Journal of Virology, November 2000, p. 10260-10268, Vol. 74, No. 21
Department of Molecular Biology and Genetics,
Cornell University, Ithaca, New York 14853
Received 2 May 2000/Accepted 27 July 2000
Purified retrovirus Gag proteins can assemble in vitro into
virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the
protease domain forms spherical particles resembling immature virions
lacking a membrane but that a similar protein missing the p10 domain
forms tubular particles. Thus, p10 plays a role in spherical particle
formation. To further study this shape-determining function, we
dissected the p10 domain by mutagenesis and examined VLPs assembled
within Escherichia coli or assembled in vitro from purified
proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to
restore efficient spherical assembly to a p10 deletion mutant. Random
and site-directed mutations were introduced into this segment of
polypeptide, and the shapes of particles formed in E. coli
were examined in crude extracts by electron microscopy. Three
phenotypes were observed: tubular morphology, spherical morphology, or
no regular structure. While the particle morphology visualized in crude
extracts generally was the same as that visualized for purified
proteins, some tubular mutants scored as spherical when tested as
purified proteins, suggesting that a cellular factor may also play a
role in shape determination. We also examined the assembly properties
of smaller Gag proteins consisting of the capsid protein-nucleocapsid
protein (CA-NC) domains with short N-terminal extensions or deletions.
Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low.
Deletion of the N-terminal residue(s) abrogated assembly. Taken
together, these results imply that the N terminus of CA and the
adjacent upstream 25 residues play an important role in the
polymerization of the Gag protein.
The internal structure of
retroviruses is determined by Gag, a polyprotein that is cleaved by the
viral protease (PR) late in assembly, leading to virus maturation.
While the viral envelope proteins and the viral enzymes are essential
for infectivity, Gag alone can form virus-like particles (VLPs) closely
resembling real virions in morphology and other physical properties.
Immature virions are composed of about 1,500 Gag molecules
(34) that form a hollow sphere enclosed by the viral
membrane. The molecules are arranged radially in a parallel array, such
that the N-terminal end of Gag is outside, in contact with the inner
surface of the membrane, while the C-terminal end is inside, in contact
with RNA. This arrangement is maintained after maturation. Thus, the N-terminal domain of Gag gives rise to the membrane-associated protein
(MA), a middle domain gives rise to the capsid protein (CA) that forms
the shell of the mature core, and a domain near the C terminus gives
rise to the nucleocapsid protein (NC) in the center of the core. In
addition to these three canonical proteins found in all retroviruses,
proteolytic processing of Gag also leads to other proteins and short
peptides that are specific to the viral genus or species.
The morphology of immature virions is very similar for all retrovirus
genera. The protein shell shows characteristic radial density
variations that can be seen by cryoelectron microscopy (cryo-EM)
(9, 37). The radial density pattern is likely to correspond
to positions of globular versus extended polypeptide domains of Gag
from the C to the N terminus. In contrast, the morphology of the cores
of mature virions is distinctive for different viruses. For example,
the cores of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), have a cone-shaped appearance, while those of C-type viruses,
such as Rous sarcoma virus (RSV) and Moloney murine leukemia virus
(M-MuLV), have a polyhedral appearance. Neither immature nor mature
retroviruses are icosahedral, nor are they strictly uniform in size
(9, 34, 37). In some cases mutations in Gag have been
reported to lead to the budding of VLPs with tubular morphology
(14, 20, 28, 38), but the mechanism for and significance of
tube formation are not understood.
Retroviral VLPs can be formed spontaneously in vitro from Gag proteins
purified from Escherichia coli (4, 5, 6, 8, 12, 16, 17,
18, 19, 23, 35) or translated in vitro (26, 31, 33).
