Download Imaging of viruses by atomic force microscopy

Journal
of General Virology (2001), 82, 2025–2034. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Imaging of viruses by atomic force microscopy
Yu. G. Kuznetsov, A. J. Malkin, R. W. Lucas, M. Plomp and A. McPherson
University of California, Irvine, Department of Molecular Biology and Biochemistry, Room 560, Steinhaus Hall, Irvine,
CA 92697-3900, USA
Introduction
Two techniques that have proven particularly useful for
elucidating the detailed structures of viruses have been electron
microscopy (EM) and X-ray crystallography. These techniques
have led to our current descriptions of icosahedral and helical
viruses and the delineation of capsomere arrangements and
protein subunit distributions on their surfaces (Horne & Wildy,
1961 ; Caspar & Klug, 1962). For this, EM relied principally on
heavy metal staining or shadowing of virus particles dried in a
vacuum (Finch & Holmes, 1967 ; Horne & Wildy, 1963). More
recently, however, cryogenic EM has permitted more detailed
analysis of virus particles and, in many cases, even their interior
architecture in a more nearly physiological state is visible
(Baker et al., 1999). On the other hand, X-ray diffraction has
yielded beautifully detailed models of virus particles, though
generally lacking in nucleic acid structure, at near atomic
resolution (Harrison, 1990). The major drawback of X-ray
diffraction is that the virus must be obtained in a crystalline or
some other highly ordered form suitable for analysis
(McPherson, 1998).
It is unlikely that any probe technology such as scanning
transmission electron microscopy or atomic force microscopy
(AFM) can, at this time, compete with cryogenic EM or X-ray
crystallography as a means of delineating virus structure,
particularly the interior structure. Nonetheless, AFM may have
its place in accurately determining the dimensions of virus
particles, their mechanical properties and the architecture of
their surfaces. In the best of cases, it may be capable of
revealing capsomere arrangements and perhaps even the
distribution of capsid protein subunits within the capsomeres.
With future improvements in AFM technology, images may
begin to overlap with those of X-ray crystallography.
AFM was invented by G. Binnig in 1982 (Binnig et al.,
1986) and has been used in the visualization of biological
structure for about the last ten years. Its great advantages over
other imaging methods are that it can be carried out in a fluid
environment, including physiological medium, and that it does
not disturb the specimen from its natural state. Furthermore, it
spans a range of dimensions from a nanometre up to a hundred
Author for correspondence : Alex McPherson.
Fax j1 949 824 1954. e-mail amcphers!uci.edu
microns, which is only marginally accessible by other techniques.
AFM technology
Fig. 1 is a schematic diagram of an atomic force microscope
for imaging in fluids. The principles that define it are rather
simple, surprisingly so given its acuity, but the instrument
depends on associated advanced technologies for its application. In practice, an exceedingly sharp probe is brought
into atomic contact with the surface of the sample object, in
vacuum, air or liquid, and it is then scanned in a systematic
manner over the surface of the object. The stylus, similar to a
very sharp phonograph needle with a tip radius of about 5 to
40 nm, extends from the end of a short cantilever, typically
100 to 250 µm in length. To minimize the force between the
tip and sample in ‘ contact ’ mode, the cantilever has a low
spring constant of less than 1 N\m.
Scanning using contact mode is accomplished by translating
the stylus, which is under piezoelectric control, along a series
of raster lines. As the probe tip moves over the surface, it
interacts through aggregate atomic forces with topological
features on the surface. As a consequence of these interactions,
the probe is deflected. Cantilever displacements are amplified
by corresponding deflections of a laser beam reflected into a
split photodiode from the upper surface of the probe.
Photoelectric circuitry then converts the deflections into height
information. The resulting data, recorded as a digital image,
can then be presented in a number of different visual formats.
AFM may be operated in either ‘ height ’ mode or
‘ deflection ’ mode. In height mode, the sample surface is
maintained at a constant distance from the tip of the probe by
the piezoelectric positioner below by a feedback mechanism.
The cantilever deflection in this case is very small, tending to
zero. In deflection mode, the sample is stationary and cantilever
deflection data are collected directly.
Microfabricated cantilevers exert a force on the substrate
surface and, as one might anticipate, the resolution of the
technique depends on the degree of force employed. The
greater the force between the probe and the surface, the more
sensitive the probe is to surface variations. On the other hand,
the greater the force, the more the probe will disturb the
surface. Sample damage thus becomes an issue.
