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J Virol, June 1998, p. 4798-4810, Vol. 72, No. 6
Heinrich-Pette-Institut, D-20251 Hamburg,
Germany
Received 10 December 1997/Accepted 3 March 1998
Expression of retroviral Gag polyproteins is sufficient for
morphogenesis of virus-like particles with a spherical immature protein
shell. Proteolytic cleavage of Gag into the matrix (MA), capsid (CA),
nucleocapsid (NC), and p6 domains (in the case of human
immunodeficiency virus [HIV]) leads to condensation to the mature
cone-shaped core. We have analyzed the formation of spherical or
cylindrical particles on in vitro assembly of purified HIV proteins or
inside Escherichia coli cells. CA protein alone yielded cylindrical particles, while all N-terminal extensions of CA abolished cylinder formation. Spherical particles with heterogeneous diameters or
amorphous protein aggregates were observed instead. Extending CA by 5 amino acids was sufficient to convert the assembly phenotype to
spherical particles. Sequences C-terminal of CA were not required for
sphere formation. Proteolytic cleavage of N-terminally extended CA
proteins prior to in vitro assembly led to the formation of cylindrical
particles, while proteolysis of in vitro assembly products caused
disruption of spheres but not formation of cylinders. In vitro assembly
of CA and extended CA proteins in the presence of cyclophilin A (CypA)
at a CA-to-CypA molar ratio of 10:1 yielded significantly longer
cylinders and heterogeneous spheres, while higher concentrations of
CypA completely disrupted particle formation. We conclude that the
spherical shape of immature HIV particles is determined by the presence
of an N-terminal extension on the CA domain and that core condensation
during virion maturation requires the liberation of the N terminus of
CA.
Retroviruses are released by budding
from the plasma membrane of the infected host cell. Bud formation
occurs either from preformed spherical particles (type B and D viruses,
spumaviruses) or concomitant with assembly of the inner virion
structure (type C viruses, lentiviruses [reviewed in references
16 and 59). Early immature retroviral
particles always contain a spherical protein shell, approximately 80 to
100 nm in diameter, which is formed by assembly of the Gag- and Gag-Pol
precursor polyproteins. Soon after budding, an extensive morphological
rearrangement called maturation occurs, which is necessary for the
virion to become infectious. Maturation requires proteolytic processing
by the viral proteinase (PR) and leads to condensation of the spherical immature protein shell to a centrally located electron-dense structure, the mature core (reviewed in reference 55). Mature
retroviral cores consist of a ribonucleoprotein (RNP) complex with two
molecules of genomic RNA associated with the nucleocapsid (NC) protein
and with the replication enzymes reverse transcriptase (RT) and
integrase (IN), encased in a protein shell formed by the capsid (CA)
protein (for nomenclature of retroviral proteins, see reference
36). In human immunodeficiency virus (HIV),
additional viral and cellular proteins which are presumed to function
during virus entry (e.g., cyclophilin A [CypA] [12, 38,
54]) are also packaged into virus particles.
The HIV Gag polyprotein (Pr55gag) is divided
into the matrix (MA), CA, NC, and C-terminal p6 domain (Fig.
1). The N-terminal MA domain is
cotranslationally myristoylated (6, 21) and closely apposed
to the inner surface of the lipid envelope (17) and contains
signals for intracellular transport of the polyprotein (33).
A homomultimer of CA forms the cone-shaped capsid shell of the mature
virus (17), while NC is involved in packaging of the genomic
RNA (reviewed in reference 2) and p6 plays a role in
release of the budding virus (20). In addition, there are
short intervening sequences on the polyprotein called spacer peptides
(23), which are released by PR-mediated cleavages at their N
and C termini during maturation (41).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
N-Terminal Extension of Human Immunodeficiency Virus Capsid
Protein Converts the In Vitro Assembly Phenotype from Tubular to
Spherical Particles
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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FIG. 1.
Expression and purification of the 
MA-CA protein. (A)
Schematic representation of the genetic structure of HIV-1 and of the
region encoded in the expression plasmid. At the top, the coding region
of the HIV-1 genome is shown, with the different open reading frames
depicted as boxes. The gag reading frame and the 5' terminal
part of the pol reading frame are expanded below. The
HIV-specific region encoded by plasmid pET MA-CA is shown at the
bottom. The numbers indicate the amino acids of MA flanking the
deletion. (B) Coomassie blue-stained SDS-polyacrylamide gel
representing purification of MA-CA. Lanes: 1, lysate from induced
bacteria; 2 and 3, soluble and insoluble material after high-speed
centrifugation; 4 and 5, supernatant and precipitate following
precipitation by addition of ammonium sulfate to 25% saturation; 6 and
7, supernatant and precipitate following a second ammonium sulfate
precipitation after anion-exchange chromatography. Molecular mass
standards (in kilodaltons) are indicated on the left; the position of
MA-CA is marked on the right.
