Tài liệu Báo cáo khoa học: The capsid protein of human immunodeficiency virus: intersubunit interactions during virus assembly doc

MINIREVIEW
The capsid protein of human immunodeficiency
virus: intersubunit interactions during virus assembly
Mauricio G. Mateu
Centro de Biologı
´
a Molecular ‘Severo Ochoa’ (CSIC-UAM), Universidad Auto
´
noma de Madrid, Spain
Introduction
During HIV-1 morphogenesis [1,2], the capsid protein
(CA; or p24) participates in two distinct assembly
events. The first occurs inside the cell and involves the
Gag polyprotein, of which CA constitutes a part. A
spherical capsid comprising up to 5000 Gag subunits is
formed through self-association around a dimer of the
viral RNA genome, which is encapsidated along with
several viral and cellular proteins. Assembly-competent
Gag molecules are bound to the plasma membrane
and may directly interact with molecules of the viral
envelope polyprotein, which are embedded in the
membrane. Thus, condensation of the capsid drives its
coating by an envelope polyprotein-containing lipid
bilayer. As a result of this morphogenetic process, an
immature, non-infectious HIV-1 particle buds from the
infected cell.
Keywords
capsid; conformational stability and
dynamics; human immunodeficiency virus;
molecular recognition; protein association;
protein conformation; protein structure–

function relationships; virus assembly
Correspondence
M. G. Mateu, Centro de Biologı
´
a Molecular
‘Severo Ochoa’, Universidad Auto
´
noma de
Madrid, Cantoblanco, 28049 Madrid, Spain
Fax: +34 91 1964420
Tel: +34 91 1964575
E-mail:
Website: />mkfactory.esdomain/webs/CBMSO/
plt_LineasInvestigacion.aspx?
IdObjeto=19&ChangeLanguage=2
(Received 23 February 2009, revised 12
August 2009, accepted 20 August 2009)
doi:10.1111/j.1742-4658.2009.07313.x
The capsid protein (CA) of HIV-1 is composed of two domains, the N-ter-
minal domain (NTD) and the C-terminal domain (CTD). During the
assembly of the immature HIV-1 particle, both CA domains constitute a
part of the Gag polyprotein, which forms a spherical capsid comprising up
to 5000 radially arranged, extended subunits. Gag–Gag interactions in the
immature capsid are mediated in large part by interactions between CA
domains, which are involved in the formation of a lattice of connected Gag
hexamers. After Gag proteolysis during virus maturation, the CA protein
is released, and approximately 1000–1500 free CA subunits self-assemble
into a truncated cone-shaped capsid. In the mature capsid, NTD–NTD
and NTD–CTD interfaces are involved in the formation of CA hexamers,
and CTD–CTD interfaces connect neighboring hexamers through homodi-

merization. The CA–CA interfaces involved in the assembly of the imma-
ture capsid and those forming the mature capsid are different, at least in
part. CA appears to have evolved an extraordinary conformational plastic-
ity, which allows the creation of multiple CA–CA interfaces and the occur-
rence of CA conformational switches. This minireview focuses on recent
structure–function studies of the diverse CA–CA interactions and interfaces
involved in HIV-1 assembly. Those studies are leading to a better under-
standing of molecular recognition events during virus morphogenesis, and
are also relevant for the development of anti-HIV drugs that are able to
interfere with capsid assembly or disassembly.
Abbreviations
CA, capsid protein of HIV-1; cryoEM, cryoelectron microscopy; cryoET, cryoelectron tomography; CTD, C-terminal domain of CA; EM,
electron microscopy; H–D, hydrogen–deuterium; MA, matrix protein; MHR, major homology region; MLV, murine leukemia virus; NC,
nucleocapsid protein; NTD, N-terminal domain of CA; RSV, Rous sarcoma virus.
6098 FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS
The second capsid assembly event occurs upon bud-
ding of the immature virion. The viral protease-medi-
ated processing of Gag into several independent
proteins and peptides leads to the disassembly of the
spherical capsid inside the virion. Some of the folded
domains of Gag are then able to reassemble as inde-
pendent proteins. The matrix protein (MA; the Gag
N-terminal domain), remains associated with the viral
membrane, forming a discontinuous inner layer. The
nucleocapsid protein (NC) remains associated with the
viral RNA to form the nucleocapsid. In addition, some
1000–1500 subunits out of a larger number of CA mol-
ecules, released as a two-domain protein, self-assemble
into a truncated cone-shaped, hollow structure, namely
the mature HIV-1 capsid. The mature capsid and the