CACF
0001-7690 # 2001 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
Yu. G. Kuznetsov and others
Fig. 1. A schematic diagram of an atomic force microscope used for imaging viruses in liquids. AFM fluid cells, sealed by O
rings, are probed by a silicon nitride, ultra-sharp stylus, which moves in a raster manner over the surface of specified objects. A
reflected laser beam amplifies and reports deflections of the stylus, attached to a cantilever, to a split photodiode.
Problems arising in imaging in contact mode from unfavourable probe–surface interactions, particularly lateral force,
have been overcome to some extent by the development of
what are known as tapping mode instruments (Hansma et al.,
1994). In ‘ tapping ’ mode, the probe tip is not in continuous
contact with the surface (referred to as contact mode), but
oscillates rapidly up and down as it is scanned over the surface,
essentially tapping its way gently, as with a blind man’s cane,
but firmly and rapidly, and sensing the height of features it
encounters.
In tapping mode, the feedback mechanism adjusts, through
the piezoelectric positioner, the vertical height of the sample
surface in order to keep the amplitude of the freely oscillating
probe constant. Tapping mode minimizes the contact between
the cantilever tip and the sample surface and it greatly reduces
lateral forces. As with contact mode, cantilevers made of
silicon nitride, which have a low spring constant, are used for
tapping mode operation in liquids. For some specimens, it is
preferable to examine virus samples dried in air. For tapping
mode imaging of air-dried samples, a stiffer cantilever made of
silicon is used, which has a high spring constant of about
50 N\m.
This tapping mode approach has proven to be a significant
blessing to biological researchers, as it has allowed the
characterization of samples that would otherwise be too soft or
too fragile to withstand contact mode examination. Operating
in a liquid environment presents some complications due to
fluid dynamics, but these are not severe. A constraint that
sometimes presents obstacles is that the specimen under study
must be fixed to, or made to adhere firmly to, the substrate
surface of the fluid cell, which may be glass, cleaved mica,
plastic or any other hard material. To achieve this, it may be
necessary to treat the substrate with various reagents in order
to induce better adhesion of samples. If this condition is not
CACG
met, the specimen will move due to interaction with the probe
and no useful information will be gathered.
AFM can be applied to scan fields ranging in size from less
than 20 nm up to about 150 µm and with a spatial resolution
on soft biological materials, in the best of cases, of about 2 to
3 nm, with a height resolution as great as 0n5 nm. Its
application extends over the range lying between individual
macromolecules, which are accessible by X-ray crystallography, macromolecular assemblies, amenable to EM, and living
cells, which can be seen using light microscopy (Allen et al.,
1997 ; Bustamante & Keller, 1995). Because visualization is
carried out in a fluid environment, specimens suffer no
dehydration, as is generally the case with EM, and they require
no fixing or staining. Indeed, specimens can be observed over
long periods as long as they stay relatively immobilized. For
the most part, specimens seem to be oblivious to the presence
of the probe tip.
The measurement of particle size and the dimensions of
features of individual particles must be treated with some care.
Lateral sizes of individual particles adsorbed onto mica appear
considerably larger than might be expected because the image
obtained is the convolution of the AFM tip shape with that of
the particle. That is, the tip is not infinitely sharp and it has
finite width. Thus, the curved surface immediately adjacent to
the absolute tip causes vertical displacement of the cantilever
and, therefore, gives rise to edges in the image before, as well
as after, the absolute tip encounters the object. This does not,
however, affect the total vertical displacement of the cantilever.
As a consequence, single objects visualized by AFM appear
broader than their true dimensions but yield an accurate and
precise vertical dimension. For roughly spherical particles, such
as icosahedral viruses, although their diameter appears greater
than is in fact the case, the vertical height of the particles gives
a remarkably accurate value for their true diameter.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
Review : Atomic force microscopy
Fig. 2. (a) STMV from solution has been adsorbed onto mica and visualized as a field of 17 nm diameter particles. (b) Surface
of a cubic STMV crystal. (c, d) Small areas on the surface of an orthorhombic STMV crystal, where only the barest indications of
capsid substructure are visible.