In vitro assembly with purified proteins has been studied most
extensively for HIV-1 and RSV. For HIV-1, CA, when alone, assembles
into tubes (8, 17) at a high concentration, high ionic
strength, and appropriate pH. CA-NC also assembles into tubes but at
low ionic strength this process requires the presence of RNA (5,
17). Under some conditions, this protein forms a mixture of
tubular and cone-shaped particles resembling authentic mature cores
(12). In contrast, longer Gag proteins containing long
upstream amino acid sequences in addition to the CA and NC domains
generally assemble into spherical particles, although in some cases
these particles are much smaller than bona fide immature cores (4,
18, 35). For HIV-1, CA-NC proteins carrying even very short
N-terminal extensions form spherical particles instead of tubes, but
the assembly process in this case is quite inefficient and the
particles are not uniform in size (18, 35). It has been
reported recently that the morphology of HIV-1 VLPs is determined not
only by the linear structure of the Gag protein but also by the pH of
the assembly reaction (19). A protein with the structure
Like the equivalent HIV-1 protein, purified RSV CA-NC assembles into
tubes in the presence of RNA (5). A longer RSV Gag protein
missing only the PR domain, i.e., with the structure MA-p10-CA-NC, gives rise in the presence of RNA to spherical particles of a size and
morphology that are indistinguishable from those of immature, detergent-treated RSV particles collected from cells expressing a
PR-defective mutant (6). A similar protein with an internal deletion excising the p10 domain of Gag forms tubes instead of spheres
in vitro (6). This result suggests that the p10 domain plays
a critical role in directing Gag into the spherical mode of
polymerization. Using both assembly in vitro and assembly in E. coli cells as assays, we have dissected the p10 domain to locate the minimal sequence necessary for spherical shape determination. In
addition, we have analyzed a collection of random and targeted mutations in this minimal segment to try to gain an understanding of
the sequence requirements for its function as a shape determinant.
Role of p10 in spherical conformation.
To obtain VLPs for
analysis by transmission electron microscopy (TEM), we employed two
methods. In the first method, Gag proteins were purified as described
previously (5, 6) after expression in E. coli,
using ammonium sulfate precipitation and phosphocellulose chromatography as the major steps. To initiate assembly in vitro, purified protein in 0.5 M NaCl, pH 7.5, was mixed with 12% (wt/wt) total E. coli RNA which had been prepared by standard
methods (1), and then the mixture was either dialyzed
(5, 6) or diluted to 0.1 M NaCl, pH 6.0, and examined by TEM
after 30 min. In the second method, crude lysates from cells induced to express viral protein were centrifuged for 5 min at 10,000 × g to remove debris, and then VLPs formed in E. coli
and liberated in soluble form were viewed directly by negative staining
of the extract with uranyl acetate (5, 6). Previous in vitro
assembly studies had shown that VLPs formed by the proteins CA-NC,
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Role of the Rous Sarcoma Virus p10 Domain in Shape
Determination of Gag Virus-Like Particles Assembled In Vitro and
within Escherichia coli
and
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MA-CA-NC, where a part of the MA domain is deleted, forms spherical
particles at pH 8 but tubular particles at pH 6. This observation,
together with a similar pH dependence for monoclonal antibody
reactivity to CA, suggests that the underlying mechanism distinguishing
between tubular and spherical polymerization may be a conformational
change in Gag (19).
MBDPR, and dp10 (Fig. 1, lines 1, 2, and 5) were either spherical, with a diameter of 60 nm, or tubular,
with a diameter of 30 nm and a variable length, which was confirmed in
the present study. We constructed a new, shorter protein, p10-CA-NC
(Fig. 1, line 3), which also forms spherical VLPs. For these and all
other proteins described here, with the exceptions noted below, the
morphologies of the VLPs were indistinguishable by the two methods of
analysis. This result is shown for a representative pair of proteins,
the tube-forming CA-NC and the sphere-forming p10-CA-NC (Fig.
2A). We further confirmed spherical
particle assembly for p10-CA-NC in E. coli by thin-section
EM (Fig. 2B). Thin-section results showing tubes in E. coli
expressing CA-NC were reported previously (5). For all cases
examined, the relative efficiencies of particle formation scored by the
two methods were qualitatively similar (Table
1).
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FIG. 1.
Diagrammatic representation of assembly results. The
rectangle shows the structure of the Gag protein, with vertical lines
representing cleavage sites and numbers representing the number of
amino acid residues from the N terminus, using the standard numbering
for the Prague C strain of RSV (32). Horizontal bars
indicate the structures of the proteins studied for their assembly
properties from both in vitro and particle extraction experiments.
Black bars represent viral sense sequences. Cross-hatched bars indicate
either antisense RSV sequences or sequences from other retroviruses.
