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Journal of Virology, October 2001, p. 9543-9548, Vol. 75, No. 19
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.19.9543-9548.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Analysis of Mason-Pfizer Monkey Virus Gag Particles
by Scanning Transmission Electron Microscopy
Scott D.
Parker,1
Joseph S.
Wall,2 and
Eric
Hunter3,*
Department of Medicine, Division of
Infectious Diseases,1 and Department of
Microbiology,3 University of Alabama at
Birmingham, Birmingham, Alabama 35294, and Department of
Biology, Brookhaven National Laboratories, Upton, New York
119732
Received 6 April 2001/Accepted 29 June 2001
 |
ABSTRACT |
Mason-Pfizer monkey virus immature capsids selected from the
cytoplasm of baculovirus-infected cells were imaged by scanning transmission electron microscopy. The masses of individual selected Gag
particles were measured, and the average mass corresponded to 1,900 to
2,100 Gag polyproteins per particle. A large variation in Gag particle
mass was observed within each population measured.
| |
TEXT |
The retroviral assembly
process begins within the infected cell as retroviral Gag polyproteins
associate with one another to enclose the RNA viral genome within a
spherical particle (an immature capsid) with an electron-dense rim and
a lucent center (19). Despite extensive study, the fine
structure of an immature retroviral particle and the nature, of the
molecular interactions that drive particle assembly remain unresolved.
While the structures for the matrix and capsid proteins have been
determined for several different retroviruses (5, 10, 11, 13, 14,
16, 17, 23), the structure of the complete Gag polyprotein
precursor remains unknown. A hexagonal lattice of Gag polyproteins
beneath the plasma membrane has been visualized for human
immunodeficiency virus (HIV), and a similar arrangement was recently
observed for Mason-Pfizer monkey virus (M-PMV) Gag particles assembled
in bacteria (M. V. Nermut, P. Bron, D. Thomas, M. Rumlova, T. Ruml, and E. Hunter, submitted for publication), suggesting that the
spherical retroviral immature capsid assembles in a manner that
involves at least local symmetry (1, 2, 18-20). However,
high-resolution cryo-electron microscopy (cryo-EM) images of both
murine leukemia virus and HIV type 1 immature particles have failed to
identify icosahedral symmetry within immature retroviral particles.
While these particles were found to be ordered with radial symmetry and
distinct layers, they were heterogeneous in size (9, 32). In addition, measurements of the masses of individual Rous sarcoma virus (RSV) virions by quantitative dark-field scanning transmission electron microscopy (STEM) have demonstrated a significant standard deviation in viral mass (30, 33). Taken together, these
views of immature retroviral particles without a well-defined size or mass are inconsistent with a well-ordered icosahedral structure and
suggest an alternative assembly mechanism.
In order to define the mass and population diversity of immature
capsids of a retrovirus that assembles in the cytoplasm without a
requirement for membranes, we analyzed M-PMV immature Gag particles by
STEM. M-PMV Gag particles, which do not include viral membranes or
surface glycoproteins, can be extracted by detergent cell lysis and
purified by a series of sucrose gradients. The mass measurements generated by STEM demonstrate that M-PMV particles are heterogeneous in mass.
Isolated M-PMV Gag particles contain predominantly Gag and Gag-Pro
polypeptides.
A recombinant baculovirus expressing the M-PMV
gag, pro, and pol reading frames, with an A18V
point mutation in the matrix domain of the corresponding Gag
polyprotein, has been described previously (22).
Spodoptera frugiperda (Sf9) cells were grown at 27°C in
suspension in Grace's supplemented insect medium in a 1-liter spinner
flask to a density of 0.5 × 106 to 1.0 × 106 cells per ml. At 60 h postinfection with
recombinant baculovirus, the cells were harvested, concentrated by
centrifugation at 100 × g, and resuspended on ice in
20 ml of hypotonic cell lysis buffer, which consisted of 10 mM Tris (pH
7.6), 1 mM EDTA, 1% Triton X-100 (Sigma-Aldrich Corp., St. Louis,
Mo.), 1 mM phenylmethylsulfonylfluoride, and 2 µg of leupeptin
(Sigma)/ml. After 30 min, the cell lysate was centrifuged at
3,000 × g for 10 min to remove nuclei and cell debris.
