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Journal of Virology, March 2001, p. 2753-2764, Vol. 75, No. 6
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.6.2753-2764.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Characterization of Rous Sarcoma Virus Gag
Particles Assembled In Vitro
Fang
Yu,1
Swati
M.
Joshi,1,
Yu May
Ma,1
Richard L.
Kingston,2,
Martha N.
Simon,3 and
Volker M.
Vogt1,*
Department of Molecular Biology and Genetics,
Cornell University, Ithaca, New York 148531;
Department of Biological Sciences, Purdue University, West
Lafayette, Indiana 479072; and Biology
Department, Brookhaven National Laboratory, Upton, New York
119733
Received 17 August 2000/Accepted 19 December 2000
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ABSTRACT |
Purified retrovirus Gag proteins or Gag protein fragments are able
to assemble into virus-like particles (VLPs) in vitro in the presence
of RNA. We have examined the role of nucleic acid and of the NC domain
in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and
have characterized these VLPs using transmission electron microscopy
(TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs
of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient
assembly. The percentages of nucleic acid by mass, in the VLPs varied
from 5 to 8%. The mean mass of VLPs, as determined by STEM, was
6.5 × 107 Da for both RNA-containing and DNA
oligonucleotide-containing particles, corresponding to a stoichiometry
of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the
VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two
domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to
the C-terminal domain of CA was 33 nm, considerably smaller than that
of equivalent human immunodeficiency virus type 1 particles. Deletions
of the distal portion of NC, including the second Zn-binding motif, had
little effect on assembly, but deletions including the charged residues
between the two Zn-binding motifs abrogated assembly. Mutation of the
cysteine and histidine residues in the first Zn-binding motif to
alanine did not affect assembly, but mutation of the basic residues
between the two Zn-binding motifs, or of the basic residues in the
N-terminal portion of NC, abrogated assembly. Together, these findings
establish VLPs as a good model for immature virions and establish a
foundation for dissection of the interactions that lead to assembly.
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INTRODUCTION |
In retroviruses, the Gag polyprotein
directs the assembly and budding of virions from the plasma membrane.
Even in the absence of other viral proteins, expression of Gag leads to
budding of particles resembling real virions. In wild-type viruses, Gag
is cleaved late in the budding process by the viral protease (PR) to
yield the mature proteins MA, CA, and NC that are common to all
retroviruses plus other small proteins or peptides particular to the
retrovirus species. For most retroviruses, including human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and
murine leukemia virus (MuLV), assembly and budding occur concomitantly. These processes are not obligatorily coupled, however, since for the B-
and D-type viruses assembly of immature viral cores takes place in the
cytoplasm, followed by transport of the intact cores to the membrane
and subsequent envelopment and proteolytic maturation. From deletion
analyses, it appears that budding is dependent on the function of three
short amino acid sequences in Gag, sometimes called assembly domains.
The M (membrane binding) sequence, comprising approximately the
N-terminal half of MA with its critical basic amino acid residues and
including the N-terminal myristate group present in most retroviruses,
directs binding and/or transport of Gag to the membrane (59, 61,
74, 78, 85; reviewed in reference 52). The I
(interaction) sequence(s), comprising one or more clusters of basic
residues in NC, somehow promotes tight interactions between Gag
molecules, presumably by virtue of its binding to RNA (11, 18,
24). The L (late) motif, found either between MA and CA or near
the C terminus of Gag, is believed to facilitate the pinching off and
release of the budding virions (2, 21, 36, 40, 60, 63, 64, 79, 80, 81). Although budding of membrane-enveloped particles requires primarily these sequences in Gag, the particles thus produced
may be grossly aberrant in morphology or size unless a functional CA
sequence and the associated C-terminal spacer (SP) sequence are present
(1, 51, 53).
Despite a wealth of data on the molecular genetics, biochemistry, and
structural biology of Gag proteins, the principles of retrovirus
assembly are incompletely understood. For example, the protein-protein
interactions in assembly, the role of RNA in virion structure, the
nature and timing of the morphological change underlying maturation,
and the mechanism of protein-lipid interaction are aspects of assembly
that remain to be clarified. Since budding cannot be synchronized, the
steps in this process are difficult to follow biochemically.
Retroviruses are not icosahedral and are heterogeneous in size
(32, 82), and therefore a conventional three-dimensional
reconstruction of immature or mature virions from cryoelectron
micrographs cannot be done.
Retrovirus-like particles (VLPs) can assemble spontaneously in vitro
from purified Gag protein or fragments of Gag, as studied most
extensively for Mason-Pfizer monkey virus (50), RSV
(14, 15, 46), and HIV-1 (13, 35, 41-44, 58,
77). Assembly of VLPs from Gag protein translated in crude
extracts also has been reported (55, 67, 68, 71). To be
competent to assemble into regular structures, the Gag protein must
contain at least an intact CA sequence. Under most conditions,
productive in vitro assembly also requires NC and RNA, although CA as
an isolated protein can assemble into tubular particles (29, 35,
41, 42, 49, 77). The morphology of the VLPs is either spherical, resembling immature cores in virions lacking an active protease (PR),
or tubular. In the case of HIV-1, conical particles resembling mature
cores also are formed under some conditions (35), but in
the presence of tubular particles. Several factors have been identified
that promote Gag polymerization into one or another macromolecular
shape. In HIV-1, addition of amino acid residues to the N terminus of
CA-NC redirects the assembly outcome from tubes to spheres (43,
77). Deletion of the 14-amino-acid SP1 peptide between CA and NC
also causes this shape change, as does alteration of the pH of the
assembly reaction (44). In RSV, deletion of the p10 domain
from Gag leads to tubular particle formation (15). The
critical sequence element for this effect is the segment of 25 amino
acid residues comprising the C-terminal segment of p10
(46).
Because it is a well-defined system, in vitro assembly promises to be a
useful approach by which to unravel the protein-protein and
protein-nucleic acid interactions of Gag that lead to virion formation.