Retroviral Gag proteins alone are sufficient to form immature spherical particles (48), which are released by budding but do not mature, do not contain viral glycoproteins, and are noninfectious. Accordingly, expression of Gag alone with recombinant viral expression systems (9, 18, 25, 28-30, 46, 49) or after transfection of expression plasmids (40, 50) consistently yielded the release of virus-like particles. The presence of RNA, heterogeneous in size and of both viral and cellular origins, within these particles has been reported (18, 49), but the role and significance of RNA in the assembly process are currently not clear. C-terminal deletions of Gag, removing p6 and most of NC, did not prohibit the formation of immature particles (25, 28, 29, 46), while further truncations extending into the spacer peptide at the C terminus of CA (SP1; Fig. 1) abolished particle formation (18, 25). Furthermore, large internal deletions within MA, removing most of its globular core (24, 39, 43), had no apparent effect on the morphology of immature or mature particles (11, 52). It appears likely, therefore, that the C-terminal segment of Gag and the globular domain of MA are not essential for assembly of immature HIV-like particles.
Assembly of retrovirus particles can occur at different sites within the cell, suggesting that no specific subcellular environment is required (44, 57). Formation of spherical nonenveloped HIV- or simian immunodeficiency virus (SIV)-like particles was observed in the cytoplasm or in the nucleus following baculovirus-mediated overexpression of the respective polyprotein (9, 46). Recent evidence suggests that assembly of retrovirus-related structures can even occur in a test tube following in vitro translation (47, 51) or bacterial expression of complete retroviral polyproteins or individual domains thereof. Klikova et al. (31) reported that Escherichia coli expression of Mason-Pfizer monkey virus Gag yielded spherical unenveloped particles similar to the immature virus, both inside bacterial cells and upon incubation of purified proteins. Formation of tubular structures has been observed following in vitro assembly of purified bacterially expressed fragments of Rous sarcoma virus (RSV) or HIV Gag polyproteins. In vitro assembly of RSV and HIV CA-NC led to RNA-dependent formation of hollow cylinders with a diameter of 55 nm and heterogeneous length (7). Morphologically identical structures were observed when purified HIV CA protein was used, but cylinder formation occurred at a 20-fold-higher molar concentration of protein, in high salt, and independent of RNA (22). These results suggested that at least for HIV, CA alone determines the shape of cylinders, which are stabilized by weak hydrophobic interactions. The NC domain probably aligns and concentrates the protein on the RNA and thereby facilitates efficient assembly at lower protein concentrations (7, 22). Campbell and Vogt (8) recently reported that in vitro assembly of larger fragments of RSV Gag yielded spherical particles if the p10 region (located upstream of CA on the RSV Gag polyprotein) was present. Gag fragments lacking p10 yielded cylindrical particles similar to CA-NC. These authors concluded that RSV p10 contains a shape-determining region which facilitates interactions required for spherical particle formation (8).
To define the requirements for assembly of immature and mature HIV-like particles, we constructed bacterial expression vectors for segments of the HIV Gag polyprotein. Here, we report that CA alone formed cylindrical particles in vitro and inside bacterial cells while N-terminal extensions abolished cylinder formation. Spherical particles with heterogeneous diameter or amorphous protein aggregates were observed instead. Cylinder formation was restored if proteins were cleaved by HIV PR before in vitro assembly. These results show that N-terminal extensions of CA convert its assembly products from tubular to spherical particles and suggest that liberation of the CA N terminus is required for core condensation during maturation.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Expression plasmids. DNA manipulations were carried out by standard methods. All plasmids were derived from the prokaryotic expression vector pET11 3xc (Novagen, Madison, Wis.), which carries a T7 expression cassette. HIV-1 coding sequences were amplified from the proviral clone pNL4-3 (1) by PCR. For construction of the CypA expression vector, plasmid pGEX-2T/hu-cyclophilin A (3) was used as a template. By choosing appropriate primer sequences, we introduced a translational start codon at the 5' end (part of the newly introduced NdeI site) and two stop codons (TGA/TAG) at the 3' end of the coding sequences and also introduced novel restriction sites at the 5' end (NdeI) and at the 3' end (BamHI).
The amplified fragments were cleaved with NdeI and BamHI and ligated into pET11 3xc that was also cleaved with NdeI and BamHI, resulting in the expression plasmids pET MA-CA, pETMA-CA, pET N15MA-CA, pET MA100CA, pET
MA115CA, pET MA120CA, pET MA128CA, pET CA, pET CA13 (see Fig. 5A),
and pET-CypA. These plasmids encode recombinant proteins with predicted
molecular masses of 40.5 kDa (MA-CA), 31 kDa (MA-CA), 27 kDa
(N15MA-CA), 29 kDa (MA100CA), 27.5 kDa (MA115CA), 27 kDa (MA120CA), 26 kDa (MA128CA), 25.6 kDa (CA), and 24 kDa (CA13), all containing an
N-terminal Met residue in addition to the sequence of the respective
viral protein. In the case of CA and MA-CA, N-terminal sequence
analysis of purified proteins indicated removal of the N-terminal
methionine.
