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INTRODUCTION |
The products of the human immunodeficiency virus
(HIV-1)1 assembly are 125-nm
diameter, enveloped, immature, and mature virus particles (1). The
principal internal protein component of HIV-1 virions is encoded by the
viral gag gene. Immature HIV-1 particles are composed of
unprocessed 55-kDa Gag proteins (Pr55Gag) and appear to have an
electron dense layer of material juxtaposed to the inner faces of their
lipid envelopes (1, 2). However, during or just after virus particle
release from cells, Pr55Gag proteins are processed by the HIV-1
protease into the major mature Gag proteins matrix (MA), capsid
(CA), nucleocapsid (NC), and p6. Proteolytically processed HIV-1
particles adopt a mature morphology, in which electron-dense
material reorganizes into a central cone- or rod-shaped
structure (1).
Complete or partial structures for the MA, NC, and CA Gag proteins have
been determined in nuclear magnetic resonance spectroscopy (NMR) and
x-ray crystallographic studies (3-10). The matrix protein, which
interacts with the HIV-1 envelope (Env) protein complex (SU/TM or
gp120/gp41) and serves a membrane binding function, forms
characteristic trimers in three different crystal forms (5). The NC
domain possesses two Cys-His finger motifs, which coordinate zinc ions
and are important in RNA binding (10). The capsid domain, which is
crucial to virus particle assembly (11), has proven more difficult.
However, partial structures of HIV-1 CA have shown that it is composed
primarily of
helices (6-9), an unexpected contrast with the
B-barrel or jellyroll structures, which constitute the capsid proteins
of a number of other animal viruses.
While NMR and x-ray structures of individual HIV-1 Gag proteins provide
clues as to potential interprotein contacts within HIV-1 particles,
much remains obscure. Several reports have suggested that mature and
immature HIV-1 virions show icosahedral symmetry (1, 2), but high
resolution support for this suggestion is needed. Analysis of mature
virus cores has been hampered by difficulties in their isolation,
although the recent demonstration that RNA-Gag (CA plus NC) complexes
can be assembled in vitro (11) may lead to an accurate model
for mature HIV-1 cores. However, the most developed model for the
structure of HIV-1 is based on the immature virus form. In particular,
examination of negatively stained membranes and immature HIV particles
produced from a baculovirus vector led Nermut et al. (2) to
propose a "fullerene-like" particle structure. By this model,
Pr55Gag proteins at viral or cell membranes appear to form hexamer
rings surrounding protein-free "holes." Although first order
diffraction reflections at 65 Å
1 were barely
discernible, results of averaging five subimages, assuming 6-fold
rotational symmetry, suggested an arrangement in which only one Pr55Gag
monomer would be shared by two adjacent hexamer units (2). More recent
cryo-EM analysis of disrupted HIV-1 virus-like particles also showed
that Pr55Gag proteins appear to form a cage-like structure, although in
this case, cage hole-to-hole distances appeared to be on the order of
48 Å (13). In our current study, we describe a method for the analysis
of HIV-1 capsid proteins, assembled in vitro on a lipid
monolayer. Our results show that membrane-bound HIV-1 CA proteins
organize into a hexamer-trimer cage-like network that explains previous
structural results in a consistent model.