nucleocapsid that it contains constitute the viral core.
As a result of this dramatic structural rearrangement,
the immature virion turns into a mature, infectious
virion.
During the viral cycle, CA is present in different
structural environments: as a part of Gag, as an
unassembled protein, and as an independent protein
forming the mature capsid. Remarkably, the CA poly-
peptide appears to have evolved an extraordinary con-
formational plasticity, which allows the creation of
diverse CA–CA interfaces and other CA–ligand inter-
faces during HIV-1 morphogenesis. The present mini-
review summarizes the results of recent studies
regarding the diverse CA–CA interactions and inter-
faces involved either in the assembly of the immature
HIV-1 capsid, or in the reassembly of CA into a
mature capsid. The detailed description of the structure
and conformational dynamics of these interfaces,
together with a quantitative dissection of their energet-
ics, is providing deeper insights on the molecular recog-
nition events responsible for the assembly and stability
of the retrovirus capsid, and of viral capsids in general.
Moreover, the successful rational or semi-rational
design of anti-HIV drugs that are able to impair capsid
assembly or disassembly [3] may critically depend on
the availability of a sufficiently detailed structural and
energetic definition of those interfaces.
Identification of CA–CA interfaces in
the immature HIV-1 capsid
Although the full-length Gag protein can be isolated in

soluble form as a homodimer or homotrimer, its
atomic structure has not been determined. However,
the atomic structures of the separate Gag domains
from HIV-1 (and other retroviruses) have been solved
by X-ray crystallography and ⁄ or NMR spectroscopy.
The N-terminal domain of CA (NTD) and the C-ter-
minal domain of CA (CTD) are small, globular and
mainly helical. NTD contains a-helices 1–7 of CA, and
is connected by a flexible linker to CTD, which
contains a small 3
10
-helix, an extended strand and
a-helices 8–11 of CA (corresponding to helices 1–4 of
CTD) [4–7].
Structural, biochemical and mutational analyses of
the immature HIV-1 capsid have been carried out on
immature virions, or on capsid-like particles that can
be assembled in vitro from full-length or truncated
Gag molecules in the presence of nucleic acid and ⁄ or
other components [8–16]. Electron microscopy (EM) of
negatively stained immature capsid-like particles [8], as
well as cryoelectron microscopy (cryoEM) [14,15] and
cryoelectron tomography (cryoET) [17,18] of immature
capsid-like particles or virions, has revealed a layered
organization, which can be interpreted based on bio-
chemical evidence and the superposition on the elec-
tron density maps of the atomic structure of each
domain. The Gag subunits are radially extended, with
the N-terminal domain, MA, associated with the inner
layer of the viral membrane, and the remaining

domains forming the innermost protein layers accor-
ding to their positions in Gag: MA, CA-NTD,
CA-CTD, the spacer peptide SP1, NC, the spacer
peptide SP2 and the C-terminal peptide p6, with the
NC domain associated with the viral RNA.
CryoEM imaging of immature HIV-1 capsids [14,15]
has shown that Gag is organized as a lattice of hexa-
mers. Pseudoatomic models obtained by fitting the
atomic structures of both CA domains in cryoET maps
of immature HIV-1 virions [17,18] indicate that poly-
merization of Gag occurs mainly through the forma-
tion of an intermediate layer of cup-shaped structures,
each made of one hollow hexameric ring of CA
domains placed immediately above a stem formed by
six SP1 segments (Fig. 1).
Mutational analyses are consistent with this view.
Several Gag domains and spacer peptides were shown
to be involved in the assembly and stability of the
immature HIV-1 capsid, but CA plays a major role
[19–24]. However, the specific CA–CA interfaces
involved in Gag hexamerization and the joining of
neighboring hexamers in the immature HIV-1 capsid
lattice are still unclear. NTD has been shown to be
non-essential for the assembly of the immature capsid
[19]. On the other hand, in a comprehensive muta-
tional analysis [23], it was found that mutations that
impaired immature capsid assembly are located both
in NTD (helices 4–6) and in CTD (the loop between
helices 7 and 8 and helix 9).
Helix 9 forms a major part of the dimerization inter-

face in CA and in the free CTD [6,7]. Mutational
M. G. Mateu Capsid protein interfaces in HIV-1 assembly
FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS 6099
analyses [23] indicate that this interface could be
involved in the structural organization of the immature
capsid. However, a mutant CTD with a single amino
acid deletion has been recently crystallized as a
domain-swapped dimer [25]. The domain-swapped
interface did not involve helix 9, but instead the major
homology region (MHR), a highly conserved, 20-
amino acid stretch folded as a strand-turn-helix-8
motif. Because of this unusual conservation, numerous
mutational analyses have focused on this stretch, and
have revealed that the MHR is important for many
different steps in the HIV-1 life cycle, including the
assembly of both the mature capsid, where it forms
part of the NTD–CTD interface (see below), and of
the immature capsid [20,23]. Because the MHR also
forms a major part of the domain-swapped dimeriza-
tion interface in the mutant CTD structure, it has been
proposed that this interface could participate in the
assembly of the immature HIV-1 capsid [25]. The
structural and functional studies performed to date
may be not discriminating enough to clearly favor the
involvement of either the domain-swapped or the non-
swapped CTD dimerization interfaces (or both) in the
assembly and stability of the immature HIV-1 capsid.
In summary, in a model [17] of the immature HIV-1
capsid that may be consistent with the experimental
results achieved to date: (a) the capsid lattice is formed