Expression and purification of recombinant proteins.
Induction of Escherichia coli BL21 DE3 cells was performed
essentially as described previously (22). CypA was purified
by a published procedure (37). Purification of CA-derived
proteins was modified from our published procedure (22).
Bacterial cells were resuspended in cold lysis buffer (50 mM
morpholineethanesulfonic acid [MES; pH 6.5], 10 mM MgCl2,
1 mM EDTA, 1 mM dithiothreitol [DTT], 100 mM NaCl) and disrupted by
cell disintegration (glass beads; Biomatik) and subsequent sonication
(Branson sonifier B12). After consecutive centrifugation at 3,000 × g for 5 min, 27,000 × g for 15 min, and
246,000 × g for 60 min, CA proteins were precipitated from the soluble fraction by the addition of ammonium sulfate to 25%
saturation, redissolved in buffer containing 50 mM Tris-HCl (pH 8.0),
30 mM NaCl, 1 mM EDTA, and 1 mM DTT, and applied to DEAE-cellulose
(Whatman DE52) equilibrated with the same buffer in a batch procedure.
CA proteins were collected from the unbound material by the addition of
ammonium sulfate to 50% saturation followed by centrifugation. In some
cases, proteins were further purified by cation-exchange chromatography
with a POROS SP 20/M column on a BioCAD Sprint Perfusion
Chromatography System (PerSeptive Biosystems). Proteins were applied in
50 mM MES (pH 6.0)-50 mM NaCl-1 mM EDTA-1 mM DTT and eluted with a
salt gradient. For the MA120CA and MA128CA proteins, lysis was
performed in 50 mM Tris-HCl (pH 8.9)-1 mM EDTA-1 mM DTT-1 M NaCl,
high-speed centrifugation and anion-exchange chromatography were
omitted, and the ammonium sulfate precipitates were redissolved in 1 M
salt buffer (pH 6.0) and diluted to 50 mM NaCl before undergoing
cation-exchange chromatography. All purified proteins were collected by
ammonium sulfate precipitation, redissolved to a concentration of
approximately 3 mg/ml in 30 mM MES (pH 6.0)-1 mM EDTA-1 mM DTT-100
mM NaCl (or lacking NaCl in the case of CA and MA-CA), and stored
frozen at 
70°C.
Analysis of expression products. Protein samples were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels containing 15% polyacrylamide (30:1 ratio of acrylamide to N,N-methylenebisacrylamide) and stained with Coomassie blue. The protein concentration was determined by the method of Layne (34). Purity was assessed by scanning of Coomassie blue-stained gels with a Desaga CD50 densitometer.
In vitro assembly.
Protein stock solutions were diluted to
the appropriate concentration with storage buffer (30 mM MES [pH
6.0], 1 mM EDTA, 1 mM DTT, 100 mM NaCl) and dialyzed overnight at
4°C against assembly buffer (50 mM Tris-HCl [pH 8.0], 1 M NaCl)
unless otherwise indicated. For PR treatment before assembly, MA-CA
was diluted to 3 mg/ml with storage buffer and active-site titrated
HIV-1 PR (32) was added at a molar ratio of 1:10
(enzyme:substrate). After a 6-h incubation, the specific PR inhibitor
Ro31-8959 (45) was added to a concentration of 5 µM and
samples were dialyzed against assembly buffer and analyzed by
negative-stain electron microscopy. For PR-treatment after in vitro
assembly, MA-CA was first dialyzed overnight against 50 mM Tris-HCl
(pH 7.0)-0.65 M NaCl and PR was added subsequently at a molar ratio of
1:10 (E:S). Incubation was performed at 4°C on the grid, and PR
treatment was stopped at various time points by the addition of
Ro31-8959 to a final concentration of 5 µM and subsequent preparation
for negative staining. For analysis of the effects of CypA on in vitro
assembly, purified CA or MA-CA was mixed with purified CypA at molar
ratios between 1:1 and 10:1 and dialyzed against assembly buffer as
indicated above.
Electron microscopy analysis. The procedure for analysis of assembly products within induced bacteria will be described elsewhere (26). Briefly, induced E. coli BL21 DE3 cells were collected by brief centrifugation, resuspended and fixed in 4% paraformaldehyde-2% glutaraldehyde in phosphate-buffered saline (PBS), incubated for 5 min on ice, washed with PBS, and collected by brief centrifugation. The wet bacterial soft pellet was drawn into cellulose capillary tubes by capillary action as described previously (27). Subsequently, bacterial cells were postfixed within the capillaries for 30 min with 1% OsO4 in PBS, washed with water, stained for 30 min in 1% uranyl acetate in water, and dehydrated in a graded series of ethanol. The capillary tubes were embedded in ERL resin for sectioning. Ultrathin sections were counterstained with 2% uranyl acetate and lead citrate. Micrographs were obtained with a Philips CM120 transmission electron microscope at 80 kV.