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EXPERIMENTAL PROCEDURES |
Lipid Monolayer Incubations and Electron Microscopy--
The
His-HIVCA protein has been described previously (11) and consists of
the HIV-1 coding region with an amino-terminal histidine tag and a
3.3-kDa COOH-terminal extension. The His-HIVCA protein was purified and
analyzed as described previously (11, 15). For monolayer incubations,
we followed our standard protocol (14) with the subphase containing
0.5-2.0 mg/ml His-HIVCA in subphase buffer at pH 7.8 or 8.3. Subphase
solutions were overlayered as described (14) with 1:1 hexane:chloroform
containing 200 µg/ml phosphatidylcholine (Avanti Polar Lipids) plus
50 µg/ml nickel-charged DHGN
(1,2-di-O-hexadecyl-sn-glycero-3-(1'-2''-R-hydroxy-3'-N-(5-amino-1-carboxypentyl)iminodiacetic acid)propyl ether). After overnight incubation at 25-30 °C, arrays were lifted onto lacey grids, washed 30 s in distilled water, and
either negative-stained in 1.33% uranyl acetate or plunge-frozen in
liquid ethane. Monolayer arrays were viewed and photographed at the
Portland Veterans Affairs Hospital JEOL JEM12OOEX, the University of
Oregon Philips CM12, or the EMBL-Heidelberg Philips CM200-FEG as
described previously (14). Crystalline areas on micrographs were
identified by optical diffraction, and images were scanned and
digitized using either the EMBL-Heidelberg Perkin-Elmer 1010GM flat-bed
scanner at 2.63 Å/pixel or an Optronics CCD mounted on a dissecting
microscope at 5.25 Å/pixel. Since CM200-FEG images used in averages
were taken at 900 nm defocus, corresponding to a first contrast
transfer function zero of approximately 15.1 Å, contrast transfer
function correction was not applied in our analyses.
Image Analysis--
Image analysis steps employed both the MRC
(16, 17) and SPIDER (18) image analysis packages. Initially, ordered
areas, identified by optical diffraction, were scanned, converted to MRC image files, boxed using BOXMRC, Fourier-transformed, and viewed as
diffraction patterns on SPECTRA (19). Diffraction patterns were indexed
by hand, masked, and back-transformed to yield crude filtered images
(14, 17). For more thorough analysis, SPIDER (18) real space operations
were used. Twenty 168.3 Å × 168.3 Å windows, corresponding to
approximately two p1 unit cells from the initial filtered images, were
picked using the SPIDER operation WI from scanned negative 8347 that
had been Gaussian low pass filtered to 32.9-Å resolution. The filtered
windows were aligned using the AP RA and AP SA operations and averaged
using the AD command. The averaged image then was used as a reference with the programs CC and PK D to pick three sets of cross-correlation peaks: 100 image windows from negative 8347; the best 280 cross-correlation peaks from five digitized negatives; and 1000 windows
from the five negatives. Windows from each data set were aligned to a
black and white contrast version of the averaged reference window.
Aligned windows in each set were averaged using the SPIDER AD
operation. For statistical analysis, data sets were halved, averaged,
and compared with obtain Fourier ring correlation (FRC) indices at different rings of resolution using RF M. Image averages subsequently were filtered using FQ to 24.0 Å resolution in accordance with FRC
results.
For rotational averaging, a rotational series of the averaged, negative
8347 data set was generated using RT, and rotated images were compared
with the unrotated original using RF M, identifying rotational
correlation peaks. The original plus its 60, 120, 180, 240, and 300°
rotations were averaged to give a rotationally averaged image: the FRC
of the 0, 120, and 240° average versus the 60, 180, and
300° average was 0.90 at 36.0 Å resolution and 0.62 at 22.4 Å resolution. To simulate the lower resolution obtained in previous
studies, the 6-fold rotationally averaged image was Gaussian low pass
filtered to 52.6 Å resolution using the FQ operation. For alignment of
HIV-1 matrix protein trimers onto His-HIVCA arrays, the MA trimer unit
from the Brookhaven Protein Data Bank (accession number 1H1W) was
imaged on MIDAS, rotated so that the 3-fold axis was normal to the
projection and the predicted internal face of the virus (4, 5), and
scaled. The scaled MA trimer image was converted to SPIDER image
format, used to generate a 120° rotational series of windows at 1°
intervals, and each rotated trimer was cross-correlated to the six-fold
rotationally averaged His-HIVCA image. The best alignment had the
highest value cross-correlation peak. For display purposes, all images
were converted from either MRC or SPIDER image formats to TIFF files
and displayed using Adobe Photoshop software.