by Gag hexamers that are stabilized mainly by a six-
helix bundle of SP1 peptides; (b) the hexamers are
joined through CTD homodimerization (with the inter-
face involving either helix 9 or the MHR); and (c)
NTD residues participate to some extent in intra-
hexamer and ⁄ or inter-hexamer interactions (Fig. 1).
This model remains to be validated and extended to
higher resolution, and it may be far from complete. In
addition to CA, other Gag domains and other proteins
are known or suspected to play important roles in the
assembly and stabilization of the immature HIV-1 cap-
sid. Furthermore, a defect-free, continuous lattice of
hexamers could not form spherical particles. In an
early EM study of immature capsid-like particles of
HIV-1, a fullerene-like icosahedral shell was proposed
[8], although later studies showed no evidence of icosa-
hedral symmetry [14]. Closing the immature capsid
through 12 pentameric CA rings (that can be consid-
ered as lattice ‘defects’) cannot be excluded, but sub-
stantial regions lacking ordered Gag were visible in
detailed electron cryotomographs, suggesting that the
immature HIV-1 capsid may be not fully closed [17].
Very recent studies by cryoET suggest that released
immature virions include an incomplete but continuous
Gag layer covering approximately two-thirds of the
virion membrane [18,26]. Curvature in the hexameric
lattice is mediated by incorporation of defects of dif-
ferent geometries into the lattice, with no evidence of a
preference for pentameric defects [18]. It was also sug-
gested that late-budding structures with complete Gag

capsids may not be normal precursors of extracellular
HIV-1 virions [26].
Identification of CA–CA interfaces in
the mature HIV-1 capsid
CA multimers with the same structural organization as
authentic mature capsids have been obtained by self-
assembly of CA in vitro, even in the absence of any
other biomolecule, and subjected to structural, bio-
chemical and mutational analyses [27–37]. CryoEM
studies of mature HIV-1 capsid-like particles assembled
in vitro revealed that these are composed of an array of
CA hexamers. Fitting the atomic structures of NTD
and CTD on the cryoEM density map indicated that
NTD connects the CA subunits in each hexamer, and
CTD connects each hexameric ring to six neighbors
through homodimerization [30] (Fig. 1). Similar arrays
of CA hexamers were observed in cryoEM images of
authentic cores isolated from HIV-1 virions [38].
Recently, a CA mutant was used to obtain large,
spherical capsid-like particles that collapsed when
deposited on EM grids. The flattened particles behaved
as 2D CA crystals, and could be used to obtain a
detailed 3D map by electron cryocrystallography [39]
(Fig. 2A). Fitting the atomic structures of NTD and
CTD in this map confirmed and substantially refined
the mature HIV-1 capsid lattice model. Three different
protein–protein interfaces were observed in the CA
Immature Mature
Top
Side

Fig. 1. Proposed models for the organization of the hexameric lat-
tices in immature and mature HIV-1 capsids [17]. NTD, CTD and
SP1 domains of Gag are respectively colored cyan, yellow and
magenta. Scale bar = 8 nm. Figure reproduced with permission
[17].
Capsid protein interfaces in HIV-1 assembly M. G. Mateu
6100 FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS
lattice: the interface between NTD domains in each
hexamer, the dimerization interface between CTD
domains belonging to neighboring hexamers, and
another interface between CTD and NTD domains
belonging to neighboring subunits of the same hex-
amer [39] (Fig. 2B–D). This structural model and the
CA–CA interfaces involved are supported by muta-
tional analyses and biochemical evidence [23,32–34,36]
(Fig. 2E, F).
In vitro polymerization of CA normally leads to
open cylindrical structures, although closed, cone-
shaped particles resembling authentic mature capsids
can be also obtained. Despite the difference in shape,
the cylindrical capsid-like particles are organized with
the same hexameric lattice as the cone-shaped, authen-
tic mature capsids [30,38,39]. A molecular model for
the architecture of the mature HIV-1 capsid has been
proposed that follows the principle of a fullerene cone:
the body is composed of curved arrays of CA hexa-
mers, and the ends are closed by inclusion at defined
positions of 12 CA pentamers (acting as lattice
‘defects’) [29,30].
Very recently, cryoEM analyses of two in vitro

assembled capsids of another retrovirus, Rous sarcoma
virus (RSV), were described [40]. Both capsids are icos-
ahedrally symmetric: one is composed of 12 CA penta-
mers, and the other of 12 pentamers and 20 hexamers.
Pseudoatomic models using the atomic structures of
both CA domains revealed three distinct CA interfaces
similar to those observed within and between hexamers
A
B
C
D
EF
Fig. 2. Structure of CA and the CA hexamer
in the mature HIV-1 capsid [39]. (A) Electron
density map obtained by electron cryocrys-
tallography of the mature HIV-1 capsid lat-
tice; (B, C) Pseudoatomic models of the
CA monomer and hexamer, obtained by
fitting the atomic structures of NTD (green)
and CTD (blue) on the cryocrystallography
electron density map. (C) Showing a top
view of the hexamer, with one monomer
outlined white. (D) View of the hexamer as
in (C), but each CA monomer is depicted in
a different color; (E, F) Mapping on a CA
hexamer (top view and slabbed side view)
of mutations to alanine, according to their
effect on the assembly of mature HIV-1
capsid-like particles: (i) green or blue dots,
mutations in NTD or CTD, respectively, that