We have studied several parameters influencing RSV VLP assembly in
vitro, using a soluble RSV Gag protein missing N- and C-terminal
domains. The results showed that the appearance and density of
VLPs are not affected by the length or type of nucleic acid and that
the ratio of protein mass to nucleic acid mass in the purified
particles is constant. The mean mass of the VLPs implies a
stoichiometry of about 1,200 Gag molecules per particle. By
cryoelectron microscopy (cryo-EM), the VLPs, although lacking an
external lipid bilayer, otherwise closely resemble immature particles
formed in vivo. The structure of the Zn-binding motifs can be disrupted
without impact on assembly, and the distal parts of NC are dispensable
for the assembly process.
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MATERIALS AND METHODS |
DNA constructs.
Nucleotide sequences and numbers refer to
the RNA sequence of the Prague C strain of RSV. All plasmids were
constructed by using common subcloning techniques and propagated in
Escherichia coli DH5
. After confirmation by restriction
enzyme digestion and sequence analysis, the plasmids were moved into
E. coli strain BL21 DE3(pLysS) for protein expression. The
expression plasmid encoding the Gag protein 
MBDPR was available
from previous work (15, 46) and is based on the vector
pET3xc (Novagen). The N terminus of this protein begins at amino acid
residue 84 of MA, and the C terminus is the last residue of NC.
The pET3xc.dNC series of plasmids was created using pET3xc.MBDPR
as the starting plasmid. pET3xc.dNC7 was made by replacing the
SacII (nucleotide [nt] 1806 in RSV)-KpnI (nt
4995 in RSV) fragment from pET3xc.MBDPR with two self-annealing
oligonucleotides designed to delete all of NC and the nine amino acids
(SP) between CA and NC from MBDPR. pET3xc.dNC6 was created by
replacing the SacII-KpnI fragment with two
self-annealing oligonucleotides designed to delete all of NC from
MBDPR but to leave the SP peptide. To generate pET3xc.dNC1
through pET3xc.dNC5, PCR was performed using pATV-8K as a
template, a forward primer in CA, and a reverse primer with a
KpnI site that created the desired C terminus of the
protein. The PCR product was digested with SacII and
KpnI and subsequently subcloned into
pET3xc.MBDPR. The mutants dNC2-A to dNC2-E
were created by replacing the SacII-KpnI fragment
of dNC2 with a PCR-derived fragment carrying Ala
substitutions in clusters of basic amino acid residues. In
dNC2-A, all six K or R residues between the two CCHC motifs
were mutated. In dNC2-B and dNC2-C, the three Cys
residues and one His residue in the first CCHC motif were mutated. The
following residues in the proximal segment of NC were mutated in the
other constructs: dNC2-C, R5, R7, R16, and R18;
dNC2-D, R5, R7, and R16; dNC2-E, R5 and R7.
Protein and RNA purification.
E. coli BL21
DE3(pLysS) cells were grown and induced for protein expression as
previously described (14, 15). Purification of protein
GagMBDPR was done using a modification of the method previously
described (15). The frozen bacterial pellet was
resuspended on ice in buffer A (20 mM Tris [pH 7.5], 0.5 M NaCl, 10%
glycerol, 1 mM EDTA, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride) at 25 ml/liter of cell culture, and the cells were broken by
sonication. In some experiments, 0.1% Nonidet P-40 was added to buffer
A. Insoluble debris and nucleic acids were removed by addition of 0.3%
(wt/vol) polyethyleneimine, followed by centrifugation
(19). The protein was precipitated by adding 1 volume of
room temperature saturated ammonium sulfate to 3 volumes of
supernatant. After 30 min at 0°C, the precipitate was collected by
centrifugation for 10 min at 10,000 rpm in an SS34 rotor. The
precipitate was resuspended in buffer B (20 mM Tris [pH 7.5], 1 mM
EDTA, 10 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) plus
0.5 M NaCl at 2 ml/liter of cell culture and incubated on ice for 30 min. Buffer B was slowly added until the final NaCl concentration was
0.1 M. Insoluble protein was removed by centrifugation for 2 min at
7,000 rpm in an SS34 rotor, and the supernatant was immediately applied
to batch phosphocellulose (Whatman P11) at a volume ratio of 10:1. The
resin with bound protein was washed with buffer B plus 0.1 M NaCl and
then with buffer B plus 0.3 M NaCl. The volume of the washes was
approximately 3 ml/ml of packed resin. Protein was eluted with the same
buffer plus 0.5 M NaCl and then with the same buffer plus 1.0 M NaCl.
The eluted protein was precipitated with 50% saturated ammonium
sulfate, resuspended, and dialyzed overnight against buffer B plus 0.5 M NaCl at 4°C. Storage was at 
80°C. The concentration of the
purified protein was determined by spectrophotometry
(A280 and A260) as
described below. Purity was gauged by Coomassie blue staining after
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).
For purification of proteins dNC1 through dNC5, frozen cells were
resuspended in buffer A and broken by sonication. Insoluble
debris was
removed by centrifugation at 10,000 rpm for 10 min
in an SS34 rotor,
and soluble protein was precipitated with 25%
saturated ammonium
sulfate. The precipitate was resuspended in
buffer B plus 0.5 NaCl and
dialyzed overnight at 4°C.
Proteins dNC6 and dNC7 were purified using a different scheme. Frozen
cells were resuspended in buffer A without NP-40 and
broken by
sonication. Protein was precipitated with 34% saturated
ammonium
sulfate from the soluble crude cell extract. The precipitate
was
resuspended in buffer B plus 0.1 M NaCl at 5 ml/liter of cell
culture and dialyzed overnight against the same buffer at 4°C.
Insoluble material was removed by centrifugation, and the supernatant
was applied to a DEAE-cellulose (DE-52) column. The resin was
washed
with buffer B plus 0.1 M NaCl. The flowthrough and wash
of DEAE were
pooled and precipitated with 50% saturated ammonium
sulfate. The
precipitate was resuspended in buffer B plus 0.1
M NaCl and dialyzed
overnight at 4°C.