Regions A, B, and C in p10 are segments of approximately 20 aa residues
each. All constructs were placed into the pET3XC vector by common
cloning techniques, propagated in E. coli DH5 
cells,
confirmed by sequencing, and transformed into BL21(DE3)/pLysS cells for
protein expression and purification. MBDPR, dp10 (called
p10.52 in reference 25), and CA-NC have been
described previously (5, 6, 25). MBDdp10 combines the
deletions of MBDPR and dp10. DNA segments encoding the C-terminal
62 aa residues of M-MuLV p12 or HIV-1 MA or various segments of p10
(AB, BC, and C) were amplified by PCR from the appropriate viral
clones, using primers encoding a SpeI site, and inserted
into the unique SpeI site in MBDdp10. Lines: 1, CA-NC; 2, MBDPR; 3, p10-CA-NC; 4, p10TR-CA-NC; 5, dp10; 6, MBDdp10; 7, C-terminal 62 aa of MMLV p12; 8, C-terminal 62 aa of HIV-1 MA (BH10
strain); 9 to 11, insertions of antisense sequences derived from DNA
coding for p10; 12, segment C; 13, segment AB; 14, segment BC; 15, segment C plus the last 15 aa of segment B; 16, segment C plus the last
10 aa of segment B; 17, segment C plus the last 5 aa of segment B. The
proteins shown in lines 7 to 14 contain the inserted dipeptide Thr-Ser
between the wild-type Gly and Pro residues five residues from the C
terminus of p10. The proteins shown in lines 15 to 17 contain the
wild-type sequence at this location. The 25-aa sequence corresponding
to the minimal insertion sufficient for spherical particle formation is
shown at the bottom, with the boundary between p10 and CA marked. s,
formation of spheres; t, formation of tubes; 
, no regular assembly;
TR, Thr-Arg insertion. Assembly for all of the proteins shown was
tested both in vitro with purified protein and in E. coli
lysates, with the same results, shown in the column marked
"Morphology."
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FIG. 2.
Morphology of CA-NC and p10-CA-NC particles assembled in
vitro and within E. coli. (A) Electron micrographs of
negatively stained particles. Samples were adsorbed onto Formvar- and
carbon-coated grids for 2 min and stained with 2% uranyl acetate (pH
5.2) for 20 s. Upper left, CA-NC protein assembled into tubes in
vitro; upper right, CA-NC tubes from crude extracts; lower left,
p10-CA-NC protein assembled into spheres in vitro; lower right,
p10-CA-NC spheres from crude extracts. Bars = 100 nm. (B)
Thin-section electron micrograph of VLPs formed within E. coli cells expressing p10-CA-NC protein. Cells were pelleted and
fixed for 2 h in 0.1 M sodium maleate (pH 5.2)-3% glutaraldehyde
and then washed in 0.1 M sodium cacodylate, pH 7.4. The samples were
postfixed for 2 h in 1% OsO4-0.1 M sodium cacodylate
(pH 7.4), quickly rinsed in 0.1 M sodium maleate (pH 5.2), and then
washed extensively in the same buffer. The samples were then stained
with 1% uranyl acetate-0.1 M sodium maleate (pH 6.0), washed in 0.1 M
sodium maleate (pH 5.2), and serially dehydrated with 50, 70, 95, and
100% ethanol and 100% propylene oxide. Pellets were embedded in 50%
propylene oxide-standard Spurr, and thin sections were stained with
2% uranyl acetate and then Reynold's lead citrate.
TABLE 1.
Characteristics of Gag protein mutants
p10.52 (25), 52 of the 62 residues in p10
are replaced with the dipeptide sequence Thr-Ser, corresponding to a
SpeI site, leaving 5 wild-type residues at each end. The dp10 protein includes the 84-aa membrane binding domain at the N
terminus of Gag, which represents about one-half of MA. While the
membrane binding domain, as well as MA itself and MA-p2-p10, is soluble
in E. coli extracts (27; A. Barbera and
V. M. Vogt, unpublished observations), previous in vitro assembly
experiments indicated that in the context of larger Gag proteins
extending through CA, this domain leads to poor solubility of Gag
proteins in E. coli, thus complicating studies on in vitro
assembly (6). Hence, in order to establish a more tractable
system for studying the effects of p10 and to investigate the
generality of the effect of p10 deletion on VLP morphology, we combined
the deletions of dp10 and MBDPR into a single construct,
MBDdp10 (Fig. 1, line 6). The resulting purified protein was found
to assemble into tubes with an efficiency similar to that of dp10
(Table 1), suggesting that the N-terminal membrane binding domain does
not contribute to morphology.