Many viruses with dimensions ranging from 17 to 150 nm
whose capsid sizes were accurately established by EM, light
scattering or X-ray crystallography have now been examined.
Images of these particles consistently showed them to have
lateral dimensions of about 2n5 times their actual diameter,
which serves as a reasonable rule of thumb. In all cases,
however, heights measured by AFM were accurate to within a
few per cent. Therefore, precise free particle diameters must be
based on vertical measurements.
In the case of virus particle crystals, the situation is
different. With virus crystals, each particle is embedded in a
lattice composed of similar members. As the AFM tip passes
over the surface, the tip never approaches the ‘ bottom ’ of a
single virion (i.e. a surface equivalent to the flat mica substrate)
before it encounters a neighbouring virion. Thus, the height of
the particle in crystalline form does not give a valid measure of
its true diameter. However, it is a simple matter to measure the
centre-to-centre distances of the virus particles in the lattice
and, because the virions are, in general, closely packed, these
distances do yield precise diameters of better than a few per
cent. Fourier transformation of the lattice arrays (Kuznetsov et
al., 1997) can improve these values.
More distinct images of virus particles can generally be
obtained from virus crystals, rather than from single, free
particles adsorbed onto mica or glass. This is not due to any
averaging process or Fourier filtering, but seems to be an
inherent property of the technique. Presumably, the immobilization of particles and their physical stability is responsible
for the quality of the crystal’s images. When crystals cannot be
obtained, slowly drying the virus particles onto the mica
substrate may induce them to form aggregates and clusters,
often ordered into closely packed, two-dimensional para-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
CACH
Yu. G. Kuznetsov and others
crystals. These sometimes serve nearly as well as crystals. In
these semi-ordered arrays, the centre-to-centre distance of
particles also provides a reasonably accurate estimate of
diameters.
Examples of AFM images of viruses
Fig. 2 (a) is an image obtained when the smallest of the
spherical viruses, satellite tobacco mosaic virus (STMV), is
simply adsorbed onto fresh cleaved mica and imaged in the
fluid cell under buffer. The field is filled with more or less
spherical particles, each of 17 nm diameter based on height
measurements. These correlate exactly to those known from Xray diffraction analysis (Larson et al., 1998 ; Ban et al., 1995) and
quasi-elastic light scattering studies (Malkin et al., 1993). In Fig.
2 (b), swollen STMV virions are visualized in cubic STMV
crystals and, in Fig. 2 (c), normal particles in the orthorhombic
crystal lattice (Koszelak et al., 1989) can be seen. In both of
these lattices, individual virus particles are readily discriminated
and a hexagonal outline, characteristic of icosahedrons in
projection, is evident. Centre-to-centre distances of particles in
the two lattices are 18 and 17 nm, respectively, in agreement
with data from X-ray crystallography. Fig. 2 (d) is a highmagnification AFM image of STMV in the orthorhombic
lattice. While some suggestions of substructure are evident on
the 17 nm particle surfaces, no recognizable detail of the
icosahedral architecture is present.
A T l 3 plant virus, brome mosaic virus (BMV), is seen in
Fig. 3 (a, b) as individual virus particles adsorbed onto mica
(Bancroft & Horne, 1977). Virions adhere well to the substrate
Fig. 3. (a, b) BMV adsorbed onto a freshly cleaved mica substrate under buffer. Capsomeres are evident on the surfaces of
free particles. (c) AFM of BMV crystals permits the visualization of capsid structure. Capsomeres and even depressions at their
centres are evident, reflecting the toroidal clusters of coat protein subunits. (d) T l 1 icosahedral particles of 17 to 18 nm
diameter, derived after CaCl2 treatment of native T l 3 BMV, can be crystallized and visualized by AFM. Even on these very
small particles, pentameric capsomeres derived from the native T l 3 virus are recognizable on the T l 1 surface.
CACI
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
Review : Atomic force microscopy
Fig. 4. TYMV particles embedded in the surface of a virus crystal exhibit an icosahedral array of capsomeres ; at higher
magnification, some even present pentagonal and hexagonal shapes.
Fig. 5. (a, b) Individual virions of CaMV on a mica substrate. Even at this magnification, some capsid substructure is seen.