Total
E. coli RNA was purified by phenol extraction using
standard procedures (
5). Tobacco mosaic virus (TMV) RNA or
RNA
associated with the
MBD
PR particles was purified by digesting
virus or particles with proteinase K in 1% SDS-10 mM Tris-HCl
(pH
8.0)-1 mM EDTA at 37°C for 1 h, after which phenol-chloroform
extraction and ethanol precipitation were performed. RSV RNA was
synthesized in vitro by transcription with T7 RNA polymerase according
to instructions from the manufacturer (Promega Corp.), using plasmids
that were engineered to contain the desired RSV sequences. All
purified
RNA was stored in diethyl pyrocarbonate-treated water
or as an ethanol
precipitate at
20°C.
Analysis of particles assembled in vitro.
When the dialysis
method was used for in vitro assembly, protein (typically 1 mg/ml) and
RNA or DNA (typically, a 10% [wt/wt] ratio of nucleic acid to
protein) were mixed in buffer B plus 0.5 M NaCl and dialyzed for 2 h at room temperature against buffer C (assembly buffer) (50 mM
morpholine ethanesulfonic acid [pH 6.0], 0.1 M NaCl). For some
experiments, assembly buffer D was used instead (50 mM morpholine
ethanesulfonic acid [pH 6.5], 0.1 M KCl, 10 µM ZnCl2),
with similar results. For the direct dilution method of in vitro
assembly, protein (5 mg/ml) and RNA were mixed in buffer B plus 0.5 M
NaCl and diluted with 4 volumes of buffer C without NaCl. Reaction
mixtures were incubated at room temperature for 30 min. Particles
formed under these conditions were negatively stained with 2% uranyl
acetate (pH 5) on Formvar-carbon-coated grids. To determine the density
of the particles produced and to assess the efficiency of in vitro
assembly, the assembly reaction mixture was layered onto a 10 to 60%
(wt/wt) linear sucrose gradient in buffer C. The gradient was
centrifuged for 4 h at 50,000 rpm in a Beckman SW60 rotor.
Fractions were collected from the top of each tube and analyzed by
SDS-PAGE.
The apparent binding site size of
MBD
PR protein on RNA or DNA was
estimated from the measured
A260/
A280 values of
purified
protein, purified nucleic acids, and VLPs dissolved in 0.1%
SDS
in pH 6.0 buffer. The measured
A260/
A280 ratio was 0.7 for the
purified protein and 2.0 for RNA and varied for
oligonucleotides
depending on base composition. The molar extinction
coefficient
for protein at 280 nm was calculated from the number of
tryptophan,
tyrosine, and cysteine residues to be 4.9 × 10
5. The molar extinction coefficients for oligonucleotides
at 260
nm were calculated from the published values at 260 nm for dGMP,
dCMP, dAMP, and TMP (
5). For RNA, it was assumed that the
A260 nm is 1.0 for 40 µg/ml (
5).
For analysis by scanning transmission EM (STEM) in vitro assembly with
MBD
PR protein was carried out either with
E. coli RNA
or with an 80-nt oligonucleotide and with assembly buffer
C. The
preparations of VLPs at about 1 mg of protein per ml were
adsorbed to a
3-nm carbon-supported grid, upon which TMV had been
adsorbed previously
to provide an internal standard. The grids
were processed and analyzed
as described previously for wild-type
RSV virions
(
76). In brief, the mass of each VLP was determined
by
summing the number of scattered electrons over the
particle,
subtracting the background scattering from the support film,
and
calibrating with TMV particles nearby on the
grid.
For analysis by cryo-EM, VLPs were assembled with
MBD
PR protein
and
E. coli RNA at a protein concentration of 1 mg/ml.
Solutions
containing the particles were applied to perforated
carbon-coated
EM grids, which were briefly blotted to remove excess
liquid and
then rapidly plunged into liquid ethane. Specimens were
examined
using a Philips CM200 FEG transmission electron microscope
equipped
with a Gatan cryostage. Images were recorded on Kodak SO-163
photographic
film at a nominal magnification of ×27,500 under low-dose
conditions
(<20 electrons/Å
2). Micrographs were
digitized on a Zeiss-SCAI scanner using a
step size of 7 µm. The
program I.C.E. (
45) was used to box images
of the
particles. To calculate the spherically averaged density
distribution
for each particle, images of the particles were first
circularly
averaged. The origin for averaging was chosen so that
the variance from
exact circular symmetry was minimized. The spherically
averaged density
distribution was then calculated from the circularly
averaged
projections through the inverse Abel transform (
72,
75).
Inversion of Abel's integral equation was done using the
method
described in reference
28, which is stable in the presence
of high levels of noise in the experimental data. Spherically
averaged
density distributions have previously been calculated
from cryo-EM
images of immature retroviral particles using a Fourier-Bessel
method
(
32,
44). The mathematical equivalence of the two
procedures
for calculating spherically averaged density distributions
follows
from the fact that the Fourier transform of the Abel transform
is the zero-order Hankel (Fourier-Bessel) transform (
25).
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RESULTS |
Nucleic acid requirement for assembly.
For in vitro assembly
studies, it is imperative to have a purified soluble Gag
protein. The full-length RSV Gag protein with an inactivating
mutation in the PR domain remains entirely in inclusion bodies in
E. coli and is refractory to solubilization after
lysis. A deletion mutant of Gag missing the PR domain is also
found in inclusion bodies and can be solubilized and assembled in
vitro, but this protein is still prone to nonspecific aggregation (15). However, further deletion of the 84-amino-acid
membrane-binding domain leads to a protein that remains in the
soluble fraction of E. coli lysates and can be readily
purified and maintained in soluble form at several milligrams per
milliliter. We have used the latter protein, MBDPR (Fig.
