World Journal of AIDS, 2011, 1, 111-130
doi:10.4236/wja.2011.14017 Published Online December 2011 (http://www.SciRP.org/journal/wja)
111
HIV-1 Assembly, Release and Maturation
Laurence Briant*, Bernard Gay, Christian Devaux, Nathalie Chazal
Centre d’Études d’Agents Pathogènes et Biotechnologies pour la Santé (CPBS), UMR5236 CNRS, Université Montpellier 1-Mont-
pellier 2, Montpellier, France.
Received July 29th, 2011; revised September 29th, 2011; accepted October 11th, 2011.
ABSTRACT
Late steps of HIV-1 life cycle are determinant for optimal dissemination of the virus to new target cells. These steps
include assembly of structural precursors, budding of the new particle and maturation into fully infectious virions. Each
step is finely tuned and timely regulated to allow the appropriate assembly of structural components, the efficient re-
cruitment of viral and cell partners and the timely regulated proteolytic processing of the protein precursors. Despite
the huge number of studies devoted to the definition of molecular mechanisms regulating these steps, a number of ques-
tion remains to be answered before they are clearly apprehended. The elucidation of the role played by each viral pro-
teins, nucleic acids as well as host-encoded factors will provide new clues in the understanding of the retroviral assem-
bly/maturation process and will allow further development of new antiviral compounds. This review reports the most
recent progress as well as the questions that remain to be answered in the field of HIV-1 assembly, release and matura-
tion. Finally, we also describe the data available on the design and use of new antiretroviral drugs targeting these spe-
cific steps of the retroviral replication.
Keywords: HIV-1, Assembly, Maturation, Release, Gag, Antiviral Molecule
1. Introduction
HIV-1 assembly, release and maturation are steps of
primary importance for the production of infectious par-
ticles capable to disseminate in new target cells. Notably,
the capacity of HIV-1 to produce fully mature proteins
addressed to appropriate cellular compartments, the
competence of these viral proteins to multimerize and to
interact with viral nucleic acids and the faculty of these
complexes to recruit cellular partners required for effi-
cient release in the extracellular space is of crucial im-
portance for viral dissemination. Even they have at-
tracted a considerable effort since the discovery of HIV-1,
the mechanisms involved in the assembly of infectious
particles remain incompletely defined. The understand-
ing of protein-protein and protein-nucleic acids contacts
as well as interactions with cellular lipids engaged during
retroviral assembly more than ever represents a major
challenge for the future development of new HIV-1 in-
hibitors.
2. The Viral Partners of HIV-1 Assembly
HIV-1 gene transcription generates a 9 kb RNA that has
three fates: firstly, multisplicing that gives rise to regula-
tory proteins (Tat, Rev, Nef, Vpr, Vif); secondly, singly
spliced RNAs that support translation of envelope glyco-
proteins (Env). The third species is an unspliced RNA
that both serves as a matrix for the translation of Gag
structural polyprotein and of a GagPol precursor sup-
porting viral enzyme production and that forms a dimer
of genomic RNA encapsidated into newly assembled
virions. The infectious HIV-1 particle is a spherical par-
ticle of 120 - 150 nm surrounded by a lipid bilayer de-
rived from the host cell plasma membrane in which is
inserted the viral envelope composed of the gp41 trans-
membrane protein non covalently attached to the gp120
surface glycoprotein. The viral envelope is linked to a
spherical shell composed by the matrix (MA) protein. In
the center of the mature particle, a cone shaped core re-
sulting from assembly of the mature capsid protein (CA)
contains a dimeric viral genomic RNA associated with
the nucleocapsid protein (NC). The annealing of a
tRNAlys3 to the primer binding site (PBS) in genomic
RNA is required for viral infectivity. In addition, the
conical capsid contains Vpr, Nef, Vif accessory proteins
and the viral enzymes integrase and reverse transcriptase.
Finally, a huge variety of membrane or cytosolic proteins
and RNA derived from the host cell are incorporated into
HIV-1 particles [1].

HIV-1 Assembly, Release and Maturation 112
3. A Molecular Model of Gag Assembly
The production and release of an infectious HIV-1 parti-
cle is orchestrated by the Gag polyprotein precursor
which is the major component of HIV-1 assembly. In-
deed, Gag polyprotein is sufficient for in vitro assembly
and in vivo production of virus-like particles (VLPs) [2].
Such particles contain approximately 5000 Gag mole-
cules [3]. Interaction of Gag with cellular membranes
and the endocytic machinery, oligomerization at the as-
sembly site, contacts between Gag and viral proteins and
nucleic acids and finally, maturation of Gag are determi-
nant for the production of fully infectious particles. Gag
polyprotein is comprised of four major structural do-
mains: p17 matrix (MA), p24 capsid (CA), p7 nucleo-
capsid (NC), p6 spaced by two linker peptides SP1 and
SP2 (Figure 1). When addressed at the plasma mem-
brane, Gag polyproteins are radially organized with the
MA domain tightly associated with the inner face of the
lipid bilayer, while the C-terminus is oriented towards
the center of the nascent particle, [4,5]. Gag molecules
assemble into a fullerene-like model with threefold
symmetry and form a hexagonal network of rings that
can be visualized as patches of electron dense material
beneath the cell membrane in electron microscopy imag-
ing [3,6]. Multiple cooperative contacts between adjacent
Gag polyproteins, lipids or nucleic acids allow the for-
mation of an organized lattice that determines the capac-
ity to produce an assembled viral particle. The function
of each domain of Gag during the assembly process has
been decrypted.