The cleared cell lysate was loaded onto a 10-ml cushion of 35%
(wt/vol) sucrose in 10 mM Tris (pH 7.6) buffer on top of 3 ml of 75%
(wt/vol) sucrose in water in an SW28 centrifuge tube (Beckman
Instruments, Fullerton, Calif.). The immature Gag particles were
separated from the cell lysate by centrifugation at 25,000 rpm in an
SW28 rotor (Beckman) for 2 h at 4°C and removed from the 35%
sucrose-75% sucrose interface, followed by dialysis against a
10 mM Tris (pH 7.6) buffer at 4°C to remove the sucrose. The dialyzed
sample was concentrated by centrifugation at 20,800 × g in a refrigerated microcentrifuge for 30 min, and the
pellet was resuspended in 100 µl of cell lysis buffer with 500 mM
NaCl. The resuspended Gag particles were further purified by
centrifugation through a 5 to 20% (wt/vol) sucrose velocity gradient
as described previously (22). Gradient fractions (0.5 ml
each) were removed from the top of the gradient by a piston gradient
fractionator (BioComp Industries, New Brunswick, Canada). A volume
consisting of 5 µl (1%) of each gradient fraction and a resuspension
of the gradient pellet was analyzed by polyacrylamide gel
electrophoresis, and the gel was stained with Coomassie blue as
described previously (27) (Fig.
1A). In Fig. 1, bands that correspond to
Pr78Gag and Pr95Gag-Pro, as well as two smaller
bands, are identified in the gel. Both smaller bands (Fig. 1B) are
M-PMV Gag products, since they are found in a Western blot probed with
rabbit anti-M-PMV Gag polyclonal serum. Moreover, trypsin digestion of
each band after removal from an sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) gel generated several peptide fragments
with a mass equal to that predicted for a tryptic digestion of the
middle and the C-terminal regions of the Pr78Gag
polyprotein, when measured by matrix-assisted laser desorption ionization-time of flight mass spectroscopy (data not shown). The
smaller bands were not distinctly visible when 35S-labeled
cell lysates containing assembled Gag particles were run directly on
sucrose velocity gradients (without first being pelleted through a
sucrose cushion; data not shown) and thus may represent Pr95 and/or
Pr78 cleavage by cellular proteases after assembly and during the
process of particle preparation for STEM. It is possible, but less
likely, that the 50- and 54-kDa Gag proteins are present in the cell
and are included with full-length Gag polyproteins during the particle
assembly events. If so, an adjustment would be required in the
calculation of the number of Gag polyproteins per particle, which is
discussed below. As described previously (22), a
significant portion of the Gag polyproteins are found in the gradient
pellet, due to aggregation and/or to a persistent association with
cellular elements. The Gag polyproteins distributed through the middle
fractions of the gradient are those particles separated from the
cytosol which were used for STEM imaging.
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FIG. 1.
SDS-PAGE of velocity gradient fractions during the
purification of M-PMV Gag particles. (A) Pr78Gag and
Pr95Gag-Pro are shown in a Coomassie blue-stained gel with
each fraction labeled from top (fraction 1) to bottom (fraction 23).
The results for the resuspension of the pellet at the bottom of the
tube is also shown (lane labeled "pellet"). (B) Western blot of an
SDS-PAGE gel (using one gradient fraction containing Gag particles)
with anti-Gag antiserum demonstrates that all major bands in the gel
are Gag-related products.
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M-PMV Gag particle mass.
For STEM analysis, each fraction from
the velocity gradient was dialyzed and concentrated as described above,
with a final resuspension in 100 µl of 10 mM Tris buffer (pH 7.6).
For each gradient sample (fractions 8 through 18), a set of STEM
digital images were made (30 to 35 images per sample), using methods
previously defined at the Brookhaven National Laboratory STEM facility
(31). Purified Gag particles were freeze-dried with
tobacco mosaic virus (TMV) particles (as an internal control) onto a
thin (2 to 3-nm) carbon film and imaged by a 40-keV electron beam on a
low-temperature stage (
150°C). In this process, the electron beam
is focused to 0.25 nm at a series of points (pixels) within a 512- by
512-nm grid, with each pixel center separated from the next by 1 nm. At
each pixel, the scattered electrons at large (40- to
200-milliradian) and at small (15- to 40-milliradian) angles are
measured by annular dark-field detectors. The number of electrons
scattered at any one point within the sample is proportional to the
mass, and the value is multiplied by an established mass coefficient to
determine the mass at each point on the grid. The dose used in imaging
is less than 500 electrons/nm2, and the loss of mass within
the sample during imaging is less than 1%. The mass values are used to
generate a digital image, and the summation of the mass values for an
entire particle, after subtraction of an averaged background
value, yields the mass of each individual Gag particle. TMV particles
have a constant known value for mass per unit length, and the
experimental measurement of this value within each sample ensures that
the mass measurements are accurate.