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RESULTS AND DISCUSSION |
To circumvent difficulties that have limited the structural
analysis of HIV-1 particles, we have adapted the lipid monolayer method
of two-dimensional protein crystallization (22) for analysis of HIV-1
Gag protein interactions. To do so, lipid monolayers were made from
phosphatidylcholine (PC) plus a dialkylglycerol derivative, DHGN, which
carries a nickel-chelating head group that binds histidine-tagged
(His-tagged) proteins (14). The HIV-1 Gag protein chosen for analysis,
His-HIVCA, was an HIV-1 (HXB2) capsid protein derivative with an
amino-terminal His tag (11). Our rationale for using the matrix-deleted
His-HIVCA protein was that the His tag-DHGN interaction should
substitute for the membrane binding function of MA. Furthermore,
studies have shown that large HIV-1 matrix deletions are
compatible with virus particle assembly (19, 20). Although His-HIVCA
also lacks the NC and p6 COOH-terminal domains of Pr55Gag, this did not
prevent his-HIVCA from mimicking interactions observed for the
full-length protein (see below).
The recombinant His-HIVCA protein was produced in Escherichia
coli and purified to greater than 95% homogeneity by two rounds of nondenaturing nickel-chelate chromatography (15). As a conformation check, His-HIVCA and HIV-1 CA isolated from virus particles were subjected in parallel to a partial proteolytic treatments, and each
protein yielded a characteristic COOH-terminal 14-kDa partial trypsin
fragment. When His-HIVCA proteins were used in monolayer incubations
with PC plus DHGN, protein arrays with small patches of crystallinity
were observed with either stained samples (Fig. 1A) or samples frozen in
vitreous water. While such regions could be difficult to detect in
cryo-EM micrographs (see Fig. 1B), they were evident in low
pass filtered cryo-EM images (Fig. 1C). In small regions,
His-HIVCA proteins were ordered well enough to obtain distinct
diffraction patterns, as shown in Fig. 1D. Despite sample
drift for this negative, the reflections at 26.1 Å
1 are
clearly evident, and the primitive (p1) unit cell of a = 74.8 Å, b = 126.2 Å,
= 89.3° is reminiscent
of the a = 79.2 Å, b = 137.5 Å,
= 89.7° unit cell observed for capsid proteins of another retrovirus,
the Moloney murine leukemia virus (M-MuLV) (1, 14). Interestingly,
back-transformation of masked diffraction patterns from small ordered
regions yielded images (Fig. 1E) showing a cage-like
structure of proteins (light) surrounding protein-free cage holes
(dark), similar to those seen for M-MuLV CA (14) and HIV-1 Pr55Gag
(2).

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Fig. 1.
Two-dimensional arrays of His-HIVCA on lipid
monolayers. A, negatively stained His-HIVCA array formed on
PC plus DHGN. The contrast on the scanned EM negative was inverted so
that protein-free regions are dark. The scale bar
is 53.6 nm. B, unfiltered cryo-EM image of His-HIVCA arrays.
Protein-free regions appear dark, and the size
bar indicates 50 nm. C, the image from B was
Gaussian low pass filtered to 32.9 Å resolution using the SPIDER
operation FQ. The size bar indicates 50 nm. D,
the power spectrum from a 135 × 135-nm His-HIVCA array in
vitreous ice was calculated from the scanned image and can be indexed
as a primitive (p1) unit cell with a = 74.8 Å,
b = 126.2 Å, = 89.2°. The circled
2, 4 and 2,4 reflections are at 26.1 Å 1. E,
the calculated diffraction pattern from an ordered section of negative
8347 was displayed, indexed, and masked using SPECTRA (18) and
back-transformed to yield the filtered image, in which protein-free
areas appear dark, while electron-dense areas are bright. The thick black lines indicate p1 unit
cell dimensions of 74.8 and 126.2 Å, while the thin black
line shows the box format used to pick windows to average for the
cross-correlation reference in F. F, white spots
indicate cross-correlation peaks from B, using a reference
averaged from 20 168.3 × 168.3-Å negative 8347 windows that were
boxed using the two-unit cell box from E as a
guide.