For negative staining of in vitro assembly products, 5-µl samples of dialyzed protein solutions were applied to Parafilm and covered with a UV-irradiated Formvar/carbon-coated grid (200-mesh size) for 5 min. In some cases, purified antibodies to HIV-1 CA were bound to the grid to immobilize spherical particles. An immunoglobulin G fraction was prepared from rabbit polyclonal antiserum against CA (40) by successive precipitation with caprylic acid and ammonium sulfate as described previously (53). Purified antibodies were redissolved in PBS and incubated with UV-irradiated grids for 30 min at room temperature (protein concentration, 0.9 mg/ml). Subsequently, the grids were washed three times with 50 mM Tris-HCl (pH 7.2) and immediately used for binding of in vitro assembly products (1 h at room temperature). After binding, the grids were washed three times with Tris-HCl (pH 7.2) and three times with water and stained with 1% uranyl acetate for 5 min. Excess stain was removed by touching the grid to a filter paper. After being stained, the grid was air dried for 5 min and analyzed with a Philips CM 120 transmission electron microscope.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Purification of N-terminally extended CA proteins and in vitro
assembly of spherical particles.
Previously, we had shown that in
vitro assembly of purified HIV-1 CA protein with or without the
C-terminally adjacent NC domain led to the formation of tubular
particles (22). These hollow cylinders are likely to share
structural features with mature HIV capsids, which exhibit a
cone-shaped morphology in thin-section electron microscopy (16,
17) and cryoelectron microscopy (13). To extend the in
vitro assembly system to the analysis of spherical HIV core-like
particles corresponding to the immature virion, we constructed
expression vectors for extended CA proteins. Previous studies with
recombinant baculoviruses for expression had shown that the C-terminal
p6 domain of Gag and most of the NC domain are not essential for
assembly and extracellular release of spherical particles (25,
46). Since the presence or absence of NC also did not alter the
in vitro assembly phenotype (22), we reasoned that the shape
of assembly products may be determined by the N-terminal MA domain.
Furthermore, a large internal deletion within the MA domain of HIV Gag
(amino acids 16 to 99) did not change the morphology of the immature
spherical protein shell (11). We therefore made a construct
(pET MA-CA) for expression of a protein containing the first 15 amino acids and the last 33 amino acids of the MA domain fused to CA
(MA-CA, containing a deletion from amino acids 16 to 99 of MA [Fig.
1A]). Following expression in E. coli BL21 DE3 cells, the
specific product (31 kDa) accumulated to about 10% of the total
E. coli protein after induction (Fig. 1B, lane 1) and
reacted with specific antisera against CA in immunoblot analysis (data
not shown).
MA-CA protein was purified essentially as described for HIV-1 CA
(22). Following high-speed centrifugation (Fig. 1B, lane 2),
precipitation with ammonium sulfate (lane 5), and anion-exchange chromatography, approximately 20 mg of MA-CA per liter of induced bacterial culture was obtained at a purity of >95% (lane 7). For in
vitro assembly, purified CA and MA-CA proteins were diluted to a
concentration of 3 mg/ml and dialyzed overnight against assembly buffer
(1 M NaCl [pH 8]). Samples from both reactions were analyzed by
negative-stain electron microscopy. Assembly into long and hollow
tubular structures with an external diameter of 50 nm was observed for
CA (Fig. 2A), while in vitro assembly of
MA-CA yielded spherical particles of heterogeneous size but no
cylinders (Fig. 2B). Both CA-derived cylinders and MA-CA-derived
spheres exhibited a regular substructure. The diameter of spherical
particles ranged from 20 to 100 nm, with approximately 50% of
particles showing a diameter of about 60 nm. Some incomplete and broken
spheres were observed, with stain penetrating into the interior of the particle, which indicated that they were hollow with a wall thickness of 5 to 6 nm (data not shown; see below). The MA-CA-derived
particles appeared mostly circular with occasional flat or angular
facets but without evidence for icosahedral symmetry (Fig. 2B).
Approximately threefold fewer spherical particles were observed
compared to CA-derived tubular structures, and we therefore used
antibody-coated grids for immobilization of in vitro assembly products
in most experiments. No difference in the shape or morphology of
spheres was observed when grids with or without antibodies were
analyzed in parallel (data not shown). Spherical particles were first
observed after dialysis for 15 min. Large precipitates but no spherical or tubular particles were detected when MA-CA or CA was not dialyzed but immediately adjusted to 1 M salt.
