but they are less rounded and they are
smaller. The length and the width of the envelope, measured at
the level of the membrane, are both ca. 60 nm smaller than the
corresponding dimensions in the IMV particle (Table 1). It is
a frequent occurrence that one or two adjacent corners of the
envelope are flattened, giving the very characteristic shape of
the particle marked by an asterisk. Some of the envelopes are
not fully intact but the general shape is preserved (arrowhead).
The spikes seem to be packed closely on the envelope in a
disordered or an ordered way. When they are ordered, they
form a hexagonal lattice of 9.7-nm spacing. One example in
which a hexameric arrangement is clearly visible just at a
corner of the particle is circled. The order seems to improve in
specimens kept at 4°C for longer periods. The spikes protrude
from the membrane by ca. 20 nm. Seen from the side at high
resolution, the spikes seem to have the shape of a hollow tube,
resulting, when many spikes are tightly aligned, in a periodic
striation of half dimension (ca. 4.8 nm). The spikes are easily
lost from the core envelope. In many preparations, large
regions of the envelopes are devoid of spikes or small clusters
of few spikes are scattered over the envelope. In some cases,
the spikes are lost during preparation of the thin vitrified layer
and are oriented by the thin film surface. Such a case is
illustrated in Fig. 8. This orientation effect, which has been
observed frequently with other viruses (5, 6), is due to the fact
that spike proteins tend to adsorb to the surface of the thin
liquid film in which the particles are imprisoned for a fraction
of a second before vitrification. They remain in the vicinity of
the particle from which they originate, because they have not
had time to diffuse far away. The spikes are probably attached
to the liquid surface through their hydrophobic tails and
therefore all have the same orientation. In this projection, the
spike proteins of the core envelope appear as hollow cylinders
with an outer diameter of ca. 10 nm.
Shape and structure of the virion. The results presented
above demonstrate that the native freshly prepared IMV
particle is more faithfully represented in Fig. 3 than in Fig. I or
2. The difference between these two representations is mainly
due to preparation artifacts. The major features deduced from
the present work are schematized in Fig. 9. The 30-nm-thick
surface domain is osmotically sensitive and is delimited by two
membranes which can be recognized as thin zones of high
density. In aging preparations, the material seems to be more
segregated in this compartment, and the membranes fre-
quently appear with the typical double leaflet aspect of a unit
membrane. The palisade forming a regular, ca. 5-nm striation
is attached outward from the core envelope. The core appears
dense and homogeneous.
One difficulty in deducing the three-dimensional shape of
the virion comes from the fact that the sample is not homoge-
intracellular maturation. Differences between particles ob-
served on the micrographs also result from the fact that
different projections are observed. It appears however that the
virion is not randomly oriented in the thin vitrified layer. Most
particles have one long and one short dimension with a ratio of
It probably contains particles at various stages of
VOL. 68, 1994
DUBOCHET ET AL.
FIG. 9. Schematic view of an IMVparticleas it is deduced from the
presentwork. S, surface domain; OM, outer membrane; P, palisade;
CE, coreenvelope; C, core. Magnification, x200,000. Bar, 100 nm.
ca. 1.3. Intact particleswith two equally long or equally short
axes are practically nonexistent. From this fact,
concluded that the long axis of the virion is always approxi-
mately parallel to the planeof the thin vitreous film. This fact
is not surprisingsince the dimension of the virus is large and
with the kind of electronmicroscopeused in thisstudy, it is not
possibleto observe vitrified films thick enough for free orien-
tation of the virion. As aconsequence, a whole set ofprojec-
tions are never observed and important information concern-
the third dimension
compensatefor this lack of databy recording projectionsfrom
strongly tilted grids and large-angle stereographic views. The
results have been disappointing because of the lack of easily
observable structural features on the surface of the virion and
because its roundish shape does not favor three-dimensional
reconstruction (a sphere looks the same in all directions).