1), as a model for RSV Gag. In previous
experiments, partially purified MBDPR assembled efficiently
into spherical VLPs in vitro and assembly was blocked or reversed by
treatment of the protein with RNase (15). UV absorption
showed that the protein had an
A260/A280 ratio of about 1.3, implying contamination with RNA, which was inferred to be acting
as a scaffold for assembly.
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FIG. 1.
Diagrammatic representation of proteins. The rectangle
at the top represents the RSV Gag protein, with vertical lines marking
the sites of proteolytic cleavages during maturation. Numbers indicate
amino acid residues from the N terminus. The MBDPR protein is
missing the N-terminal membrane-binding domain, as well as the
C-terminal PR domain. Black bars represent proteins examined in this
study. The mutant NC proteins all have the same N-terminal portion as
MBDPR but are truncated in the NC domain as indicated. +, lysine
or arginine residue; Cys-His, 14-amino-acid segment including the
conserved zinc-binding motif; A, alanine substitution for a lysine,
arginine, cysteine, or histidine residue.
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In order to investigate the nucleic acid requirement for assembly, we
removed residual RNA from
MBD
PR by first treating
the soluble
cell extract in the presence of a high salt concentration
with
polyethyleneimine, a synthetic polycationic reagent, prior
to ammonium
sulfate precipitation (
19). Following further
chromatography
on phosphocellulose, the protein had an
A260/
A280 ratio of 0.7,
consistent with the absence of nucleic acid. After addition of
E. coli RNA at a high salt concentration and dialysis for 2 h
against the low-salt assembly buffer,
MBD
PR formed abundant
VLPs
that could be visualized by negative-staining EM (Fig.
2A).
In the absence of added RNA, no
regular structures were observed
(data not shown). The particles
had a morphology similar to that
of authentic immature virions and had
a mean external diameter
of about 70 nm (Table
1). The majority of particles appeared
intact and nearly spherical, although incomplete or broken particles
also were apparent. Faint striations were visible at the particle
circumference. These observations are consistent with analysis
of the
particles using cryo-EM (see below). We interpreted the
open particles
to be the result of incomplete assembly. To provide
a more quantitative
measurement of assembly, assembly reaction
mixtures were submitted to
centrifugation in an isopycnic 10 to
60% (wt/wt) sucrose gradient
(Fig.
3A and B). In the presence
of RNA,
almost all of the protein banded at a density of 1.22
to 1.25 where
VLPs could also be readily found by EM (data not
shown). In the absence
of RNA, the protein remained near the top
of the gradient. These
results support the previous conclusion
(
15) that nucleic
acid is an absolute requirement for VLP formation
in this system.
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FIG. 2.
Negative-staining EM of VLPs. MBDPR protein was
assembled with E. coli RNA, and particles were visualized by
negative-staining EM. TMV was added as an internal standard. Original
magnifications: A, ×92,000; B, ×200,000; C, ×290,000. The
structures visible in panel B are representative of
incomplete particles seen at variable frequency in the assembly
reaction mixtures.
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FIG. 3.
Assembly assay by sucrose gradient
centrifugation. MBDPR protein at 1 mg/ml was assembled with
either E. coli RNA, DNA oligonucleotides, or heparin. The
reaction mixtures were centrifuged to equilibrium through a 10 to 60%
(wt/wt) sucrose gradient, and fractions were analyzed by SDS-PAGE and
staining. A, protein alone; B, with E. coli RNA; C, with an
oligonucleotide of 22 nt; D, with an oligonucleotide of 12 nt; E, with
an oligonucleotide of 8 nt; F, with heparin. M, markers.
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Most of the previous work on in vitro assembly in the RSV system has
been carried out with total
E. coli RNA (
15,
46),
which is a collection of diverse RNA species. We addressed
the
size and sequence dependence of assembly with
MBD
PR by
evaluating
the products obtained with defined nucleic acids including
homopolymers
(Table
1). The VLPs formed with different sizes of RSV
RNAs transcribed
in vitro or with other RNAs were indistinguishable by
morphology
or size. To quantify the relative mass of RNA, the VLPs were
collected
by centrifugation and then dissolved in SDS and the
A280 and
A260 values of
the resulting solutions were determined. For RNA-containing
VLPs, the
A260/
A280 ratio ranged
from about 1.35 to 1.40, implying
that the RNA made up 7 to 7.5% of
the mass of the protein. This
estimate is based on the predicted 280-nm
extinction coefficient
of
MBD
PR (
A280 = 0.94 at 1 mg/ml) derived from number of tryptophan,
tyrosine, and
cyteine residues and on the approximation that RNA
at 40 µg/ml has an
A260 of 1.0. This percentage of RNA corresponds
to 12 to 13 nt per protein molecule. The constant mass of RNA
per VLP
implies that the smaller the size of the RNA, the larger
the number of
RNA molecules in a VLP. Single-stranded DNA oligonucleotides
also
promoted assembly, as did linear double-stranded plasmid
DNA. For DNA
oligonucleotides, the
A260/
A280 ratio varied
from
0.98 to 1.48, depending on the oligonucleotide (Table
1). Using
the extinction coefficients for the relevant nucleotides, we calculated
that the size of the apparent binding site for the different
oligonucleotides
ranges from 7 to 10
nt.
While assembly was observed for all of the nucleic acids tested, in
some cases many more VLPs were evident than in others.
Two assays were
used to quantitate assembly efficiency, i.e.,
centrifugation in sucrose
gradients (Fig.
3) and counting of VLPs
on EM grids. By the former
assay, assembly with a 22-mer oligonucleotide
was nearly as
efficient as with
E. coli RNA but smaller oligonucleotides
promoted assembly less well, with barely detectable protein banding
at
the position of VLPs with an 8-mer. The assembly efficiency
was
corroborated by counting VLPs by EM. These results are summarized
in
Table
1. Even heparin was an effective substitute for nucleic
acid.
We drew the following conclusions about the role of nucleic acids in
assembly. First, polyanions of diverse sorts promote
VLP formation by
MBD
PR. Thus, it is apparently electrostatic
interactions with the
basic NC that are required for the Gag protein
to polymerize into VLPs.