(c, d) At higher magnification, the capsomeres on the virus surface are apparent.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
CACJ
Yu. G. Kuznetsov and others
Fig. 6. (a) Damaged virions of CaMV are adsorbed onto cleaved mica in physiological media. Loops of double-stranded DNA
can be seen emerging, spreading onto the substrate from most of the particles. (b) TMV helices are seen unravelling at one or
both ends and loops of single-stranded RNA complete with protein subunits are seen displayed on the mica. (c) Degraded
virions of MoMLV are seen to release closed circular strands of RNA onto the mica substrate.
and are stable to repeated scanning. The measured heights, and
therefore the diameters of the BMV virions, are 28 nm,
consistent with data from X-ray crystallography (Lucas et al.,
2001), but the lateral diameter in the AFM images is about
70 nm, or about 2n5 times the correct value. Even on free virus
particles, substructure is evident and capsomeres consisting of
either five or six protein capsid subunits can be seen. Further
inspection indicates the presence of a depression or hole at the
centres of the capsomeres. BMV particles incorporated into the
lattice of an orthorhombic form of their crystals can be seen in
Fig. 3 (c). Here, the 28 nm centre-to-centre distances agree with
height estimates and with data from other techniques. The
capsomeres on the virion surfaces are again well-resolved and
the depressions at their centres visible.
If BMV native particles of T l 3 are exposed to CaCl ,
#
they dissociate into capsid protein. The protein can then, upon
removal of the CaCl , be induced to reassociate into T l 1
#
empty capsids (Yamazaki & Kaesberg, 1963 ; Zhao et al.,
1995 ; Cuillel et al., 1981). Fig. 3 (d) is an AFM image of the
surface lattice of their tetragonal crystals (Lucas et al., 2001).
Heights and inter-particle distances are in agreement with the
18 to 19 nm diameters deduced from X-ray crystallography.
Unlike STMV, large, pentameric capsomeres on T l 3 BMV
protrude from the surfaces of the smaller T l 1 particles (Fig.
3 a–c).
Fig. 4 shows a small area on the surface of a turnip yellow
mosaic virus (TYMV) crystal (Malkin et al., 1999 ; Hirth &
Givord, 1988). Groups of six crystallographically related T l
3 particles of 28 nm diameter gather around large, open
channels with widths that are roughly equal to a virion
diameter, presumably filled with solvent. Even at this resolution, each of the virions appears as a roughly spherical
‘ cluster of grapes ’. In many cases, individual grapes or
substructures in the clusters can be seen to exhibit hexagonal
CADA
or pentagonal outlines characteristic of icosahedral capsomeres.
The capsomeres are well-resolved from one another on the top
surfaces of the virions and have dimensions consistent with
those obtained from X-ray crystallography, about 5 to 6 nm
(Canady et al., 1996).
Comparison of the capsomeres of TYMV and the overall
architecture of the virion with that of BMV, also T l 3 but
from another virus family (Tymovirus versus Bromovirus),
shows them to be strikingly different. Whereas the capsomeres
for BMV are wide and have crown-like structures with
depressions at their centres, those for TYMV are closed and
dense. Hence, it is possible, using AFM, to discriminate the two
virus families on the basis of capsid structure.
Fig. 5 (a) shows a larger, spherical plant virus, cauliflower
mosaic virus (CaMV), adsorbed onto mica. Height measurements suggest its diameter to be 48 nm, though its width in the
AFM images is about 100 nm. X-ray crystallography indicates
it to be 52 nm (Gong et al., 1990). With AFM, even at a
relatively low magnification, quilting on the virion surface
suggests the emergence of substructure. At higher magnifications, the underlying architecture begins to develop and
individual, large capsomeric units become visible on the virion
surface (Fig. 5 b–d). These have centre-to-centre distances of 10
to 12 nm and are organized into hexameric patterns.
As virus particles become larger, their properties become
more mechanically fragile. Particles are more easily distorted
from spherical form and, in some cases, breakage or rupture of
the particles is seen. AFM may be useful in assessing the
damage caused by various procedures. As seen in Fig.