An automated selection routine was used to compare the particles in the
digital images with a theoretical model of a Gag particle:
a hollow
sphere with internal and external diameters of 640 and
900 Å,
respectively. Particles that fitted the model and also
satisfied
requirements for spherical symmetry and low backgrounds
relative to
particle mass were selected for mass measurements.
Two dark-field
images containing M-PMV Gag particles are shown
in Fig.
2. By conventional thin-section electron
microscopy, intracellular
M-PMV Gag particles appear as a dense ring
with a relatively hollow
core. While most of the particles selected for
mass measurement
have similar appearances ("empty" particles), some
have additional
mass within the center, with a higher average mass
value ("full"
particles). The full particles were found to be a
minor component
(16% of all particles) in the middle fractions of the
velocity
gradient (fractions 8 through 12), where most of the Gag
particles
are found, while in the fractions associated with a higher S
value
(fractions 13 through 18), up to 50% of selected particles had
a
significant central density (data not shown). The full particles
are
inconsistent with viral particles imaged by thin-section electron
microscopy in other expression systems (
15,
24,
26) and,
because they segregate into the lower fractions of the velocity
gradient that are associated with a higher S value, may represent
the
introduction of additional material into the center of the
particle
during assembly rather than artifacts of particle preparation
for
imaging. Thus, particles taken from the lower fractions (fractions
13 through 18) of the velocity gradients were not used for mass
analysis.
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FIG. 2.
STEM images. Two representative dark-field STEM images
that depict the complete Gag particles (full and empty) and damaged or
incomplete particles found during image analysis are shown. TMV is also
shown as an internal control for mass measurements.
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In two independent preparations with clean backgrounds and a large
number of particles available for analysis (388 Gag particles
in the
first sample and 348 in the second sample), the average
mass for all
particles selected for mass measurement ranged from
164 to 174 MDa,
with a standard deviation in mass of 20 to 21
MDa. A histogram of
particle mass measurements from each of these
samples demonstrates the
considerable diversity in particle preparation
(Fig.
3A). The difference in mass between empty
and full particles
taken from all fractions of the velocity gradient is
also shown
(Fig.
3B). Mass-per-unit-length measurements for the TMV
standards
(13.4 ± 0.8 and 13.6 ± 0.9 kDa/Å) were within
5% of the predicted
value (13.1 kDa/Å) for both sets of samples.
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FIG. 3.
Distribution of particle mass. (A) Particle frequency is
plotted versus particle mass (in megadaltons) for all particles used
for mass measurements from gradient fractions 8 through 12 from two
independent samples. These data were used in the calculation of the
average mass for M-PMV Gag particles. (B) Particle frequency is plotted
as a function of particle mass for both empty and full particles found
within velocity gradient fractions 8 through 18 from sample 1.
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Protein and RNA content of M-PMV Gag particles.
In order to
estimate the number of Gag and Gag-Pro polyprotein molecules that
participate in a particle assembly event, the protein and RNA contents
were measured in each of the velocity gradient fractions used for STEM
imaging. The RNA content for each sample of Gag particles was extracted
from particle lysates with RNA columns, using a QIAamp viral RNA kit
(Qiagen, Valencia, Calif.). The viral RNA was quantified by UV
absorption at 260 nm, with a conversion factor of 40 µg per ml per
optical density unit, using a Beckman DU 530 spectrophotometer. The
protein content of the same samples was estimated by a modified Lowry
assay using a DC protein assay kit (Bio-Rad Laboratories, Hercules,
Calif.) after establishing an approximate absorbance-to-concentration curve with purified bovine serum albumin. The average Gag/RNA mass
ratio was 30.1:1, which yielded a calculated average genomic RNA
content of 3.2%, ranging from 2.1 to 4.2% (data not shown).