Second, since the ratios of RNA to protein
are similar for diverse
nucleic acid sizes, each Gag protein must
contact nucleic acid,
presumably in a similar way. We operationally
defined the nucleic acid
binding site size as the molar ratio
of nucleotides to proteins in the
VLPs, i.e., 7 to 13 nt (Table
1). By this definition, the binding site
is larger than that
determined for the mature retroviral NC protein in
most reports,
about 5 to 8 nt (
30,
47,
48,
83), although
in one report
this size is up to 14 nt for HIV-1 NC extended at its C
terminus
(
48). Third, there is a minimum size of
oligonucleoide that
can support efficient assembly. The minimum size is
larger than
the binding site
size.
Assembly optimization and kinetics.
To establish the optimal
conditions for in vitro particle formation, the effects of salt and pH
on assembly were examined. Constant concentrations of protein (1 mg/ml)
and E. coli RNA (10%, wt/wt) were incubated at different pH
values and salt concentrations. The reaction mixtures were centrifuged
for 30 min in a microcentrifuge, and fractions of the supernatant and
pellet were analyzed by SDS-PAGE. Soluble proteins were expected to be
in the supernatant, and VLPs were expected to be in the pellet. Samples
were also visualized by EM. VLP formation was found to be pH and ionic
strength dependent (data not shown), as reported less systematically
previously (12). Few particles were formed above 0.3 M
NaCl. The effect of a pH change (from 5.5 to 8.0) was less dramatic
than that of an ionic strength change, but pH influenced both the
intactness of particles and their clumping (Table
2). At pH 6.0, particles were more aggregated than at pH 7.0, as judged by EM and by increased turbidity, visible by eye or measured as A350. The number
of single, unaggregated VLPs counted on EM grids increased as the pH
was raised from 6.0 to 7.0. Aggregates formed at pH 6.0 could be
dissociated into individual particles by increasing the pH or salt
concentration. At pH 7.0, relatively more incomplete particles were
observed. At pH values above 7.0, assembly was poor, with very few
particles detected at pH values above 8.0.
The kinetics of assembly represent an important parameter for the
reaction, but one that is challenging to measure. Fully
formed VLPs
each contain over a thousand Gag proteins, implying
a
multitude of possible intermediates. Quantitating
complete and
partially complete particles in various stages
of assembly is
difficult using EM, since completed and partially
completed VLPs
cannot always be distinguished. Moreover, depending on
the nucleic
acid used and on the pH, a variable fraction of the
assembly products
are nonspherical aggregates. As a first step to
approach the kinetics
of in vitro assembly, we set up a simple
light-scattering assay
at a wavelength at which the protein and nucleic
acid components
do not absorb.
MBD
PR protein and
E. coli RNA were diluted rapidly
in a cuvette to the standard pH and
salt concentration of assembly,
and then the increase in
A350 was measured as a function of time
(Fig.
4). By this assay, assembly was very
rapid. At a concentration
of 0.5 mg of protein per ml and at 22°C,
the reaction was complete
in a few minutes, with a half-time of less
than 1 min. Assembly
could be slowed by lowering the protein
concentration (data not
shown) or by decreasing the temperature. At
4°C, the extent of
the reaction was similar, with a plateau
A350 of about 1.5, but
the rate was decreased at
least 10-fold, with a half-time of about
7 min. Visualization of
products by EM showed that by 5 min at
22°C and by 60 min at 4°C,
abundant VLPs had formed. Quantitative
interpretation of these
results was hindered by the different
scattering properties of
incomplete particles, complete particles,
and other aggregates
that may form. Nevertheless, the simplicity
and robustness of a
light-scattering assay make it a potentially
powerful tool with
which to study assembly.
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FIG. 4.
Kinetics of assembly. MBDPR protein at 0.5 mg/ml
was diluted to 0.1 M NaCl and pH 7.0 in the presence or absence of
E. coli RNA, and the course of the assembly reaction was
followed in a spectrophotometer at an optical density (OD) of 350 nm. The half-time represents the estimated time of half-maximal optical
density. Reaction temperatures are indicated.
|
|
Mass of VLPs by STEM.
Understanding retrovirus structure
requires knowledge of the stoichiometry of Gag in virions. We
previously reported mass measurements of infectious RSV particles
by STEM (76). STEM allows the quantitation of
electrons scattered from small objects, such as virus particles, on an
EM grid. Since scattering is proportional to mass, with suitable
standards, it is possible to accurately determine the mass of
individual particles. The mean mass of infectious RSV was found
previously to be 250 MDa, with a significant variation in the mass of
the particles, indicating that they are not all the same size. This
conclusion is consistent with cryo-EM size measurements of HIV
(32) and RSV (R. Kingston, unpublished data). Based on a
number of assumptions about virion composition, the mean mass of RSV
was used to calculate that virions contain ~1,500 Gag molecules
(76).
We used STEM to determine the mass of VLPs formed in vitro in the
presence of RNA or of a DNA oligonucleotide. To more closely
mimic
intracellular ionic conditions, in this case, the assembly
buffer
contained KCl instead of NaCl and included a low concentration
of
Zn
2+ ions to bind to the Cys-His motifs in NC. In addition,
a slightly
higher pH of 6.5, instead of 6.0, was used to reduce
the tendency
of VLPs to aggregate without greatly compromising particle
integrity
(Table
2). The DNA-containing and RNA-containing particles
had
similar mean masses of 6.7 × 10
7 and 7.0 × 10
7 Da (standard deviations, 0.9 × 10
7
and 1.1 × 10
7 Da), respectively (Fig.
5). These values correspond to ~1,200
MBD
PR molecules per particle. In contrast to the
calculations
of Gag stoichiometry for infectious virions, the
calculation for
VLPs requires few assumptions, since only two
macromolecular components
are used in the assembly reaction.