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MA-CA protein occurred in buffer of neutral to alkaline pH, at high
ionic strength, and at a protein concentration of 3 mg/ml or more (100 µM), similar to the conditions determined for CA (22).
Virtually no particulate structures were observed at pH 8 and 0.2 M
salt, and the efficiency of sphere formation was only 5 to 10% at 0.5 M salt compared to 1 M salt. No ordered structures were observed if
MA-CA protein was incubated at pH 6 and 0.1 M salt, while assembly
of spherical particles occurred when the salt concentration was raised
to 1 M, albeit at a threefold-lower efficiency than that at pH 8 and 1 M salt. Thus, in vitro assembly of MA-CA-derived spheres appeared
less sensitive to pH and more sensitive to salt concentration than did
assembly of CA-derived tubular particles (22). No
particulate structures were observed when assembly reactions (in 1 M
salt buffer [pH 8]) were performed at a protein concentration of 0.2 mg/ml MA-CA. Increasing numbers of particles were detected when the
protein concentration was raised above 1 mg/ml. Addition of 5 µM
Ca2+ had no effect on the efficiency of assembly or on the
morphology of spherical particles. Similarly, addition of RNA at 3%
(by weight) of protein or pretreatment with RNase A (0.2 mg/ml for 1 or
2 h) did not alter the yield or structure of assembly products. Spherical particles were unaltered following incubation in 0.5% Triton
X-100 for 150 min.
Effect of PR treatment and of cyclophilin A on in vitro
assembly.
To analyze the effect of PR treatment on in vitro
assembly, we incubated MA-CA with purified HIV-1 PR at a molar ratio
of 1:10 (E:S) either before or after performing the assembly reaction. Incubation of in vitro-assembled spherical particles (Fig.
3B) with PR for 10 min led to cleavage of
most of the MA-CA protein to MA and CA (Fig.
3A, lane 2). Spherical particles were completely disrupted under these
conditions, and no ordered structures were observed on the grid (data
not shown). Incubation of assembly products with PR for 1 h or
longer resulted in complete cleavage of MA-CA (lane 3) and formation
of long and irregular rod-like protein conglomerates with similar
overall dimensions to those of CA-derived cylinders (Fig. 3C). However,
these structures were not cylindrical and did not exhibit regular
substructures. Cleavage of MA-CA before in vitro assembly, on the
other hand, resulted in efficient formation of cylindrical structures
which were morphologically identical to CA-derived tubes (Fig. 3D). In
a separate experiment, MA-CA and PR were mixed and the entire sample
was dialyzed against assembly buffer during the cleavage reaction.
Dialysis for 6 h led to the formation of tubular structures which
were slightly more heterogeneous in diameter than CA-derived cylinders
(external diameters ranging from 40 to 60 nm [Fig. 3E]).
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MA-CA in the presence of CypA at a molar ratio of 1:1 also
completely disrupted particle formation (data not shown). Virtually no
spherical particles but small unstructured protein aggregates of
variable sizes were observed at a molar ratio of 3:1 (MA-CA to CypA
[Fig. 4E]), while assembly efficiency was not significantly altered when CypA was added at a molar ratio of 10:1. However, assembly products were considerably more heterogeneous in size and shape (Fig.
4D). Larger particles with a diameter up to 200 nm, as well as very
small particles, were observed. Furthermore, only 50% of particles
appeared spherical and various altered morphologies (elliptical,
indented, teardrop shaped [Fig. 4D]) were observed. Very
rarely, cylinders with similar dimensions to those of CA-derived particles, which had never been observed in the absence of CypA, were
also detected. We conclude that CypA affects the in vitro assembly of
both spherical and tubular particles, leading to a complete loss of
particle formation if equimolar amounts of CypA are used and to altered
assembly properties at substoichiometric concentrations. The effects of
CypA can be blocked by the immunosuppressive drug cyclosporine, and
assembly of both tubular and spherical particles was restored if 10 µM cyclosporine was added to an assembly reaction mixture containing
equimolar amounts of CypA and CA or MA-CA (data not shown).
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In vitro assembly of CA proteins with short N-terminal
extensions.
To determine whether the MA-CA protein contains a
specific shape-determining region giving rise to assembly of spherical particles, we constructed additional bacterial expression vectors for
N-terminally extended CA proteins. Besides MA-CA, containing the entire
MA and CA sequence, the following extended CA proteins were expressed
in E. coli: N15MA-CA (containing the first 15 amino acids of
MA fused to the N terminus of CA), MA100CA (from amino acid 100 of MA
to the C terminus of CA), MA115CA, MA120CA, and MA128CA. N15MA-CA and
MA100CA contain the N-terminal or C-terminal segment of MA that is also
present in the MA-CA protein. In parallel experiments, we also
analyzed the in vitro assembly of CA and of a truncated CA protein
lacking the N-terminal 13 amino acids of CA (CA13), which correspond
to a tightly folded 
-hairpin in the three-dimensional structure
model of CA (14, 19). A schematic representation of the
HIV-specific regions encoded by the various constructs and of the
expected expression products is shown in Fig.