did not impair assembly; (ii) orange, yellow
or red dots, mutations that impaired
assembly and are respectively located in
the NTD–NTD, CTD–CTD or NTD–CTD
interfaces. Figure reproduced with
permission [39].
M. G. Mateu Capsid protein interfaces in HIV-1 assembly
FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS 6101
in the model of the HIV mature capsid. This study
provides support for the fullerene model, and shows
how pentamers can be accommodated in the retroviral
capsid.
Structural and energetic
characterization of CA–CA interfaces
in the mature HIV-1 capsid
From the results reviewed above, it could be concluded
that the picture of CA–CA and other protein–protein
interfaces in the immature HIV-1 capsid remains
blurred because of uncertainties on the structural ele-
ments involved, and the possibility of transient interac-
tions. By contrast, three different CA–CA interfaces
have been clearly identified in the mature HIV-1 cap-
sid, and mapped with relative accuracy. Some of the
knowledge acquired to date on the detailed structure
and energetics of intersubunit interfaces involved in
the quaternary structure of the mature HIV-1 capsid is
reviewed below.
The CTD–CTD dimerization interface
A high-resolution structural description of the CTD–
CTD interface that connects neighboring hexamers in

the mature HIV-1 capsid has been obtained by X-ray
crystallography of dimeric CTD [6,7]. This interface is
essentially formed by the parallel packing of helix 9
from each monomer, but also involves interactions
between residues in the 3
10
-helix of one monomer and
residues in helices 9 and 10 of the other monomer. No
MHR residues are involved. The structural description
of the dimerization interface in the isolated CTD dimer
is fully consistent with descriptions of the CTD–CTD
interface in the mature HIV-1 capsid. These latter
descriptions derive from: (a) pseudoatomic models of
capsid-like particles [30,39]; (b) analyses of the effect
of CA mutations on the assembly of mature capsid-
like particles (Fig. 2E,F) and ⁄ or the formation of viral
cores; the results obtained showed that residues located
in helix 9 (among others), impaired mature capsid
assembly both in vivo and in vitro [23,34]; and (c)
hydrogen–deuterium (H–D) exchange experiments
analysed by MS. The residues buried in the intersub-
unit interfaces could be identified by their slower H–D
exchange in the assembled particle, relative to the free
CA protein. Again, residues in helix 9 (and others)
were involved in intersubunit interactions [33].
In the atomic structure of CTD, the dimerization
interface involves some 22 amino acid residues from
each monomer, and it would bury approximately
1800 A
˚

2
of solvent-accessible area if no induced fit had
occurred; approximately two-thirds of this area is con-
tributed by nonpolar side chains [6,7]. The isolated
CTD domain dimerizes with essentially the same affin-
ity as full-length CA, and likely exhibits all of the ener-
getically significant CTD–CTD interactions in CA [7].
This provided a very unusual opportunity for a quanti-
tative thermodynamic dissection of a protein–protein
interface in a virus capsid [36,41,42]. The individual
energetic contribution of each interfacial side chain to
CTD–CTD association was determined by alanine
scanning mutagenesis on free CTD, using analytical
gel filtration chromatography to determine the equilib-
rium constant of the association step for each single
mutant. It was found that removal of the side chain
interactions of any one of almost half the residues at
the interface (Ile150, Leu151, Arg154, Leu172, Glu175,
Val181, Trp184, Met185 and Leu189) destabilized the
CTD–CTD association by over 6 kcalÆmol
)1
, leading
in each case to essentially monomeric CTD (even at
high protein concentrations) [42]. The CTD dimeriza-
tion interface is formed by a central area of energeti-
cally critical, mostly hydrophobic residues, surrounded
by a ring of energetically less important, polar residues
(Fig. 3).
Those structural and energetic features of the CTD
dimerization interface are typical of many protein–pro-

tein interfaces, but these generally exhibit affinities
higher than those of CA or CTD (K
d
= 10–20 lm).
In CTD, the dimerization affinity is kept low partly
because several interfacial side chains contribute each
to substantially destabilize the CTD–CTD association
[42]. Quantitative thermodynamic double mutant cycles
clearly showed that a part of this destabilizing effect is
a result of intersubunit electrostatic repulsions at the
CTD–CTD interface, including those between Glu180
from both subunits, that may be conserved in HIV
[36]. It was suggested that such repulsions could arise
as one consequence of a selective pressure to maintain
an optimum balance between capsid stability (i.e. for
structural integrity in the virion) and instability (i.e.
for viral core disintegration and RNA release in the
infected cell) [36].
This thermodynamic description of the CTD
dimerization interface may also apply to the interface
as a part of the HIV-1 capsid: a good correlation
was found between the effects on CTD dimerization
and on capsid-like particle assembly of mutations
that decreased, increased or preserved the affinity, or
showed non-additive effects [6,34,36]. The detailed
structural and thermodynamic descriptions obtained
on the CTD–CTD interface have been already used
in the field of anti-HIV research, for example for the
design of a helix-9 peptide mimic [3,43], and are
Capsid protein interfaces in HIV-1 assembly M. G. Mateu