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Because the sizes of ordered His-HIVCA patches were not large enough to
permit a thorough analysis by standard diffraction analysis methods
(16, 17), we opted to analyze His-HIVCA monolayer structures by a
modification of single particle analysis methods (18). To do so, an
image-averaged reference corresponding to approximately two p1 unit
cells (168.3 Å × 168.3 Å) was used to locate cross-correlation peaks
from five separate digitized micrographs. As expected from the low pass
filtered image (Fig. 1C), peaks tended to be located in
small ordered patches throughout the micrographs (Fig. 1F).
From cross-correlation maps, three data sets were derived. These
consisted of 100 image windows from our highest quality micrograph, 280 windows corresponding to the best cross-correlation peaks, and 1000 windows (total) from the five micrographs. All windows were aligned to
a black and white averaged reference window, after which data sets were
merged to obtain 100-image, 280-image, and 1000-image averages (Fig.
2, A-C). To evaluate results,
each data set was halved, and averages from each half set were compared with their counterparts at different rings of resolution. As shown in
Table I, for each data set, Fourier ring
correlation values fall off at resolutions better than 24.0 Å. These
results are consistent with the observation of reflections at 26.1 Å
1 (Fig. 1D), and Figs. 2, D-F,
have been filtered to this resolution limit.

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Fig. 2.
Arrangement of HIV-1 capsid proteins bound to
a lipid monolayer. Image windows from cryo-EM micrographs of
two-dimensional His-HIVCA arrays were aligned and averaged as described
under "Materials and Methods." Averages are displayed as 168.3 × 168.3-Å areas with protein-free zones dark and
electron-dense areas light. Averages were either unfiltered,
or Gaussian low pass filtered to 24.0-Å resolution, in accordance with
results from Table I. A, 100-window average, unfiltered;
B, 280-window average, unfiltered; C, 1000-window
average, unfiltered; D, 100-window average, filtered; E, 280-window average, filtered; F, 1000-window
average, filtered.
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Table I
Phase residual and Fourier ring correlation (FRC) values of averaged
data sets at different resolution ranges
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The most obvious features of the His-HIVCA structures shown in Fig. 2
are the large circular holes of 26.3 Å in the protein cage, which are
bordered by six rectangularly shaped protein units. However, each
circular hole also is surrounded by six smaller protein-free zones,
which in turn are bordered by three rectangular units. Thus,
membrane-bound HIV-1 capsid appears to form a cage consisting of
hexamer and trimer units. Since previous lower resolution studies on
in vivo derived immature HIV-1 particles employed a 6-fold
rotational averaging scheme in image reconstructions (2), we wished to
assess the results of rotational averaging for comparative purposes. As
shown in Fig. 3A (top
panel), the large cage holes in the HIV-1 capsid arrays appeared
as 6-fold rotational axes, and the averaged image correlated with its
60° rotation to about 21.3 Å (Fig. 3A, bottom panel).
Six-fold averaging increased the distinction of putative monomer units
(Fig. 3B), and when this image was filtered to 52.6 Å resolution, the four rectangular units separating each trimer hole
appeared as single electron-dense units (Fig. 3C). The
resultant image compares well with HIV-1 Pr55Gag arrays at membranes
and in disrupted immature virus particles (2, 13). In particular, the
74.2-Å hexamer to hexamer cage hole spacing that we observe (Figs. 2
and 3) is consistent with the spacing observed for Pr55Gag proteins
arranged on the plasma membranes of baculovirus vector-infected cells
(2). Since that study used negatively stained samples and was at low
resolution, we believe that trimer cage holes were not resolved, much
as they are not resolved in our low pass filtered image (Fig.
3C). In contrast, cryo-EM analysis of disrupted immature
HIV-1 particles has shown a cage hole center-to-center spacing of
approximately 48 Å (13), which is in good agreement with our
observation of trimer to hexamer cage hole spacing of 44.9 Å. Given
the above observations, it appears that membrane-bound HIV-1 CA
proteins assemble in a fashion similar to the full-length Pr55Gag
proteins. This may seem surprising, since Gag protein nucleocapsid (NC) domains, possibly through associations with RNA, exert a strong influence on retrovirus particle assembly (12, 14). However, the
similarity of our HIV-1 CA projection structure (Figs. 2 and 3), and
that of Pr55Gag proteins in virus-like particles (2), is consistent
with indications that MA, CA, and NC domains in Pr55Gag molecules stack
roughly as long rods (13). We thus hypothesize that NC and RNA
interactions do not alter the basic arrangements of membrane-tethered
capsid proteins, at least at our level of detection.