5A. Following induction of E. coli BL21 DE3, all proteins were produced equally well and
accumulated to about 10% of total E. coli protein (Fig.
5B). Full-length MA-CA protein was poorly soluble after bacterial lysis
and was not further analyzed. N15MA-CA, MA100CA, MA120CA, MA128CA, CA,
and CA13 were purified as described above and were all obtained at a
purity of 90% or better.
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MA-CA-derived particles, these smaller spheres did not exhibit any
regular substructure. MA100CA, on the other hand, produced particles
very similar to those observed for MA-CA, albeit at more than a
100-fold-lower efficiency. Occasional particles with an average
diameter of 60 nm, a circular appearance, and a regular substructure
were detected (Fig. 6B). These results indicate that N-terminal
extensions of CA by heterologous (N15MA-CA) or homologous (MA100CA)
MA-derived sequences can block in vitro assembly of cylinders and
convert the assembly phenotype to spherical particles.
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MA-CA. However, these particles
were considerably more heterogeneous in morphology, and many broken or
incomplete particles were observed (Fig. 6C and D), in contrast to the
regular assembly products of MA-CA (Fig. 2B). Many half-shells
filled with stain were observed on the grid (Fig. 6C), which permitted
accurate determination of their wall thickness. Similar to the tubular
particles analyzed previously (22), the walls of these
spheres were approximately 6 nm thick. No tubular particles typical of
in vitro assembly of CA (Fig. 6E) were ever detected in MA120CA or
MA128CA. The shortest protein analyzed in the in vitro assembly system
contained a deletion of the first 13 amino acids of CA (CA13). This
protein yielded tubular particles at similar efficiency and of similar morphology to CA itself (Fig. 6F). CA13-derived particles were slightly more heterogeneous in diameter but otherwise indistinguishable from CA-derived particles. However, more unstructured protein aggregates were observed with CA13.
Assembly of N-terminally extended CA proteins in E. coli. The Gag protein of Mason-Pfizer monkey virus and fragments of the RSV Gag protein assemble into either spherical or tubular particles inside bacterial cells, and their assembly properties in E. coli correlate with the in vitro assembly properties of the respective purified proteins (8, 31). However, these proteins contain the RNA-binding NC domain and in vitro assembly occurs in an RNA-dependent manner in low-salt buffer. To analyze whether CA and N-terminally extended CA proteins also assembled into particulate structures inside E. coli cells, the respective expression vectors were transformed into E. coli and bacterial cells were prepared for thin sectioning 3 h after induction. Fixation and further preparation were performed on bacteria drawn into cellulose capillary tubes, which obviated the need for repeated centrifugations and facilitated the handling and preparation of specimens.
Induced bacteria harboring the CA expression vector readily showed the accumulation of intracellular cylinders (Fig. 7E). These cylinders were smaller in diameter than those observed in vitro (30 nm compared to 50 nm) and were considerably more branched. Furthermore, the cylinder lumen was difficult to detect in most cases, indicating that many particles had collapsed. MA-CA, containing the entire MA and CA regions of HIV-1, did not yield any ordered structures inside E. coli cells, and only large amorphous protein aggregates were observed (Fig. 7A). Similar results were obtained for MA120CA and MA128CA (data not shown), both of which had been shown to assemble into spherical particles in vitro (Fig. 6C and D). N15MA-CA gave rise to large arrays of small irregular particles of variable sizes very similar to the in vitro assembly products (Fig. 7B). MA100CA produced predominantly unstructured protein aggregates similar to MA-CA, but occasional regular spherical particles similar to those observed in vitro were also detected (Fig. 7C). Unexpectedly,MA-CA which assembled into
regular spherical particles with much higher efficiency in vitro gave a
similar result to MA100CA in induced bacteria. Only rare spherical
particles but predominantly unstructured protein aggregates were
detected (data not shown). Induced bacteria harboring pET MA115CA
(containing the last 18 amino acids of MA upstream of CA) showed large
spiral structures (Fig. 7D). These structures most probably correspond to two-dimensional sheet-like arrays of protein. Similar spirals of
slightly different morphology as well as unstructured protein aggregates but no regular cylinders were observed for CA13 inside bacterial cells (Fig. 7F).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this report, we have shown that fragments of the HIV-1 Gag
polyprotein can assemble into hollow spherical particles in vitro
provided that an N-terminally extended form of CA is used for assembly.
Cylindrical particles were produced, on the other hand, if CA protein
with its native N terminus or with a short N-terminal deletion
(CA13) was used. Formation of spherical particles did not require
any other viral or cellular protein or nucleic acid. In vitro assembly
of spheres occurred at neutral pH and high protein and salt
concentrations, similar to the previously described in vitro assembly
of cylindrical particles from purified CA (22). These
results indicate that sphere formation is also mediated by weak
hydrophobic interactions, most probably contributed by the CA domain.