We are therefore left with the question of the third dimen-
sion of the virion. Has the virion the general shape of a
rounded barrel or that of a rounded brick? In the latter
possibility,what is the thickness of the brick? These questions
couldprobablybe solvedby observing thicker films ofsuspen-
sions which would requirethat the observations are made in a
high-voltage electron microscope.
A hint isprovided byobservation of the core envelope (Fig.
7). Here, whether the envelope
dependson whether the topand bottom sides of the observed
particlesarepartsof acylinderseenperpendicularly to its axis
or ifthey are two parallel flat surface elements. The second
possibility seems to be correct, since undistorted symmetrical
hexamericarrangementof the spike proteins can be seen even
at the edge of the core envelope (circled region in Fig. 7).
Direct observation ofstereographic pairs of tilted images also
givesthe impression that the core membrane is made of flat
surface elements (not shown).
The core envelope with its more or less regularly arranged
spikes observed in purified form in the present work (Fig. 7)
palisadethat forms the limit between the surface domain and
the core of the intact virion (Fig. 3). This is proved by the fact
it can be
missing. We havetriedto
is brick- or barrel-shaped
internal membrane with the
that the size of the core of the intact virion and that of the
purified core envelope are very close (Table 1, keeping in mind
that the surface domain is 30 nm thick) and also because the
spikes in the core envelope are in the same relative position
and show the same periodicity as the palisade.
It will be interesting to identify by immunolabelling the
proteins forming the various structures of the virion. For
example, we need to know what is the protein forming the
spikes of the core envelope? Such data, together with that of
the structure will help in elucidating the morphogenetic path-
way of the virus in the cell.
Surface tubules and dumbbell-shaped core-two artifacts
which need to be understood. We have shown that surface
tubules and probably also the dumbbell-shaped core are
artifacts due to dehydration of the virion. They are however so
remarkable and reproducible that they must also be under-
stood in terms of the underlying structure. It is amazing to note
that the more real structure revealed by cryoelectron micros-
copy has also been observed in previous studies made by
conventional methods, but it has not been considered to be
significant. (For example, see Fig. 2 and 8 of reference 16.)
Our failure to detect surface tubules by cryoelectron micros-
copy is not a consequence of the absence of P4C in our WR
strain, because these structures were also absent in the A392
isolate expressing intact P4c. The surface tubules are probably
the result of dehydration of the surface domain. The extent of
the phenomenon shows that the collapse takes place in the
whole volume of the surface domain, suggesting that in the
native state, this region has the consistency of a gel. A hint at
an explanation of the reproducible aspect and the frequent
regularity of the surface tubules is offered by the image of the
aging preparations of virions showing apparent breaking
points, sometimes regularly spaced, in the outer membrane of
the virus. The nature of these breaking points is not known, but
one can imagine that they are related to some kind of scaffold,
involved in the development of the virion.
The dumbbell shape of the core and the formation of the
lateral bodies probably result from a nonisotropic drying
collapse. We suggest that the gel-like core material formed by
a more or less homogeneous mixture of internal proteins and
DNA collapses under the effects of dehydration. The DNA
could precipitate together with part of the proteins. The
characteristic dumbbell-shaped core would derive from the
previous arrangement of the DNA. Part of the original core
material, expelled during the collapse, would form the lateral
In conclusion, we note that artifacts similar to those ob-
served in the present study are likely to take place whenever
large enveloped viruses are observed by conventional prepara-
tion methods of electron microscopy. The results of such
observations should be considered with caution unless they are
confirmed by observations in the vitrified state.
We thank G. Griffiths for useful discussions and for Fig. 2b and c, D.
Pickup for the virus isolate A392 and for information on P4c, and F.
Fenner for permission to reproduce Fig. 1.
This work was supported by the Swiss National Fonds for Scientific
Research (grant 31-26562.89 to R.W.) and by the Etat de Vaud.
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