In retrovirus Gag proteins other than those of lentiviruses, one or
more short proteins or peptides lie between MA and CA. In general,
these amino acid sequences are rich in glycine and proline residues.
For example, both RSV p10 and M-MuLV p12 are about 30% Gly and Pro.
Thus, it seemed possible that the amino acid composition of p10 might
be the characteristic responsible for spherical particle formation. To
test this hypothesis, we replaced the amino acids deleted in dp10 with
a similar-size segment from the C terminus of M-MuLV p12 or, as a
control, from the C terminus of HIV-1 MA, which is not rich in Gly or
Pro. In each case the foreign sequence was inserted into the
SpeI site of MBDdp10. Neither of these chimeric proteins
assembled into recognizable structures in vitro (Fig. 1, lines 7 and
8). We conclude that the effect of the p10 domain on particle shape is specific.
In order to delineate the sequences within p10 responsible for the
shape-determining function, we originally deleted one-third or
two-thirds of p10 in the context of p10-CA-NC. However, protein expression from these two constructs was poor, and the assembly phenotype could not be determined (data not shown). As an alternative approach, and to buffer the Gag protein against possible N-terminus effects, we inserted portions of the p10 coding sequence back into
MBDdp10. The experimental logic was to determine the minimal segment
of polypeptide sufficient to restore spherical morphology. The central
52-aa core of the p10 domain was divided approximately into thirds,
called A, B, and C. Insertion of segment C into MBDdp10 abolished
the assembly of regular structures altogether (Fig. 1, line 12).
Insertion of segment AB still gave a tubular assembly phenotype (Fig.
1, line 13). Insertion of segment BC restored efficient assembly of
spherical particles (Fig. 1, line 14). Since a single restriction site
was used for cloning, some of the inserted DNA fragments were inserted
in the antisense orientation. One of these abrogated assembly (Fig. 1,
line 10), while two others gave rise to tubes (Fig. 1, lines 9 and 11).
It should be noted that in all of the insertion mutants shown in Fig.
1, lines 7 to 14, the final protein sequence included an extra
dipeptide sequence, Thr-Ser, positioned 6 aa upstream of the start of
CA, corresponding to the SpeI cloning site. That this
dipeptide insertion itself does not perturb spherical assembly is shown
by the phenotype of the BC insertion mutant (Fig. 1, line 14).
To locate the shape-determining function more precisely, a set of
serial deletions was constructed. Stretches of 5, 10, and 15 residues
were deleted from the N terminus of the BC segment. These constructs
were prepared in such a way that the 25 to 35 aa of p10 upstream of CA
were entirely wild type, without the added dipeptide sequence from the
artificial SpeI site. Each new protein was assayed for
particle shape during assembly. In this way, the minimal region
sufficient to restore efficient spherical assembly was determined to be
the C-terminal 25 residues of p10, which encompass 5 residues from
segment B and all of segment C (Fig. 1, line 17). The sequence of this
minimal region, including the five residual residues still present in
the original dp10 deletion, is PGPALTDWARVREELASTGPPVVAM.
RSV Gag proteins with mutations in p10 were reported to be
quantitatively defective in budding in chicken cells (7). In that study, each of several 15- to 20-aa deletions throughout p10
reduced budding about 20-fold. In addition, two more localized mutations, an insertion of Thr-Arg at p10 residue 8 and a Gly-to-Arg substitution at residue 56, resulted in a temperature-sensitive budding
phenotype in chicken cells. We built the Thr-Arg insertion mutation
into the context of the p10-CA-NC expression plasmid. The resulting
protein, called p10TR-CA-NC (Fig. 1, line 4), assembled into spherical
particles in E. coli with the same efficiency as the
wild-type p10-CA-NC (Table 1).
Random mutagenesis of the minimal region.