However, meaningful comparison
of the calculated in vitro and in vivo
stoichiometry would rely
on the assumption that the VLPs are all
complete. By negative-staining
EM, about 90% of the VLPs appeared
intact, but this estimate must
be considered approximate, since
incomplete particles might appear
intact in some orientations on the
grid and since small, incomplete
sectors in the particles might not be
visible by negative-staining
EM.
View larger version (12K):
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[in a new window]
|
FIG. 5.
Mass distributions of RNA-containing and DNA
oligonucleotide-containing VLPs. MBDPR protein at 1 mg/ml was
mixed with E. coli RNA or with an oligonucleotide of 80 nt
and dialyzed against assembly buffer D. The particles formed were
adsorbed to a carbon-coated grid, and the masses of individual
particles were analyzed by STEM. N, number of particles.
|
|
Cryo-EM analysis of VLPs.
Virion ultrastructure is best
visualized in the absence of staining and fixation, since both of these
procedures can give rise to artifacts and distortions. To gain a
preliminary understanding of their structure, we analyzed unstained
VLPs by cryo-EM. In vitreous ice at liquid nitrogen temperature, the
particles appeared spherical and relatively homogeneous in size but
exhibited various degrees of closure (completeness), as also seen by
negative staining (Fig. 6). Incomplete
particles resembled sectors of a sphere, having approximately the same
curvature as the fully closed particles. The most striking feature of
the images of the in vitro-assembled particles was the track-like
striations near the particle circumference. These are characteristic of
immature retrovirus particles (32, 44, 82) and are
believed to arise from the side-by-side packing of the radially
arranged Gag proteins. Also evident in images of the VLPs was a series
of concentric electron-dense and electron-sparse annuli which result
from the several globular domains of Gag that are connected by extended
polypeptides.
View larger version (176K):
[in this window]
[in a new window]
|
FIG. 6.
Cryo-EM of VLPs. MBDPR protein at 1 mg/ml was
mixed with E. coli RNA and dialyzed against assembly buffer
C, and the resulting particles were prepared for cryo-EM as described
in Materials and Methods. Arrows highlight incomplete particles.
|
|
Spherically averaged density distributions were computed from images of
the particles using the inverse Abel transform. At
radial distances of
greater than ~20 nm, the density distributions
calculated from
different particles were largely consistent. A
typical example is shown
in Fig.
7. Based on earlier results for
HIV-1 and MuLV (
32,
44,
82), we can assign the features
in
the density distribution to the domains within the Gag polyprotein
with
some certainty. NC, in complex with RNA, gives rise to a
broad interior
peak. CA, which encompasses two spatially separated
domains in RSV
(
16,
49), as was shown earlier for HIV (
10,
33,
34,
38,
57), is associated with a double peak in the
density
distribution. Commensurate with the size of the two domains,
the outer
peak (due to the N-terminal domain) is broader than
the inner peak (due
to the C-terminal domain). Finally, the p10
domain and the truncated
matrix protein are likely to be responsible
for the weak features seen
at higher radii.
View larger version (119K):
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[in a new window]
|
FIG. 7.
Analysis of cryoelectron micrographs. (Top)
Composite view of two typical particles (left) and their circular
averages (right). (Middle) Spherically averaged density distribution
calculated from particles in the top panels. The horizontal axis shows
the distance (in nanometers) from the particle center. The
vertical axis shows the spherically averaged density (on an
arbitrary scale). The assignment of the consistent features of the
distribution to the domains within the Gag polyprotein is indicated.
(Bottom) Histogram of particle sizes. Radial distances to the innermost
of the peaks due to CA are shown.
|
|
In the absence of a lipid bilayer, there are no strong features
delineating the particle exterior. Hence, as a measure of
the particle
size, we have taken the radial distance to an interior
feature, the
inner of the two peaks due to CA. This is the sharpest
and
most conserved peak in the spherically averaged density distribution.
The mean distance to this feature is 33.0 nm (measured from 244
particles without obvious defects; standard deviation, 1.5 nm)
(Fig.
7). It is unlikely that the particle sizes are normally
distributed
when the radius is used as a size metric. The microscope
magnification
was not calibrated, so the true size distribution
may be slightly
shifted in either direction. However, the VLPs
are significantly
smaller than equivalent particles assembled
from a truncated
HIV-1 Gag polyprotein, where the mean distance
to the same feature
was ~55 nm (
44). The difference in the sizes
of HIV-1
Gag particles and RSV Gag
PR particles is also obvious
in thin
sections of virus pellets prepared from a baculovirus-insect
cell
expression system (M. C. Johnson, H. M. Scobie, and V. M.
Vogt, unpublished
data).
Role of NC sequences in assembly.
Since RNA (or a substitute
polyanion) is necessary for in vitro assembly, we assume that
interaction between NC and RNA is the first step in the formation of
VLPs in vitro. The two conserved features of retroviral NC sequences
are the Cys-His zinc-binding motifs and numerous basic amino acid
residues. To begin to define the sequences within NC which are
important for in vitro assembly, we constructed a series of
C-terminal truncations of MBDPR, named dNC1 to dNC7 (Fig.
1). One of the proteins, dNC5, has an artificial stretch of basic
residues (RKKGRKKA) replacing all of NC. The rationale for
this construct is the observation that an almost identical sequence
(RKKGRKK) was found to be able to replace NC in RSV assembly
in vivo (11).
To determine whether these NC mutant proteins were competent to
assemble, the RNA-free proteins at 1.5 mg/ml were dialyzed
against the
assembly buffer in the presence of RNA and then examined
by both
negative-staining EM and sucrose density gradient centrifugation
(data
not shown). The two proteins with the smallest deletions,
dNC1 and
dNC2, assembled into regular spherical structures at
a slightly lower
efficiency than
MBD
PR (Table
3).
Despite the
lower efficiency, however, the VLPs were indistinguishable
by
morphology. In contrast to these two deletion mutant proteins,
all
of the deletions mutant proteins dNC3 to dNC7 failed to produce
identifiable particles, with or without added RNA and at a variety
of protein concentrations.