In order to calculate the relative amounts of Pr78
Gag and
Pr95
Gag-Pro that were assembled into Gag particles in
insect cells, [
35S]methionine-labeled immature capsids
were separated from cell
lysates directly by velocity gradient
sedimentation and the ratio
was determined by quantitative
phosphorimaging of dried SDS-PAGE
gels (data not shown). The intensity
of each Pr78 and Pr95 band
in several velocity gradient fractions from
independent preparations
was measured and adjusted for a difference in
the number of methionine
residues between Pr78 and Pr95. The protein
content averaged 92%
Pr78 and 8% Pr95, with a small amount (<1%) of
Pr180
Gag-Pro-Pol. Although there are no other significant
protein bands seen in
the samples prepared for STEM, the contents of a
sample of purified
[
35S]methionine-labeled Gag particles
were separated by SDS-PAGE
and quantitatively analyzed by
phosphorimaging. The bands corresponding
to Gag polyproteins
represented 91% of the activity across an
entire lane (data not
shown), and this value was used as the percentage
of Gag relative to
the amount of all proteins within a particle
in order to calculate the
number of Gag polyproteins per
particle.
Using M-PMV particles purified and concentrated for STEM, the mass for
Pr78
Gag and Pr95
Gag-Pro was determined by
matrix-assisted laser desorption ionization-time
of flight mass
spectroscopy. Purified M-PMV Gag particles were
disrupted by mixing
them with 6 M guanidine at a 1:1 ratio and
then boiling for 5 min. The
sample was diluted 1:50 with a matrix
(sinapinic acid [Sigma]
dissolved in acetonitrile and mixed at
a 1/1 ratio with trifluoroacetic
acid), and 1 µl was pipetted
onto a smooth plate. Samples were
analyzed in the positive mode
on a Voyager Elite mass spectrometer with
delayed-extraction technology
(PerSeptive Biosystems, Framingham,
Mass.). The acceleration voltage
was set at 25 kV, and 50 to 100 laser
shots were summed. The mass
spectrometer was calibrated with bovine
serum albumin. The mass
values for Pr78 and Pr95 were 73.0 and 92.5 kDa, respectively.
The smaller Gag-related proteins discussed above
were measured
at 50.1 and 54.4 kDa. After 3% was subtracted from each
averaged
particle mass measurement (above) to account for RNA content,
and after the remaining mass was multiplied by 0.91 to exclude
protein
content unrelated to Gag, the values for the masses of
Pr78
Gag and Pr95
Gag-Pro and their relative
amounts in Gag particles were used to calculate
the number of Gag
and/or Gag-Pro molecules included in a particle
assembly event. These
values ranged from 1,900 for an average
particle mass of 164 MDa to
2,100 for an average particle mass
of 174 MDa, with a standard
deviation of 270 or 280 molecules
per particle,
respectively.
A number of assumptions that could have a small effect on the
calculations have been made in arriving at these figures. When
M-PMV
Gag particles are prepared for STEM imaging, the dominant
portion of
the protein content is composed of Pr78
Gag and
Pr95
Gag-Pro polyproteins. For reasons described above, we
have assumed that
the smaller Gag-related proteins (50 and 54 KDa) seen
in the velocity
gradient fractions are a consequence of
Pr78
Gag and/or Pr95
Gag-Pro cleavage after
particle assembly. However, if these smaller Gag
products arise before
or during particle assembly and are incorporated
into Gag particles as
truncated Gag polyproteins, the calculated
number of Gag polyproteins
per particle increases by a small amount
(40 molecules per particle, or
2% of the total). We have also
presumed that the salt and Triton X-100
detergent used during
particle purification is removed by extensive
washing of the samples
in salt-free Tris buffer prior to STEM imaging.
It is possible,
however, that salts and/or detergent remained
associated with
the Gag particles and thus increased the measured
particle mass.
The genomic RNA content of each sample of M-PMV Gag
particles
was measured after column extraction. The columns used are
designed
for the extraction of genomic RNA and may not efficiently bind
smaller RNAs (less than 200 bp), such as tRNAs, that may represent
up
to one-third of the RNA found in the viral core (
3,
4,
6,
7,
28,
29). Thus, the viral RNA content may be moderately
underestimated and the protein (Gag) content may be slightly
overestimated
in this study. If one-third of the RNA content of the
M-PMV Gag
particles has been lost and an adjusted RNA content of 4.5%
is
used in place of the measured value of 3%, the number of Gag
molecules
per particle is reduced by ~30. In all studies to date, RNA
is
a relatively minor component of the virion, and we feel that the
uncertainty in the mass of small RNAs in the Gag particle represents
a
small error in our
calculations.
In a previous STEM analysis of RSV virions and RSV Gag particles
(
30,
33), the calculated number of Gag polyproteins per
virion was significantly less than that we report here (1,500
and
1,200, respectively, versus ~2,000). In a recent cryo-EM study
of in
vitro-assembled RSV particles, the radii of the immature
particles were
significantly smaller than those observed for HIV,
murine leukemia
virus, or M-PMV (33 versus ~55 nm) (
9,
33;
S. D. Parker et al., unpublished data), suggesting that there
may be
significant differences among the different genera of retroviruses
in
the number of protein subunits used to assemble an immature
Gag
particle.