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Fig. 3.
Rotational averaging and comparison with
virus particle and atomic structures. A, top panel, the
unfiltered average from Fig. 2A was used to generate a
360° rotational series of images, which were compared with the
original, unrotated average. Fourier ring correlation (FRC) values at
33.7-Å resolution were plotted versus rotation angles and
show six peaks. Bottom panel, the averaged image from Fig.
2A was compared with 30° (filled squares) or
60° (open squares) rotated versions of itself, and results
of the comparisons were plotted as FRC indices versus resolution in reciprocal space. For the 60° comparison, the FRC was
0.5 at 21.3 Å. B, the 6-fold rotationally averaged image
from the 100-window data set is shown with dark protein-free
zones and electron-dense areas in green. The image size is
168.3 × 168.3 Å. C, to simulate lower resolution
results obtained with immature HIV-1 virus-like particles produced
in vivo (2), the rotationally averaged image from
B was filtered to 52.6-Å resolution. D, the HIV-1 matrix protein trimer structure (5) (Brookhaven Protein Data Bank
accession number 1H1W) in blue was scaled and fitted with
the His-HIVCA image (purple) to maximize projection overlap as described under "Materials and Methods." After identification of
the best-fit cross-correlation peak, the scaled matrix trimer, depicted
as a space-filling model, was oriented so that its predicted membrane-binding face (4, 5) is away from the reader. In this
representation, matrix residues 11-32 and 87-100 are oriented toward
hexamer holes, where HIV-1 gp120/gp41 envelope protein complex
cytoplasmic tails have been postulated to reside (2).
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Although the HIV-1 CA hexamer-trimer arrangement agrees with available
data on immature HIV-1 patches, it differs slightly from the
hexamer-hexamer arrangement, which M-MuLV capsid proteins form on
membrane monolayers (14). In particular, M-MuLV capsid proteins form
arrays in which roughly symmetrical hexamer cage holes are surrounded
by six skewed hexamer cage holes. Also, in contrast with HIV-1 CA
arrays, in which each monomer unit contributes to one hexamer and one
trimer, M-MuLV monomer units each contribute to one symmetrical and two
skewed cage holes (14). Furthermore, at hexamer vertices, it appears
that putative HIV-1 CA monomers coordinate with three other monomer
units, while M-MuLV monomers coordinate only with two additional
monomers. However, both M-MuLV and HIV-1 arrangements suggest that
cytoplasmic portions of the retroviral Env proteins may be accommodated
in the hexamer cage hole regions. For HIV, if this is the case, then
one might expect that the Pr55Gag matrix domains ordinarily should be
positioned between the viral membranes and the CA domains. In keeping
with this hypothesis, when HIV-1 matrix trimers as observed in
three-dimensional crystals were oriented toward viral membranes as
predicted previously (3-5) and scaled to our projection structure,
they overlaid neatly onto the capsid trimer units (Fig. 3D).
In this structure, matrix residues 11-32 and 87-100 are oriented
toward hexamer holes and could interact with the cytoplasmic tails of
HIV-1 gp120/gp41 Env protein trimers (21), which have been postulated
to localize to cage holes (2). While the extent to which our system
mimics authentic HIV assembly is unclear, we believe that our results, will help identify protein-protein interactions in HIV virions that can
be targeted for antiviral therapies.
We are grateful for the help we have received
from Lori Farrell, Sonya Karanjia, Jenny Stegeman-Olsen, Mike Yamauchi,
Yuanjui Rui, Xiumin Zhao, Brent Gowen, Russell Jones, Charles Meshul, Eric Schabtach, Dick Brennan, Darrick Carter, Mike Schmid, and Jackson
Shea.