C-terminal segments of Gag were not required for in vitro assembly of
spherical particles.
The observation that the presence of the N-terminal MA domain or a segment thereof determines the shape of the HIV core particle is in agreement with studies reporting the assembly phenotype of gag deletion mutants in tissue culture. Deletion of the entire p6 domain (28, 29, 46) and of most or all of the NC domain (25, 29, 46) did not prevent the release of HIV-like particles containing a spherical protein shell following baculovirus-mediated expression. Furthermore, large internal deletions within the HIV MA domain also had no apparent effect on the morphology of the resulting particles. A Gag protein lacking amino acids 16 to 99 of MA directed the budding of morphologically normal immature particles into the cisternae of the endoplasmic reticulum (11). A longer deletion mutant (amino acids 16 to 120 of MA) permitted extracellular release of particles (56), while deletion of almost the entire MA domain (amino acids 16 to 132, retaining only the first 15 amino acids of MA upstream of CA) led to accumulation of nonenveloped spherical particles with a similar morphology to authentic HIV (52). Lee and Linial (35) reported that replacing the entire MA domain of HIV-1 by a short signal for N-terminal myristoylation also permitted the assembly and release of spherical HIV-like particles, albeit at lower efficiency. In this case, the deletion included the first 10 amino acids of CA, indicating that the CA N-terminal region may not be essential for assembly of immature HIV particles. A similar result was obtained following baculovirus-mediated expression of a deletion mutant lacking codons 41 to 143 of the HIV gag reading frame (including the first 10 codons for CA), which led to the efficient release of immature particles with a morphology very similar to that of wild-type particles (4). While the expression products were quite different in these various studies, they all had in common that upstream sequences were present at the N terminus of CA. A similar phenotype was observed in the in vitro assembly system: spherical particles were formed when N-terminally extended CA proteins were used. These results suggest that morphogenesis of spherical particles may be controlled by the presence of additional sequences upstream of the CA domain on the polyprotein.
Only cylindrical particles, morphologically identical to CA-derived
cylinders, were obtained when the MA-CA protein was cleaved with
HIV-1 PR prior to the in vitro assembly reaction. This result indicates
that neither the synthesis as a polyprotein before cleavage nor the
presence of the MA peptide in the assembly reaction was responsible
for the assembly phenotype, but only the presence of additional
sequences at the N terminus of CA. Proteolytic separation of MA and CA
should therefore also be required for condensation of the mature
cone-shaped capsid shell. Mutation of the MA-CA cleavage site in the
context of an HIV-1 proviral clone led to noninfectious particles
containing an electron-dense RNP core and a thickening of the viral
membrane but no conical capsid shell (21). The additional
density at the membrane was most probably due to the CA domain, which
had not been removed from the membrane-attached MA domain. Recent
experiments showed that cleavage at both sides of SP1, separating NC
and SP1 from the CA domain, is also essential for virion maturation,
and it has been suggested that HIV-1 maturation is a sequential process
governed by the rate of processing at individual cleavage sites
(58).
The three-dimensional structure model of the N-terminal segment of
HIV-1 CA, obtained by nuclear magnetic resonance spectroscopy (19) and X-ray analysis (14, 42), provides a
likely explanation for the role of N-terminal extensions of CA during
assembly. The structural studies indicated that CA is a largely helical
protein with several putative interfaces for intermolecular
interaction. The two most prominent interfaces are the C-terminal
dimerization domain (15) and the N-terminal part of CA
including helices 1 and 2 (amino acids 17 to 43 of CA) and a N-terminal
-hairpin structure from amino acids 1 to 13 of CA (14,
19). In the cleaved CA protein, the N-terminal
NH2+ group of Pro1 of CA is oriented toward
helix 3 and forms a salt bridge with Asp51, which is presumed to
stabilize the -hairpin (19). Preventing the formation of
this salt bridge by substitution of the Asp codon in a proviral clone
abolished condensation of the mature cone-shaped core, and the same
substitution also prevented the formation of tubular particles in the
in vitro assembly system (55a). N-terminal extensions of CA
are predicted to block formation of the -hairpin, because additional
residues would sterically clash with the first helices and no
positively charged group would be available for interaction with Asp51
(19). Presumably, N-terminal extensions cause unfolding of
the -hairpin structure into an extended conformation, making the
MA-CA bond accessible for proteolytic cleavage. This alteration may
prevent cylinder formation, either because the -hairpin structure is
an essential determinant of the CA interface in the cylinder or because
the flexible extended N terminus interferes with cylinder formation. In
either case, the assembly of spherical particles would be determined
primarily by CA-mediated interactions and not by additional
protein-protein interactions provided by the N-terminally fused
sequences. This model can also explain why short N-terminal extensions
of only 5 amino acids (MA128CA), as well as heterologous MA-derived
sequences (N15MA-CA), can convert the assembly phenotype from cylinders to spheres. It should be noted, however, that particles derived from
MA-CA appeared more regular and were mostly intact whereas MA128CA
and other shorter proteins yielded heterogeneous particles which were
mostly incomplete. These results indicate that MA sequences may
contribute to the morphogenesis of the spherical protein shell in
additional ways besides anchoring the Gag protein to the membrane and
keeping the N terminus of the CA domain in an unfolded state. All
particles obtained in the in vitro system were smaller than the Gag
protein shells of immature HIV particles, and additional segments of
Gag and/or nucleic acids or cellular proteins may be required for the
assembly of particles of regular size.