6102 FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS
currently being used in the development of higher-
affinity inhibitors of HIV-1 assembly.
The NTD–NTD hexamerization interface
No hexamers of native CA from HIV-1 (with the
CTD inactivated) or its isolated NTD have been
obtained yet [32]. However, a high-resolution struc-
tural description of the NTD–NTD interface in the
mature HIV-1 capsid is available. The homologous
NTD from MLV did crystallize in the form of hexa-
meric rings, and the X-ray structure of this NTD
hexamer could be obtained [44]. The tertiary struc-
tures of NTD from MLV and HIV-1 are very simi-
lar, and superposition of the atomic structure of
monomeric NTD from HIV-1 on the MLV hexamer
yielded a detailed model of quaternary interactions
in the HIV-1 hexamer [45]. Most recently, the full-
length CA from HIV-1 was engineered to form
soluble, assembly-competent hexamers, and high-reso-
lution crystallographic structures of the modified CA
hexamers were obtained [46]. The modifications that
yielded the most detailed structures involved the
engineering of interprotein disulfide bridges through
the introduction of Cys residues at the NTD–NTD
interface (by mutations Ala14Cys and Glu45Cys)
and the substantial weakening of CA–CA dimeriza-
tion through the CTD–CTD interface (by mutations
Trp184Ala and Met185Ala).
The X-ray model of the NTD hexamer from MLV,
the superimposed model of the NTD hexamer from

HIV-1, the high-resolution X-ray model of the modified
CA hexamer from HIV-1, and the pseudoatomic models
of the HIV-1 hexamer obtained from cryo-EM [30] and
electron cryocrystallography [39] maps are all in very
good general agreement regarding the elements forming
the NTD–NTD interface. Helices 1, 2 and 3 of NTD are
closer to the central hole of the hexameric ring, forming
a 18-helix bundle, with helices 1 lining the hole. The
NTD–NTD interface is defined by contacts between
residues in helices 1 and 3 from a NTD monomer and
residues in helices 1 and 2 from the neighboring mono-
mer. These models are also validated by: (a) mutational
analysis, which showed that residues located in helix 1
or 2 impaired mature capsid assembly both in vivo and
in vitro [23,34] (Fig. 2E,F), and (b) H–D exchange
experiments, which identified residues in helices 1 and 2
as involved in intersubunit interactions in mature HIV-1
capsid-like particles [33].
No thermodynamic studies are available on the
NTD–NTD interface. However, in the X-ray structure
of the MLV hexamer, this interface buries a surface
of only 1100 A
˚
2
. Most of the interactions are weak
polar contacts, including some mediated by water
molecules, and a substantial hydrophobic central area
is absent. All these features differentiate the NTD–
NTD interface from the CTD dimerization interface
and many other protein–protein interfaces, and may

contribute to explain the extremely low intrinsic ten-
dency of NTD to homo-oligomerize. Similar to that
observed for the CTD dimerization interface, the
binding free energy of the NTD–NTD interface may
be also limited by intersubunit electrostatic repulsions
between charged residues in NTD, although, in this
case, those residues do not form a part of the inter-
face itself [35].
The NTD–CTD interface
The NTD–CTD interface between neighboring subun-
its within the hexameric rings that form the mature
HIV-1 capsid has been mapped based on different
observations, that are summarized below.
Fig. 3. Energetic dissection of the CTD–CTD dimerization interface
of HIV-1 [42]. A spacefilling model of one of the monomeric subun-
its in a CTD dimer is represented. The interfacial residues are color-
coded according to the effect on CTD dimerization of removing the
interactions of the side chain beyond the Cb (by mutation to ala-
nine). The effect is quantitated according to the difference in free
energy between nonmutated CTD and each mutant for association
of the CTD monomers into a dimer (DDG
a
). Red, truncation had a
dramatic effect and led to monomeric CTD at all but the highest
concentrations (DDG
a
> +6 kcalÆmol
)1
); orange, truncation had a
substantial effect (DDG