In the absence of
structural information to help guide site-directed mutagenesis, we
generated a collection of random mutations in the p10 minimal segment,
using a partially degenerate primer and amplification by PCR. In the
forward primer, a stretch of nucleotides corresponding to the 25 aa of
interest was doped such that at each position the nucleotide was
correct in 88% of the molecules and mutant in the remainder. The
backwards primer was wild type in sequence and started at the natural
NdeI site in the CA gene (Fig.
3A). The PCR product was inserted between
the unique SpeI and NdeI sites of the plasmid
encoding MBDdp10. After transformation into BL21(DE3)/pLysS cells,
125 colonies were picked for analysis. This level of doping resulted in
an average of two or three amino acid substitutions per expressed
clone, as predicted theoretically.
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MBDdp10, we were most interested in mutations
that altered the assembly phenotype back to tubes, indicating that the
function of the 25-aa segment was perturbed. In total, 18 of the mutant
proteins still had efficient spherical assembly, 15 had tubular
assembly, and 32 lacked assembly altogether. DNA sequences were
determined for all clones giving rise to tubes or spheres and for a
subset of the clones that did not lead to assembly. The mutant clones
were assigned numbers with the prefix Min, to show that they had been
created in the minimal p10 segment required to promote efficient
spherical assembly, with the wild-type segment being called Min0 (Fig.
3B).
Inspection of the collection of unique amino acid sequences and their
phenotypes (Fig. 3B) does not show an obvious pattern. For instance,
each of the prolines in the minimal region was mutated in at least one
clone, without changing spherical assembly. In fact, three of the four
prolines were changed in one mutant, Min9, without changing spherical
particle formation. Nevertheless, the prolines could not be changed
arbitrarily, since mutant Min21 (P58T) had a tubular assembly phenotype
and mutant Min32 (P40T) did not assemble at all. Similarly, the
percentages of all the nonconservative changes were similar for the
collections of mutants that formed spheres or tubes did not assemble.
We conclude from these results that since the minimal segment of p10 is
sensitive to mutation, the amino acid sequence must fold into an
ordered structure, either by itself or in conjunction with other
sequences in CA.
Since most of the mutants had more than one amino acid substitution, we
next used site-directed mutagenesis to isolate individual mutations and
thus try to determine which were responsible for the phenotype. Twelve
single amino acid mutants were constructed in this way. In the
collection of all single amino acid mutants, including those obtained
in the original screen, 10 assembled into spheres in E. coli, 3 assembled into tubes, and 3 failed to assemble into
regular structures. The three tube-forming mutants, Min21 (P58T), Min23
(M62V), and Min24 (L52F), have conservative substitutions, consistent
with the notion that subtle changes of a structural element are enough
to perturb the Gag polymerization pathway.
The three single mutants and one double mutant, Min22 (L42M and A61V),
that gave rise to tubular particles in crude extracts were also tested
for assembly phenotype in vitro, after purification of each protein.
Surprisingly, each of these mutant proteins assembled in vitro into
spherical rather than into tubular particles (Fig. 3B). This finding of
altered assembly phenotypes was unexpected, since all other Gag
proteins examined previously (all the proteins shown in Fig. 1) had the
same phenotype in vitro and in E. coli. As a control we also
tested two single mutants (Min15 and Min16) that formed spheres in
E. coli; both of them showed the same spherical phenotype as
purified proteins. Several further experiments were carried out to try
to gain evidence for the underlying difference between assembly in
E. coli and in vitro (data not shown). First, a concentrated
E. coli lysate without Gag protein was added to the in vitro
assembly reaction with several purified mutant proteins. No change from
spheres to tubes was observed. Second, for the same mutants, particles
in crude lysates were diluted in high-salt (0.5 M NaCl) lysis buffer in
order to break apart any preformed VLPs and then dialyzed against in
vitro assembly buffer (0.1 M NaCl, pH 6.0). The VLPs observed in this
experiment had the same tubular shape as those in the original crude
extracts. Third, purified Gag proteins were assembled in vitro at pH
7.0, which is similar to the intracellular pH. Although the assembly
was less efficient at this pH, the VLPs observed still had the same spherical shape as seen at pH 6.0.