The N-terminal sequence of CA corresponding to the -hairpin is not
required for the assembly of spherical protein shells (4,
35). Our in vitro assembly experiments have shown that it is also
not required for the formation of tubular particles of similar
morphology to CA-derived cylinders. Thus, the N-terminal region of CA
appears to have a decisive influence on particle morphogenesis when it
is present in the protein, although it is not required for either
cylinder or sphere formation. Most probably, this part of the protein
serves as a molecular switch which is triggered by PR-mediated
proteolytic cleavage during maturation. Gamble et al. (14)
reported that the N-terminal interface of CA occludes 800 Å2 of accessible surface area and the -hairpin
structure contributes 230 Å2 to this value. The residual
interactions mediated by the two N-terminal helices of CA may be
sufficient for cylinder formation in the in vitro system, where only a
single protein (CA13) is present. However, no cylinders were
detected for CA13 in the more complex environment inside bacterial
cells, suggesting that the -hairpin contributes to cylinder
formation. Full-length CA protein, on the other hand, also gave rise to
cylinders inside bacterial cells, although these structures appeared
less ordered and were mostly collapsed.
CypA is a cellular protein that is incorporated into HIV-1 particles at
a ratio of 1:10 (relative to CA) and is important for virus replication
(12, 38, 54). It binds to a flexible loop in CA
(14) and is incorporated into the particle via its interaction with the CA domain of Gag (38). Based on
analysis of individual steps in viral replication, it has been
suggested that CypA plays a role in viral entry, at an early step
preceding reverse transcription (5). Structural analysis of
CA-CypA cocrystals (at a CA-to-CypA ratio of 1:1) showed that CypA
binding may inhibit interactions between planar CA strips, thereby
preventing the formation of higher-order structures (14).
Given the 10-fold-lower CypA-CA stoichiometry in the virion, this
inhibition may have a destabilizing effect preparing the core for
disassembly (14). In vitro assembly of CA or MA-CA in the
presence of CypA at a molar ratio of 1:1 prevented formation of ordered
structures, as suggested by the structural studies. Furthermore, a
severe reduction in assembly efficiency and formation of significantly shorter cylinders was also observed at a CypA-to-CA molar ratio of 1:3
and no particles were detected for MA-CA at this ratio. When CypA
was added at a molar ratio of 1:10, as found in the virion, on the
other hand, there was no significant difference in the assembly
efficiency for either CA or MA-CA compared to experiments in the
absence of CypA. Significantly longer cylinders which had lost their
tendency to aggregate were observed in the case of CA. Thus, at least
in our in vitro system (1 M NaCl [pH 8]), the presence of CypA at a
concentration of 10% of the CA level does not interfere with cylinder
formation and the observed phenotype could be explained by a chaperone
function of CypA which may enhance CA reorganization, resulting in
longer cylinders in the in vitro system. It is interesting that the
same concentration of CypA altered the shape of MA-CA-derived
particles from spheres to polymorphic structures with occasional
cylinders with diameters of 50 nm, which had never been observed in the
absence of CypA. Conceivably, CypA may also destabilize the spherical
protein shell during virion maturation and facilitate the formation of
the cone-shaped core. Even minor alterations in the ordered
reorganization of the mature core may produce subsequent defects in
virus entry.
| |
ACKNOWLEDGMENTS |
|---|
We are grateful to A. Billich for the CypA vector and to J. Konvalinka for ETG. We thank C. Hegert for preparation of recombinant proteins and for sequence analysis, B. Müller for critically reading the manuscript, and I. Ellhof for expert assistance and photography. We are grateful to W. Sundquist, S. Campbell, and V. Vogt for communicating their experimental results on in vitro assembly prior to publication.
This work was supported in part by a grant from the German ministry for education and research to H.-G.K.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Heinrich-Pette-Institut, Martinistr. 52, D-20251 Hamburg, Germany. Phone: 40 48051-241. Fax: 40 48051-184. E-mail: hgk{at}hpi.uni-hamburg.de.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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