a
= +1.1 kcalÆmol
)1
); green, truncation had
no significant effect (DDG
a
< +0.3 to )0.3 kcalÆmol
)1
), or had only a
small negative effect (Lys199; DDG
a
= +0.5 kcalÆmol
)1
); violet,
mutation increased the dimerization affinity (DDG
a
= )0.4 to
)0.8 kcalÆmol
)1
). Figure reproduced with permission [42].
M. G. Mateu Capsid protein interfaces in HIV-1 assembly
FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS 6103
First, in the pseudoatomic models of capsid-like
particles of the homologous RSV [40], the equivalent
NTD–CTD interface involves the MHR, and previous
genetic analysis revealed that mutations in the MHR
causing a defficiency in assembly were compensated by
secondary mutations that also map in the NTD–CTD
interface [47–49]. Incidentally, one of these mutations
alone eliminated a positive charge from a cluster of

basic residues and increased the propensity of CA to
assemble. Thus, it has been suggested that charge
repulsion, which would be partially relieved by that
mutation, may also naturally occur at this interface, at
least in RSV [40].
Second, the inhibitory activity of isolated CTD on
the in vitro polymerization of CA from HIV-1 was
found to be relieved by addition of isolated NTD [41].
Third, crosslinking experiments and MS analysis
revealed that, in capsid-like particles, Lys70 of NTD
was in close proximity to Lys182 of CTD from a
neighboring subunit [33].
Fourth, H–D exchange experiments indicated that
the C-terminus of helix 3 and the N-terminus of helix
4 in NTD, which were not a part of the CA–CA inter-
faces already identified, were additionally involved in
intersubunit contacts [33].
Fifth, the very recent determination of the hexameric
form of a modified full-length CA (with a crosslinked
NTD–NTD interface and a substantially inactive
CTD–CTD dimerization interface) has allowed a more
complete analysis of the precise residues and interac-
tions involved in the NTD–CTD interface [46]. Most
of these involve side chains from helix 8 of the CTD
which pack against the N-terminal end of helix 4 of
the NTD of a neighboring subunit in the hexamer.
Additional contacts involve helix 11 of the CTD and
the C-terminal end of helix 7 of the NTD of the neigh-
boring subunit. The NTD–CTD interface, similar to
the NTD–NTD interface, lacks a substantial hydro-

phobic core, and mainly involves polar interactions,
including water-mediated hydrogen bonds and inter-
domain helix-capping interactions.
Finally, the pseudoatomic model of the HIV mature
capsid lattice and mutational analysis [39] (Fig. 2E,F)
are consistent with the above results.
CA–CA interactions and stability of the mature
HIV-1 capsid
Mutations generally located at or close to CA–CA
interfaces, and that either increase or decrease the sta-
bility of mature HIV-1 capsid-like particles [34]
and ⁄ or authentic HIV-1 cores [50], resulted in a loss
of viral infectivity. Thus, the mature HIV-1 cap-
sid ⁄ viral core does appear to have evolved an opti-
mum, delicate balance between stability inside the
virion and instability inside the infected cell. It is
remarkable that different electrostatic repulsions
between neighboring CA subunits through all three
identified interfaces in the mature retrovirus capsid
have been shown or suggested to occur [35,36,40].
Furthermore, covariant mutations during HIV evolu-
tion may have preserved at least a CTD–CTD elec-
trostatic repulsion that was unambiguosly revealed
using a thermodynamic cycle [36]. The low oligomeri-
zation affinity of CA and the stability balance of the
HIV capsid may be a result, in part, of the preserva-
tion of intersubunit electrostatic repulsions.
Conformational rearrangements of CA
and HIV-1 capsid assembly
Both CA domains are covalently connected through a

flexible linker, and they appear to be unusually flexible
themselves. Accordingly, there is substantial evidence
for the occurrence of different conformational rear-
rangements of CA during the assembly of both the
immature and the mature capsid of HIV-1 and other
retroviruses. Induced conformational switching in CTD
and ⁄ or NTD and ⁄ or alterations in the linker region
could explain several observations, including: (a) the
different ability of retroviral CAs and their free domains
to multimerize or oligomerize in different conditions;
(b) the different shapes of the immature and the mature
HIV-1 capsid [32], and of the mature capsids of different
retroviruses; (c) the different structural organization of
CA in the immature and mature capsids, including the
arrangement and spacing of the hexameric lattice [12]
(Fig. 1); (d) the formation of both hexamers and penta-
mers using essentially the same structural elements of
CA [40]; (e) the different interactions of CA with several
ligands and their effects on capsid assembly [3]; and (f)
the possibility of transitions between alternative inter-
faces during HIV-1 morphogenesis (see below).
Conformational changes in CTD
Biophysical and thermodynamic analysis of the process
of HIV-1 CTD dimerization (from unfolded monomer
to native dimer) [41,51] revealed a transient, folded
monomeric intermediate of low conformational stabil-
ity. This free monomer lacks a part of the structure of
the monomeric subunits in the native, stable homo-
dimer. CTD dimerization was found to involve a
substantial conformational rearrangement of the inter-