6 (a), DNA can be seen leaking from CaMV virions and
spreading onto the substrate. In Fig. 6 (b), long strands of RNA
still wrapped with subunits of protein can be seen extending
from the ends of tobacco mosaic virus (TMV). In addition, it is
evident that most TMV particles in sample preparations are
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
Review : Atomic force microscopy
Fig. 7. When virus particles are clustered into two-dimensional arrays on the mica substrate, they are more firmly immobilized
and, therefore, yield better AFM images. The hexagonal arrangement seen here is of the insect virus TIV. Although little capsid
structure is visible, the polygonal architecture of the capsid is evident.
Fig. 8. (a) A herpesvirus particle adsorbed onto freshly cleaved mica. The virus is still enveloped and rough indications of
substructure on the surface of the capsid are detected by the AFM probe through the lipid membrane. (b) A herpesvirus virion
treated with detergent to reveal the underlying polygonal capsid. The virion is damaged and flattened somewhat on the
substrate, yielding a good surface for AFM imaging. Capsomeres are clearly evident, as are axes of pentagonal and hexagonal
symmetry. Depressions at the centres of some capsomeres are also visible in some cases.
less than full-length and therefore unlikely to be infectious. In
Fig. 6 (c), circular RNA molecules originating from ruptured
virions of Moloney murine leukaemia virus (MoMLV) can be
seen (Lowey, 1985).
The largest virus to be investigated by AFM is the insect
virus tipula iridescent virus (TIV). It is known from EM to have
a diameter of about 120 nm and a complicated capsid
architecture enveloped by a membrane (Williams, 1998). If the
virus is dried from water onto mica, it forms regular hexagonal
arrays that have some degree of crystalline order. An array of
air-dried TIV is shown in Fig. 7. Though not properly
crystalline, the constraints introduced by association into an
array make it possible to measure centre-to-centre distances
that agree well with height measurements and dimensions
determined by other methods. Although the capsid structure is
not clear on these dried virions, probably because of the
membranes, the virus particles do exhibit distinctive polygonal
forms with large triangular faces.
Herpesvirus is an enveloped virus of about 100 nm with a
lipoprotein membrane surrounding a protein capsid (Kaper,
1975 ; Roizman & Batterson, 1985 ; Wagner & Hewlett, 1999 a,
b). Enveloped viruses adsorb well to a mica surface, but the
protein capsid substructure is veiled by the membrane and
obscured. If the membrane is removed by detergent, the
protein capsids frequently fail to adhere. In Fig. 8 (a), the
protein capsid can be seen through the membrane of an
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
CADB
Yu. G. Kuznetsov and others
Fig. 9. (a) A mass of rod-shaped TMV
virions adsorbed onto mica. (b, c) Some
representative cylindrical particles seen at
higher magnification. Most are not full
virions, but are sections broken from the
helical virus. (c) The 23 nm repeat is
barely discernible on the middle virion.
enveloped herpesvirus virion. Despite the membrane, some
substructure does emerge beneath the shroud as ordered units
on the capsid surface. Herpesvirus treated with detergent and
dried onto a mica substrate is seen in Fig. 8 (b). Here, the capsid
structure is clearly evident and even depressions are visible at
the centres of some capsomeres.
Viruses do not need to be icosahedral to be studied by
AFM. The classic rod-shaped TMV is, for example, seen in Fig.
9. Infectious virions are known from EM and X-ray diffraction
(Bloomer & Butler, 1986) to have lengths of about 300 nm,
cylindrical diameters of 18 nm and a helical pitch of 23 nm.
Many of the virus particles in Fig. 9 are not full-length, but are
fragments of TMV. At the same time, anomalously long TMV
particles, several times the normal length, can also be seen in
abundance, including some particles in Fig. 9. Height measurements of these segments consistently give a value of 18 nm,
which corresponds well to the known cylindrical diameter.
CADC
Apparent widths in the AFM images, however, are again about
2n5 times that value, or about 45 nm. The repeat along the
helical axis cannot be resolved unambiguously with AFM,
though there are fleeting and suggestive periodicities in some
images.
Some preliminary conclusions
AFM provides a useful approach for evaluating the
dimensions, general architecture, capsomere arrangement and,
in some cases, even the detailed capsid structure of viruses.
Because it can be applied under physiological conditions, it
captures the particles in a natural state without the distortions
imposed by drying, fixing or staining. Even on individual,
isolated virus particles, in the best of cases, capsomere structure
can be resolved and underlying motifs identified. At this time,
the method does not allow delineation of the protein subunit
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
Review : Atomic force microscopy
distribution within the capsomeres. Images suggest that viruses
can be better visualized when restrained as a member of an
ordered array, e.g. a crystal, but some advantage is gained even
when the particles are only members of semi-ordered masses,
e.g. a two-dimensional aggregate or a paracrystalline array.