To better delineate sequence elements in NC that are important for
assembly, we constructed site-directed mutant proteins
in the context
of dNC2, containing the minimal 46-amino-acid N-terminal
NC segment.
Clustered alanine substitutions were introduced to
change the four
Zn-coordinating residues, the six basic residues
between the two
Cys-His motifs, or the four basic residues in
the N-terminal part of NC
(Fig.
1). Each protein was partially
purified and tested for the
ability to form VLPs in vitro with
E. coli RNA. The results
showed that while the Cys and His residues
are dispensable for
assembly, the two clusters of six or four
basic residues, respectively,
are essential (Table
3). In a second
set of alanine mutant proteins,
only two or three of the basic
residues in the proximal segment of NC
were mutated. Both mutant
proteins failed to assemble. Taken together,
these results imply
that the positively charged residues in RSV NC are
the critical
determinants for VLP formation, as they also are for
assembly
in vivo and for nonspecific RNA binding (
11,
18,
24,
31,
62,
69,
84).
| |
DISCUSSION |
In this study, we used a partially purified soluble RSV Gag
protein, MBDPR, to more fully characterize the assembly of VLPs in vitro. By cryo-EM, VLPs closely resemble immature HIV-1 virions and
immature MuLV virions in the absence of membranes, as well as HIV-1
VLPs formed in vitro (32, 44, 82). RNA, DNA,
single-stranded oligonucleotides, and even a sulfated polysaccharide
can promote the assembly of VLPs. Such promiscuity suggests that the
determining interactions are predominantly electrostatic, between the
basic NC domain and the acidic phosphodiester backbone. This conclusion is consistent with the results of our mutagenesis of NC, which demonstrated that the Cys-His motifs are dispensable for assembly while
the clusters of positively charged amino acid residues, at least those
in the N-terminal and middle portions of NC, are essential. A more
detailed mutagenesis in the context of the full NC domain is needed to
uncover if particular Arg or Lys residues are critical in this process.
The observation that the relative mass of RNA in VLPs is constant,
independent of RNA length, implies that in formation of the particle,
each NC domain of Gag binds to a constant length of nucleic acid. This
length can be calculated using the
A260/A280 ratio of VLPs
after dissolution in SDS. From the values in Table 1, for diverse RNAs,
each Gag molecule appears to bind to about 12 to 13 nt. For DNA
oligonucleotides, this value is somewhat smaller, about 7 to 10 nt. The
binding site size for retrovirus NC proteins maximally packed on RNA
has been estimated from titration experiments with fluorescence or
fluorescence quenching to be 6 to 8 nt (47, 48, 83). We
showed recently by STEM that RSV virions contain about 1,500 of each of
the Gag molecules (76). Assuming two copies of genomic RNA
of 10 kb in infectious RSV, each NC domain of Gag would thus cover
about 13 nt during assembly in vivo, a value that is similar to the
present estimate for VLPs formed in vitro with RNA. The significance of
the difference in A260/A280 ratios for VLPs
made with RNA or with DNA oligonucleotides is unclear. Perhaps the VLPs
assembled with a heterogeneous mixture of RNAs contain stretches of RNA
not bound by protein. Thus, the Gag proteins may be bound in clusters,
reflecting independent nucleation events or secondary structures,
leaving stretches of the RNA free of protein because the growing
particle has reached a maximum density of protein molecules. On the
other hand, the difference in
A260/A280 ratios may
reflect a difference in packing on ribonucleotide as opposed to
deoxyribonucleotide polymers. A limitation of quantitative
interpretations of the
A260/A280 ratios we have
measured is that it is impossible to exclude the possibility that
nonspecific protein-nucleic acid aggregates contaminated the VLPs.
However, EM showed no evidence of such aggregates.
The lack of specificity of the RSV NC protein in promoting assembly in
vitro is in accord with the observations that retrovirus NC proteins
can bind to diverse nucleic acids (47, 48, 70, 83;
reviewed in references 23 and 66) and can act as general RNA chaperones (27, 65, 66, 73). However,
sequence-specific in vitro binding of HIV-1 NC or NC-containing
proteins to viral sequences (8, 9, 22, 37), as well as to
RNAs selected from a pool of random sequences (3, 6, 20,
56), has also been reported. NC also has a preference for some
DNA sequences over others (30). In vivo, the NC domain of
Gag is inferred to play the central role in the specific recognition of
psi, the RNA packaging sequence, and the Cys-His motifs are essential
for this packaging function (reviewed in reference 7).
However, certain NC mutants with subtle changes in the Cys-His motif
still package RNA although they lack infectivity (39).
Packaging is also affected by other mutations in NC. Notably, in the
RSV system, mutation of two basic residues downstream of the second
Cys-His motif dramatically reduces specific RNA packaging
(54). This sequence is in the part of NC deleted in our
dNC1 and dNC2 constructs, which are competent for assembly in
vitro, suggesting that the sequence-specific binding and nonspecific
binding are, in part, due to structurally distinct portions of
the protein. This notion is also suggested by the solution structures
of HIV-1 NC bound to portions of the packaging sequence (4,
26), which show phosphodiester contacts with basic residues in
the N-terminal portion of NC upstream of the first Cys-His motif. It is
perhaps not coincidental that we observed basic residues in the same
portion of RSV NC to be critical for in vitro assembly in the context of deletion mutant proteins dNC1 and dNC2. A full understanding of the
sequence-specific and nonspecific properties of NC and the NC domain
remains elusive.