acting, transient monomers that was thermodynami-
cally characterized. CTD mutant Trp184Ala is unable
Capsid protein interfaces in HIV-1 assembly M. G. Mateu
6104 FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS
to dimerize [6], even at 1 mm; biophysical and thermo-
dynamic analysis of this monomeric mutant indicated
that it could provide a good structural model for the
transient monomer involved in the dimerization of
nonmutated CTD [41]. Recent NMR studies of this
CTD mutant were consistent with those results, and
revealed that the tertiary structure of the isolated CTD
monomer is not identical to that of the monomeric
subunit in the dimer. In particular, the dimerization
helix 9 is only transiently structured, and the last two
helices are rotated by 90° compared to their position
in dimeric CTD [52].
Subsequently, the structure of another monomeric
CTD mutant, Trp184Ala ⁄ Met185Ala, was reported
[53]. In this structure, helix 9 is shortened but formed,
and the last two helices are placed as in the dimer. It
has been proposed that the structural differences found
for the two monomeric mutants may be a result of the
different pHs used in the NMR experiments [54]. The
structure of the monomer with the transiently formed
helix was determined at neutral pH, whereas that of
the monomer with the structured helix was determined
at acidic pH and, when the pH was raised, the reso-
nances belonging to that helix disappeared [53]. These
pH-dependent changes in tertiary structure are not
unusual in proteins [55,56].

In addition to its propensity to local rearrangements
and dynamics, the free CTD monomer appears to be
highly flexible overall [54]. Thus, both in vitro and dur-
ing HIV-1 morphogenesis in vivo, the energetic balance
between alternative CTD conformations could be
altered by even subtle changes in the environment
[12,31], mutation and ⁄ or ligand binding to CTD (such
as that of capsid assembly inhibitor peptides CAI and
NYAD-1) [3]. This model is consistent with the several
modes of association observed for CTD in crystal
form, including the balance between a domain-
swapped CTD–CTD interface [25] and a nondomain-
swapped interface, proposed to have a role during
HIV-1 morphogenesis.
In the context of the unusual conformational plastic-
ity of CTD, it should be noted at this point that muta-
tion of certain CTD residues, including residues
belonging to the MHR, had substantial effects on
CTD dimerization in solution [41], even though those
residues and the MHR are located away from the rele-
vant dimerization interface. Thus, it may be worth
considering that, in addition or alternatively to a direct
role in intersubunit interactions, some CA residues and
the MHR could indirectly participate in HIV-1
morphogenesis; for example, by stabilizing an assem-
bly-competent CA conformation and ⁄ or facilitating
interactions between other residues.
The lack of a perfect fitting of the closely-matching
atomic structure of the CTD dimer [6,7] on the elec-
tron cryocrystallography map of mature HIV-1 capsid-

like particles has led to the suggestion that small
conformational changes could also occur in the CTD
dimerization interface, and in the tertiary structure of
CTD itself, during assembly of the mature HIV-1 cap-
sid [39]. The detailed atomic structure and quantitative
thermodynamic description available for the CTD
dimerization interface could be then considered to
define the sterically unconstrained, minimum free
energy conformation. Steric constraints in the mature
HIV-1 capsid lattice could distort and destabilize
somewhat the ‘ideal’ CTD–CTD interface (and per-
haps other CA–CA interfaces). Such constraints could
contribute, in addition to electrostatic repulsions and
other effects, to establish the appropriate balance
between stability and instability of the viral core.
Conformational changes in NTD
During maturation, the proteolytic cleavage of Gag at
the linker between CTD and SP1 could destabilize the
proposed SP1 six-helix bundle, facilitating disassembly
of the immature capsid [17,57]. In addition, processing
of the MA-NTD linker allows the folding as a b-hair-
pin of the NTD N-terminal segment, leading to a local
conformational change in NTD. This rearrangement
has been proposed to destabilize the immature capsid
and ⁄ or create the NTD–NTD interface observed in the
mature HIV-1 capsid, thus promoting core assembly
[58]. In addition, alterations in the H–D exchange pro-
tection pattern when immature and mature virus-like
particles were compared have provided evidence for a
maturation-induced formation of the NTD–CTD inter-

face observed in the mature HIV-1 capsid [59]. Ligand
(e.g. CypA [60]) binding to NTD can also lead to
conformational rearrangements.
To summarize, conformational rearrangements
allowed by the unusual structural plasticity of both
CA domains, and their connection through a flexible
linker, may shift their association equilibria during
HIV-1 morphogenesis. The rearrangements proposed
and their detailed molecular description remain to be
further substantiated in future structural studies.
Macromolecular crowding effects on
CA–CA association and HIV-1 capsid
assembly
Both NTD and CTD of retroviruses have an intrinsi-
cally low or undetectable tendency to oligomerize in
solution, and conformational rearrangements in CA
M. G. Mateu Capsid protein interfaces in HIV-1 assembly
FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS 6105
(including those reviewed above) may not be sufficient
to allow the establishment of the relatively stable CA–
CA interactions observed during HIV-1 assembly. For
example, formation of a b-hairpin in NTD during
maturation may not provide an explanation for the
apparent inability of unmodified CA and its NTD to
hexamerize in solution because the hairpin is already
formed in them [45]. Similarly, the induction of a con-
formational rearrangement in NTD through interac-
tion with CTD is unlikely to explain why CA with the
dimerization interface inactivated through mutation is
unable to hexamerize in the same conditions where