We interpret these results to mean that some factor or condition in
E. coli favors tubular assembly for mutant proteins that are
intrinsically able to polymerize in either a spherical or a tubular
mode. The propensity to form one shape or another may be keyed by
alternative conformations of Gag, as suggested for HIV in vitro
assembly of Gag proteins (19). The more subtle mutations in
the 25-aa stretch of p10 may, then, be viewed as balancing the protein
between two alternative conformations, such that small changes in the
environment lead to different polymerization modes. We speculate that
once assembly has been nucleated in either mode, proteins will adapt
their conformation to add to the polymerized lattice in the same fashion.
Importance of residues at the immediate N terminus of CA.
In
the presence of RNA, HIV-1 CA-NC assembles into tubes in vitro (5,
17). However, CA-NC carrying a few extra N-terminal residues
forms spheres instead of tubes, although the assembly products in this
case are heterogenous in size and the assembly is not efficient
(18, 35). A model explaining this result (35)
invokes the hypothetical malformation of the 
-hairpin at the N
terminus of CA, a feature seen in the three-dimensional structure of
the N-terminal domain of CA (10, 15). The hairpin is
stabilized by a salt bridge between the amino-terminal residue Pro1 and
Asp51, both of which are highly conserved residues in CA proteins of
diverse retroviruses. It was thus hypothesized that an intact hairpin
and salt bridge are involved in the CA-CA interactions leading to tube
formation (35). In contrast, extensions of the CA N
terminus, which would prevent formation of the salt bridge and thus
presumably abrogate the formation of the hairpin or cause its
malformation or displacement, would promote a structural change that
allows CA to polymerize into spheres. A -hairpin almost identical to
that in HIV CA is found in the N-terminal CA domain of RSV
(22).
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-hairpin found there (22).
The data presented here are based exclusively on EM, a technique that
is necessarily qualitative or, at best, semiquantitative. While we have
noted gross quantitative differences in assembly efficiency, for
example, for comparisons of assembly of m-CA-NC and p10-CA-NC, we have
not reported lesser quantitative differences because of uncertainty
about their significance. However, the major conclusions from this
study do not rest on quantitative differences but rather on the obvious
distinctions in the shapes of tubes and spheres. All visual assays by
EM were carried out at least twice with independent protein
preparations or with cell extracts from independently induced E. coli cultures. With the single exception of the protein m-CA-NC,
there was no protein for which particles of both shapes were observed
together or particles of one shape were observed once and particles of
another shape were observed in a different experiment. To verify that
both tubes and spheres would be identifiable in the same extract, we
prepared mixtures of two crude extracts, one containing tubes and the
other containing spheres, at ratios of 1:1, 10:1, and 1:10. Both types of particles were readily visible in all of these mixtures (data not
shown). The observation of two shapes for the m-CA-NC protein is a
special case, given that the protein preparation was found to be
composed of molecules with two different N-terminal amino acid
sequences. Thus, overall, the scoring of particle shapes of the various
mutants should be reliable. The scoring of lack of assembly, which by
nature is a negative result, arguably is less convincing, since
assembly might take place with some alteration in conditions. However,
for these mutants, at least two independent experiments were also
carried out, with the same phenotype being recorded for each.
While the roles of several RSV Gag domains during assembly are
understood at least in part, the role of the p10 domain has remained
largely unknown. The Gag proteins of most retroviruses include one or
two small proteins derived from amino acid sequences between MA and CA,
and at least one of these proteins is typically very rich in Pro and
Gly, suggesting the lack of an overall globular fold. This prediction
is consistent with radial density measurements of immature HIV and
M-MuLV particles, which imply that part of the Gag sequence between the
membrane binding domain of MA and the N-terminal domain of CA is in an
extended conformation (9, 37). RSV p10 is just C terminal to
the sequence PPPY, comprising the core of the late domain, which plays
a key role in a late step in budding (29, 36), probably by
interacting with one of the members of the cellular family of WW
proteins (13). Three previous studies have investigated the
role of p10 in RSV assembly in vivo in transfected cells. Krishna et
al. (25) reported that deletion of part or most of p10
modestly reduced assembly in COS cells and gave rise to particles that
appeared somewhat smaller by rate zonal sedimentation. Dupraz and Spahr
(7) showed that several partial deletions of p10 reduced the
budding of virus particles as much as 20-fold in chicken cells. In
addition, they found that two of five more subtle mutations in p10 gave
a temperature-sensitive budding phenotype. On the other hand, T. Cairns
and R. Craven (personal communication) found that budding from
transfected COS cells was not significantly impaired either in the dp10
mutant or in another mutant deleting 31 aa residues of p10, in the
context of an otherwise infectious viral clone expressed in quail
cells. In addition, they found that a mutant with a deletion of 26 residues from the N-terminal portion of p10 retains infectivity. The
reason for the apparent discrepancies among these results remains
unclear, but they might be due to differences in virus strain or cell
type. In none of these cases was the morphology of the budding
particles determined.