In both studies, the distribution of particle mass is remarkable in its
breadth. M-PMV Gag particles and enveloped HIV type
1 virus-like
particles, both expressed from baculovirus-insect
cell expression
systems, have been studied and compared with similar
products from
mammalian expression systems by conventional electron
microscopy
(
12,
20,
25; Parker et al., unpublished). Although
no
dramatic differences have been observed between the products
of
different expression systems, the baculovirus system may produce
Gag
particles which are more heterogeneous than those found in
naturally
infected cells. We have assumed that any variations
in particle mass
are due to differences in the numbers of Gag
polyproteins within the
particle and that the RNA-to-protein ratios,
as well as the ratio of
Pr78 to Pr95, remain constant among all
particles measured. These
assumptions are very difficult to address
experimentally, and some of
the variation in particle mass may
be due to differences in RNA content
and/or Pr78/Pr95 ratios.
Nevertheless, high-resolution cryo-EM imaging
of a variety of
different retroviruses, including M-PMV (
9,
32,
33; Parker
et al., unpublished), demonstrates a similarly
considerable diversity
in particle size, suggesting that a variable
number of Gag polyproteins
are included in the assembly process.
Several high-resolution
microscopic studies have demonstrated a
well-ordered arrangement
of Gag domains, with a threefold axis of
symmetry near the surfaces
of both immature Gag particles and mature
virions similar to that
seen with icosahedral viruses (
1,
2,
18-20, Nermut et al.,
submitted). However, it is difficult to
define an assembly model
with a high degree of order in two dimensions
on a local level
but with large variations in particle mass and
size.
There are three possible explanations for the variable number of Gag
polyproteins found within M-PMV Gag particles. The first
possibility is
that the otherwise icosahedral Gag particles have
random defects or
holes as an artifact of assembly or the purification
process. Indeed,
small defects are found around the perimeters
of some M-PMV Gag
particles by STEM imaging. However, while particle
defects may have a
small effect on the variations seen in particle
mass, the selection
criteria by which M-PMV Gag particles are
chosen for STEM imaging make
it unlikely that large defects, in
the absence of other factors, are
present to the degree necessary
to explain the large variation in
particle mass. The second possible
explanation is that Gag particles
have strict icosahedral symmetry
with variable triangulation numbers
and that discrete classes
in particle mass are obscured in the
measurements by defects in
the Gag shell or variable amounts of mass
around a particle that
is unrelated to Gag (detergent or salt).
Particles assembled by
a truncated form of the Ty-1 capsid protein in
Saccharomyces cerevisiae have been shown by cryo-EM
to have icosahedral symmetry but variable
triangulation numbers (T=3
and T=4), and strict icosahedral symmetry
is lost as the length of the
capsid protein is increased (
21).
However, if the
triangulation numbers for M-PMV particles are
restricted to those
allowed in the model described by Forster
et al. (
8), the
large difference in predicted mass between
the classes of particles
allowed in the model should not have
been obscured by an experimental
artifact or relatively small
particle defects. The third possible
explanation is that the Gag
molecules are well ordered in two
dimensions into hexagonal or
fullerene-like arrays but are not well
ordered in the way that
Gag sheets are curved or otherwise organized in
three dimensions
to form a sphere (
8,
9). This last
explanation may be the
most likely and would explain both the negative
stain and freeze-fracture
electron microscopy studies of HIV and M-PMV
particles that have
shown the Gag polyproteins to be well organized on
a local level
in two dimensions (
19,
20) as well as the
variable size and
mass observed for Gag particles by cryo-EM and STEM
(
9,
30,
33).
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ACKNOWLEDGMENTS |
This investigation was supported by U.S. Public Health Service
research grant CA-27834 from the National Institutes of Health. The BNL
STEM is an NIH-supported Resource Center, NIH P41-PR01777, with
additional support provided by DOE, OBER.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Alabama at Birmingham, BBRB 256, 1530 3rd Ave. South, Birmingham, AL 35294. Phone: (205) 934-4321. Fax: (205)
934-1640. E-mail: Ehunter{at}UAB.edu.
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Journal of Virology, October 2001, p. 9543-9548, Vol. 75, No. 19
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.19.9543-9548.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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