The manner in which NC promotes budding in vivo in the absence of
genomic RNA clearly is largely nonspecific, just as we have found in
vitro. That is, diverse RNAs can be packaged in the absence of any RNAs
carrying the packaging sequence psi, and recent evidence suggests that
the mass of RNA in particles lacking a genome is similar to the
mass in those containing a genome (D. Muriaux and A. Rein, personal
communication). The NC domain is not an absolute requirement for
budding in vivo, since Gag proteins without NC can bud into the medium
of transfected cells, although at lower efficiency than Gag proteins
with an NC domain. However, NC-lacking particles have a lower density
than proper retrovirus particles, suggesting that NC somehow mediates
close packing of Gag molecules (11, 24, 69, 84). The
sequence elements in NC that promote proper density are called
interaction domains, or I domains, which have been inferred to consist
of clusters of basic residues (11, 18, 24, 69, 84).
However, the connection between density and basic residues in NC has
been challenged recently (17). It remains to be
established exactly how I-domain function correlates with assembly in
vitro. However, at least in one respect, in vitro assembly is more
stringent than budding. A short polybasic peptide, engineered to
replace the entire NC domain, can provide I-domain function in RSV
(11). A similar finding has been reported for HIV-1
(69). But the same peptide used by Bowzard et al.
(11) cannot substitute for NC in our in vitro assembly
assay. This discrepancy might be accounted for by Gag-membrane
interactions and possibly Gag-host protein interactions in vivo, which
themselves aid in the assembly process. Detailed dissection of the
amino acid sequences in NC that are involved in VLP formation in vitro, in dense particle formation in vivo, and in specific and nonspecific nucleic acid binding should lead to a better definition of NC function.
Perhaps the most puzzling question about the role of nucleic acid in
assembly in vitro is by what mechanism nucleic acid binding leads to
polymerization of the Gag protein. We originally hypothesized that by
binding to RNA, NC domains gather together the attached CA domains and
thereby concentrate them in space, promoting what are otherwise weak
CA-CA interactions leading to polymerization of the protein
(14). How, then, is an oligonucleotide as small as a
22-mer, which binds only about two Gag molecules, efficiently incorporated into the the VLP? According to one model, NC-nucleic acid
interaction leads to a conformational change in the upstream CA and SP
domains, facilitating CA-CA interactions. The observation that a
dimer-forming coiled-coil domain can substitute for NC in budding
assays (2, 84) can be interpreted to support this model
and suggests that Gag dimers are the building blocks in retrovirus
assembly. In an alternative model, one NC domain forms a bridge between
two oligonucleotides, functionally tying them together as if they
comprised a contiguous chain. According to this notion, a portion of
one NC domain would bind one oligonucleotide and a different portion
would bind a second. To help distinguish between these models, it is
important to fully characterize the nature and size of the
oligonucleotide binding site for the NC domain in the context of
proteins like MBDPR and of the effect of DNA sequence on binding
affinity, as has been done, at least in part, for HIV-1 NC
(30).
Mass measurement by STEM and visualization by cryo-EM provide solid
evidence that VLPs are an excellent model for immature virions formed
in vivo. Since the VLPs are composed of only two components that
contribute in a measurable way to VLP mass, the mean mass obtained, 65 MDa, should be reliable to within 5%. The only major uncertainty in
calculating Gag stoichiometry comes from the assumption that the
particles are intact. In the preparations used for STEM analysis, about
10% of the particles were judged by negative staining to be
incomplete, i.e., not completely spherical or with missing sectors.
Given the lack of knowledge of orientation on the EM grid and the
possibility that small missing sectors would not be discernible, this
value is an underestimate. However, since the distribution of mass
values does not fall off more sharply at the higher values than at the
lower values, it is impossible to say to what degree the calculated 65 MDa, corresponding to 1,200 Gag proteins, underestimates the mass of a
complete VLP. Thus, it remains uncertain if Gag stoichiometry in vitro
is different from that estimated in vivo, i.e., 1,500 (76). Considering the several assumptions required for the
latter calculation, in particular, contents of lipid and of proteins
other than Gag, we favor the hypothesis that the stoichiometry in vitro
and that in vivo are the same. In this context, it is important to note
that the radius measurements of VLPs by cryo-EM are much more tightly
clustered than the mass values (and, indeed, are more tightly clustered than the measurements of diameters of infectious RSV [Johnson et al.,
unpublished data; R. Kingston, unpublished data]). Since radii can be
determined even if a particle is not fully intact, the tighter
clustering of radius measurements is not inconsistent with the mass determinations.
In vitro assembly with purified components continues to be a promising
avenue by which to study the principles of retrovirus assembly. This
system is independent of the known and putative cellular factors
important for assembly in vivo
for example, chaperonins, membrane
lipids, viral and nonviral membrane proteins, and cellular proteins
interacting with late domains. The ability to control the concentration
of components and the other reaction conditions should facilitate a
biochemical dissection of the steps in assembly.
| |
ACKNOWLEDGMENTS |
We acknowledge Stephen Campbell for carrying out much of the
preliminary analysis of the assembly properties of the MBDPR protein; Marc Johnson and Deborah Lynn for critical reading of the
manuscript; and Alan Rein, John Wills, Rebecca Craven, and Stephen
Campbell for discussions. We thank Michael Rossmann and Timothy Baker
(Purdue University) for allowing the cryo-EM to be carried out in their
laboratories and Paul Chipman for technical assistance with this work.
The cryo-EM was performed with the support of NIH grants
GM33050 (to Timothy Baker) and AI34216 (to Michael Rossmann). The STEM
measurements were performed at Brookhaven National Laboratory and were
supported by NIH resource center grant P41-RR01777. The work at Cornell
University was supported by NIH grant CA20081 (to V.M.V.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Genetics, Biotechnology Bldg, Cornell University, Ithaca, NY 14853. Phone: (607) 255-2443. Fax: (607) 255-2428. E-mail:
vmv1{at}cornell.edu.
Present address: Department of Medicine (Infectious Diseases),
University of Massachusetts Medical School, Worcester, MA 01655.
Present address: Institute of Molecular Biology, Howard Hughes
Medical Institute, University of Oregon, Eugene, OR 97403-1229.
| |
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Journal of Virology, March 2001, p. 2753-2764, Vol. 75, No. 6
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.6.2753-2764.2001
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Abstract
Introduction