nonmutated CA is able to polymerize into capsid-like
particles [33]. Indeed, there is evidence that, in addition
to conformational changes in CA, physicochemical
conditions having an effect on the chemical activity
(‘effective concentration’) of CA may also play a major
role in the associative properties of NTD and CTD to
form the HIV-1 capsid, as reviewed below.
The concentration of CA inside the mature HIV-1
virion may be at least 3.5 mm [38] (approximately
8mm if an estimation of close to 5000 CA molecules
per virion [16] is accepted). However, in the very lim-
ited space available inside a mature HIV-1 virion,
thousands of molecules of MA, CA, SP1, NC, SP2
and p6, as well hundreds of other viral and cellular
protein molecules and two long RNA molecules, are
found. Thus, in the virion, as in the cell, macromolecu-
lar crowding effects must be in operation as a result of
the exclusion of water molecules from the large frac-
tion of internal volume occupied by the macromole-
cules themselves [61]. The chemical activity, or
‘effective concentration’ of CA in the HIV-1 virion
(upon release from Gag during maturation) must be
not a few millimolar, but much higher. Under these
conditions, protein association reactions, such as CA
assembly, must be strongly favored [61]. The available
experimental evidence summarized below is consistent
with this prediction.
In vitro polymerization of CA into mature HIV-1
capsid-like particles in the virtual absence of macro-
molecular crowding (at maximum protein concentrations

in the order of 500 lm, which is far lower than the CA
effective concentration in the virion) was achieved only
in nonphysiological conditions, such as: (a) a very high
ionic strength [32] that could screen electrostatic repul-
sions [35,36] or (b) the use of a CA–NC fusion protein in
the presence of nucleic acid [9,28,29], which could
increase the CA local concentration through multiple
NC-mediated interactions with CA–NC. Similarly, it has
been suggested that putative interactions between CA
and a NC–RNA complex could initiate HIV-1 core
assembly in vivo [38]. However, this possibility may
have to be re-evaluated if the chemical activity of CA
inside the maturing virion (as a result of macromolecu-
lar crowding) is taken into account.
By contrast to previous in vitro assembly procedures,
very efficient assembly of mature HIV-1 capsid-like
particles was recently achieved using free CA at moder-
ate concentrations, in the absence of any other bio-
molecule, and at physiological ionic strength [37]. The
procedure was simply based on the addition of inert
macromolecular crowding agents to increase the CA
effective concentration to very high values, closer to
those present in the HIV-1 virion. The capsid-like tubu-
lar and, occasionally, cone-shaped particles formed
were indistinguishable by EM from those obtained at
very high salt concentrations (and known to be orga-
nized similarly to authentic mature HIV-1 capsids).
The capsid-like particles formed at close-to-physio-
logical CA effective concentration and ionic strength
were kinetically much less stable than those formed at

high ionic strength [37]. This reinforces the view that
electrostatic repulsions could contribute to the
observed HIV-1 core instability in infected cells. In
addition, the release of the viral core and many free
CA molecules from the confined space in the virion
into the very large volume within the cell would also
facilitate uncoating by ‘dilution’ (i.e. through a dra-
matic decrease in the CA effective concentration) [50].
These observations indicate that the very high
chemical activity of CA inside the virion as a result of
macromolecular crowding may be critical for the
assembly and stability of the mature HIV-1 capsid.
The very high chemical activity of CA in the maturing
virion may promote association through the CA–CA
interfaces identified, even though both retroviral CA
domains have a low or negligible tendency to oligo-
merize in solution. Similarly (and in addition to con-
densation mediated by MA–plasma membrane and
NC–RNA binding), macromolecular crowding in the
cell may have a strong influence on the assembly of
the immature HIV-1 capsid, by promoting CA–CA
and other weak Gag–Gag interactions.
Acknowledgements
The author acknowledges J. L. Neira for collaboration
and critical reading of the manuscript, and M. del
A
´
lamo, R. Bocanegra and A. Rodrı
´
guez-Huete for

excellent experimental work. Current work in the
author¢s laboratory is supported by grants from FIPSE
(36557 ⁄ 06), the Spanish Ministry of Science (BIO2006-
00793) and the Madrid Regional Government (S-0505⁄
MAT ⁄ 0303), and an institutional grant from Funda-
cio
´
n Ramo
´
n Areces. The author is an associate
Capsid protein interfaces in HIV-1 assembly M. G. Mateu
6106 FEBS Journal 276 (2009) 6098–6109 ª 2009 The Author Journal compilation ª 2009 FEBS
member of the Instituto de Biocomputacio
´
nyFı
´
sica
de los Sistemas Complejos, Zaragoza, Spain.
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