Heights of single particles on mica and centre-to-centre
distances of particles in ordered arrays are the most reliable
measures of a virion’s dimensions. If these are used, shapes and
dimensions obtained by AFM agree well, to within a few per
cent, with results based on X-ray structure determination. The
size of the virus particle does not appear to be a limitation in
terms of resolving capsid detail. That is, 17 nm virus capsids, as
seen here for BMV T l 1 particles, and 28 nm viruses, such as
TYMV, yield as much detail as 50 or 100 nm viruses. The
favourable increase in virion curvature with increase in
diameter is probably compensated for negatively by increasing
virion deformity. Even membrane-encapsidated virions yield
some information regarding the core or protein capsid
substructure.
Unrelated T l 3 plant viruses, such as TYMV and BMV,
can be discriminated from one another on the basis of shape,
size and capsomere structure, even with current technology.
Resolution of very closely related viruses, such as cowpea
chlorotic mottle virus and BMV, would probably not be
possible at this time. Nonetheless, AFM can give some useful
information regarding the presence or distribution of different
viruses in a sample. As AFM technology progresses and better,
more acute tips are developed (Woolley et al., 2000), the
technique will become increasingly powerful and may begin to
challenge and supplement low-resolution X-ray crystallography.
The authors wish to thank Professor Edward Wagner for samples of
herpesvirus, Professor Hung Fan for Moloney murine leukaemia virus
samples and Professor Brian Federici for tipula iridescent virus. This
research was supported by grants from the NIH and contracts from
NASA.
Bustamante, C. & Keller, D. (1995). Scanning force microscopy in
biology. Physics Today 48, 32–38.
Canady, M. A., Larson, S. B., Day, J. & McPherson, A. (1996). Crystal
structure of turnip yellow mosaic virus. Nature Structural Biology 3,
771–781.
Caspar, D. L. D. & Klug, A. (1962). Physical principles in the
construction of regular viruses. Cold Spring Harbor Symposia on
Quantitative Biology 27, 1–24.
Cuillel, M., Jacrot, B. & Zulauf, M. (1981). A T l 1 capsid formed by
the protein of brome mosaic virus in the presence of trypsin. Virology
110, 63–72.
Finch, J. T. & Holmes, K. C. (1967). Methods in Virology III, 351–474.
Gong, Z. X., Wu, H., Cheng, R. H., Hull, R. & Rossmann, M. G. (1990).
Crystallization of cauliflower mosaic virus. Virology 179, 941–945.
Hansma, P. K., Cleveland, J. P., Radmacher, M., Walters, D. A., Hillner,
P. E., Bezanilla, M., Fritz, M., Vie, D., Hansma, H. G., Prater, C. B.,
Massie, J., Fukunage, L., Gurley, J. & Elings, V. (1994). Tapping mode
atomic force microscopy in liquids. Applied Physics Letters 64, 1738–1740.
Harrison, S. C. (1990). Principles of virus structure. In Fields Virology,
2nd edn, pp. 37–61. Edited by B. N. Fields & D. M. Knipe. New York :
Raven Press.
Hirth, L. & Givord, L. (1988). Tymoviruses. In The Plant Viruses,
pp. 163–212. Edited by R. Koenig. New York : Plenum Press.
Horne, R. W. & Wildy, P. (1961). Symmetry in virus architecture.
Virology 15, 348–373.
Horne, R. W. & Wildy, P. (1963). Virus structure revealed by negative
staining. Advances in Virus Research 10, 101–170.
Kaper, J. M. (1975). The chemical basis of virus structure, dissociation
and reassembly. In The Frontiers of Biology Series, pp. 74–75. Edited by
A. Neuberger & E. L. Tatum. Amsterdam : North-Holland.
Koszelak, S., Dodds, J. A. & McPherson, A. (1989). Preliminary analysis
of crystals of satellite tobacco mosaic virus. Journal of Molecular Biology
209, 323–325.