What is the significance of the two shapes of VLPs? The spherical
particles are closely similar to immature virions without a membrane,
as shown for HIV-1 (19) and RSV (R. Kingston, personal communication) by cryo-EM. That is, the in vitro-assembled particles show the characteristic radial density profile first noted for HIV-1
(9) and M-MuLV (37), in which the most prominent
features are the densities of the two structurally distinct domains of CA. We hypothesize that the tubular particles are analogues of mature
cores, but the logic for this hypothesis is indirect, relying on
comparisons with HIV-1 assembly in vitro. In HIV-1, under some conditions CA-NC forms a mixture of tubes and cones (12),
and thus the protein-protein contacts in these two structures are probably similar. Mature lentivirus cores are conical. To date, no in
vitro assembly system has produced mature cores of C-type retroviruses,
like RSV or M-MuLV, which appear to have an irregular polyhedral shape
(37; R. Kingston, personal communication). An
alternative hypothesis is that tubular particles represent the
polymerization of Gag in a manner like that observed for some mutants
expressed in vivo. For example, baculovirus expression of an HIV-1 Gag
protein with the structure MA-CA leads to the massive appearance of
membrane-enclosed tubular structures on the surfaces of insect cells
(14, 28). Similarly, deletion of the M-MuLV p12 domain leads
to budding of both tubular and spherical particles (38).
Since p12 carries the M-MuLV late domain sequence, PPPY, believed to
interact with a cellular protein, the alternative morphologies recorded
in that study may be related to interaction or lack of interaction with
a cellular factor. Conceivably, the discordance we have observed for
some RSV p10 mutants which showed spherical assembly in vitro but
tubular assembly in E. coli is also accounted for by a
factor present in E. coli cells.
What mechanisms underlie the difference in the shapes of tubes and
spheres? It has been known for several decades that capsid proteins of
plants and bacteriophages can polymerize into diverse regular shapes.
For example, cowpea chlorotic mosaic virus capsid protein forms either
spheres, sheets, or tubes, depending on pH and ionic strength
(2). The different polymerization modes observed in
retroviral Gag proteins are likely to reflect subtle differences in the
conformation of the protein, in particular the CA domain, since this
part of Gag is generally considered to be the major locus of
interactions between Gag molecules in assembly. This notion is based in
part on the observation that HIV CA forms dimers in vitro via
interactions between C-terminal domains (11). However, the
nature of CA-CA interaction in RSV is less clear, since RSV CA remains
monomeric even at concentrations as high as 20 mg/ml (22).
The first detailed model proposed to explain the tubular and spherical
polymerization modes in vitro (35) invoked the presence or
absence of the -hairpin at the N terminus of mature CA (10,
15). However, both reduction of pH and deletion of the spacer
peptide between HIV CA and NC can change the in vitro assembly product
from spheres to tubes (19). This recent result suggests that
the alternative assembly modes reflect more global conformational
changes or, alternatively, that there is some communication between the
C-terminal and the N-terminal domains of CA. The published solution
structures of retroviral CA proteins (3, 21) show little
evidence for interaction between the two CA domains. However,
structures of larger Gag proteins, encompassing CA with both upstream
and downstream sequences, have yet to be determined.
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ACKNOWLEDGMENTS |
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We thank Stephen Campbell and Yu Ma for helpful advice, Mark Berryman for help with thin sectioning, Wes Sundquist for communication of unpublished results, and Marc Johnson and Deborah Lynn for critical reading of the manuscript.
This work was supported by USPHS grant CA-20081.
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FOOTNOTES |
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* 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.
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Journal of Virology, November 2000, p. 10260-10268, Vol. 74, No. 21
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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