Kuznetsov, Yu. G., Malkin, A. J., Land, T. A., DeYoreo, J. J., Barba de la
Rosa, A. P., Konnert, J. & McPherson, A. (1997). Molecular resolution
Allen, S., Davies, M. C., Roberts, C. J., Tendler, S. J. B. & Williams,
P. M. (1997). Atomic force microscopy in analytical biotechnology.
imaging of macromolecular crystals by atomic force microscopy.
Biophysical Journal 72, 2357–2364.
Larson, S. B., Day, J., Greenwood, A. & McPherson, A. (1998). Refined
structure of satellite tobacco mosaic virus at 1n8 A/ resolution. Journal of
Molecular Biology 277, 37–59.
Lowey, D. R. (1985). Transformation and oncogenesis : the retroviruses.
In Fields Virology, 1st edn, pp. 235–263. Edited by B. N. Fields & D. M.
Knipe. New York : Raven Press.
Trends in Biotechnology 15, 101–105.
Lucas, R. W., Kuznetsov, Yu. G., Larson, S. B. & McPherson, A. (2001).
References
Baker, T. S., Olson, N. H. & Fuller, S. D. (1999). Adding the third
dimension to virus life cycles : three-dimensional reconstruction of
icosahedral viruses from cryo-electron micrographs. Microbiology and
Molecular Biology Reviews 63, 862–922.
Ban, N., Larson, S. B. & McPherson, A. (1995). Structural comparison
of the plant satellite viruses. Virology 214, 571–583.
Bancroft, J. B. & Horne, R. W. (1977). Bromovirus (brome mosaic virus)
group. In Atlas of Insect and Plant Viruses, vol. 8, pp. 287–291. Edited by
K. Maramorosch. New York : Academic Press.
Binnig, G., Quate, C. F. & Gerber, C. (1986). Atomic force microscope.
Physical Review Letters 56, 930–933.
Bloomer, A. C. & Butler, P. J. G. (1986). Tobacco mosaic virus : structure
and self-assembly. In The Plant Viruses, pp. 19–52. Edited by M. H. V. van
Regenmortel & H. Fraenkel-Courat. New York : Plenum Press.
Crystallization of brome mosaic virus (BMV) and T l 1 BMV particles
following a structural transition. Virology (submitted).
McPherson, A. (1998). Crystallization of Biological Macromolecules,
p. 586. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory.
Malkin, A. J., Cheung, J. & McPherson, A. (1993). Crystallization of
satellite tobacco mosaic virus. I. Nucleation phenomena. Journal of Crystal
Growth 126, 544–554.
Malkin, A. J., Kuznetsov, Yu. G., Lucas, R. W. & McPherson, A. (1999).
Surface processes in the crystallization of turnip yellow mosaic virus
visualized by atomic force microscopy. Journal of Structural Biology 127,
35–43.
Roizman, B. & Batterson, W. (1985). Herpesvirus and their replication.
In Fields Virology, 2nd edn, pp. 497–526. Edited by B. N. Fields & D. M.
Knipe. New York : Raven Press.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13
CADD
Yu. G. Kuznetsov and others
Wagner, E. K. & Hewlett, M. D. (1999 a). Basic Virology, pp. 337–358.
London : Blackwell Science.
virus with calcium chloride and the isolation of its protein and nucleic
acid. Journal of Molecular Biology 7, 760–762.
Wagner, E. K. & Hewlett, M. D. (1999 b). Basic Virology, p. 127. London :
Zhao, X., Fox, J. M., Olson, N. H., Baker, T. S. & Young, M. J. (1995).
Blackwell Science.
In vitro assembly of cowpea chlorotic mottle virus from coat protein
expressed in Escherichia coli and in vitro-transcribed viral cDNA. Virology
207, 486–494.
Williams, T. (1998). Invertebrate iridescent viruses. In The Insect Viruses,
pp. 31–68. Edited by L. K. Miller & L. A. Ball. New York : Plenum Press.
Woolley, A. T., Cheung, C. L., Hafner, J. H. & Lieber, C. M. (2000).
Structural biology with carbon nanotube AFM probes. Chemistry &
Biology 7, 193–204.
Yamazaki, H. & Kaesberg, P. (1963). Degradation of brome mosaic
CADE
Published ahead of print (1 June 2001) in JGV Direct as
DOI 10.1099/vir.0.17690-0
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 17:30:13