3.1. Role of the MA Domain in HIV-1 Assembly
MA is the membrane proximal domain of the Gag poly-
protein. Its function, as a domain of Gag or as a mature
structural protein, relates particularly to the trafficking of
HIV-1 complexes, membrane binding and Env incorpo-
ration. The structure of HIV-1 MA has been solved by
crystallography and NMR [2]. The first 104 residues of
MA form a globular domain composed of four α helices
centrally organized and a fifth one which is projected
away from the packed bundle of helices. This globular
core is capped by a three-stranded β sheet-enriched in
basic residues. When expressed in mammalian cells, MA
is found both in association with cellular membranes and
in a membrane-free form. Regarding the unprocessed
Gag precursor, despite detected in the cytoplasm as mo-
nomers and low-order multimers, it is almost exclusively
bound to membranes where it forms patches of assem-
bled multimers [7]. The presence of a myristic acid at the
N-terminal end of MA was established as the primary
determinant for interaction of Gag with acid phospholip-
ids [2,8]. According to the local concentration of Gag,
the conformation of the MA domain is switched from a
state where the myristate moiety is sequestrated inside
the core of the protein to a myristate-exposed conforma-
tion. When exposed, this myristate moiety is capable to
insert into membrane bilayers and together with a stretch
of basic residues spanning positions 26 - 32 in MA dic-
tates the capacity of MA the protein to interact with cel-
lular membranes [8]. This basic domain engages electro-
static interactions with the negatively charged head-
groups of acidic phospholipids in the inner leaflet of the
plasma membrane.
The nature of microdomains supporting the interaction
of Gag with cellular membranes is discussed below.
Then, the MA domain organizes in a hexameric network
of trimers [9,10]. The formation of the trimer unit is dic-
tated by weak interactions of α-helix 4 [2,11]. However,
minimal Gag constructs lacking matrix domain retain the
capacity to assemble and to generate efficient particle
formation when the matrix domain is substituted by a
myristylation signal [2]. Accordingly, MA, despite play-
ing a structural role in HIV-1 assembly cannot be con-
sidered as a driving force in viral particle formation.
Besides its function in lipid binding, MA is also re-
quired for incorporation of gp120 and gp41 envelope
glycoproteins into nascent particles [2,12]. A consider-
able number of studies reported that MA interacts with
the intracytoplasmic domain of gp41 during assembly
and proposed that these interactions, either occurring
directly or through a molecular bridge involving a cell
cofactor, promote the recruitment of Env complexes into
nascent virions (for review see [12]). Finally, in addition
to these activities, several studies have reported the ca-
pacity of the retroviral MA to bind nucleic acids (re-
viewed in [13]). The RNA binding site in MA was
mapped to overlap PI(4,5)P2 interacting domain. Dele-
tion of this sequence allows production of poorly infec-
tious viruses that can be rescued in trans by coassembly
with wild type Gag proteins [14]. Recently, inositol
phosphate binding was reported to stimulate nucleic ac-
ids chaperone activity of Gag [15]. In contradiction with
this observation, RNase treatment of Gag in vitro transla-
tion lysates reduces the selectivity of Gag binding to
PI(4,5)P2 liposomes. In this model, MA interaction
with nucleic acids is likely to increase the selectivity of
MA for PI(4,5)P2 rich cellular membranes and to pre-
vent premature intracellular assembly. However, cryo-
EM study revealed that Gag in solution undergoes a
conformational switch according to interaction with
RNA or lipids. The low affinity of MA domain-RNA
binding is proposed to be reversed by MA-lipid interac-
tion, allowing the conversion from a compact RNA-
bound Gag protein to an extended lipid-bound Gag [16].
The exact contribution of such observations needs to be

HIV-1 Assembly, Release and Maturation 113
explored. In conclusion, MA not only acts as a scaffold
that brings together Env, Gag and nucleic acids during
assembly but also regulates targeting of Gag to the cell
membranes.
3.2. Contacts in the CA Domain of Gag
The CA domain in Gag is strictly required for assembly.
The CA domain present in immature virions is organized
as a hexagonal lattice with a characteristic spacing aver-
aging 8 nm [17]. After protease processing, the mature
CA protein is released as a 231 amino acids polypeptide
that folds into two distinct globular domains connected
by a flexible linker structured as a 310 helix [18,19]. Ac-
cording to crystal structures and cryo-electron micros-
copy reconstructions, CA still forms a network of hex-
americ rings and of pentamers in the fullerene core
[20,21]. The CTD moiety of CA and the adjacent SP1
domain are required for Gag multimerization. Three sep-
arate functional domains have been identified (reviewed
in [22]): 1) CA helix 9 in the CTD moiety directs the
formation of Gag dimers in solution and in crystals
through parallel packing; 2) The Major Homology Re-
gion (MHR) represents a stretch of 20 residues (amino
acid 153 to 172 in CA) highly conserved in capsid pro-
teins from distinct retroviruses and strictly required for
HIV-1 assembly. Swapping of the MHR regions of adja-
cent Gag molecules was proposed to be directly involved
in CACTD dimerization and in stabilization of the hex-
americ network [23]; 3) The NTD moiety of CA, more
precisely α helices 1 and 2, and helices 4 and 7 stabilize
the immature lattice by six-fold symmetric interactions
between neighboring hexamers. However, the CANTD is
not strictly required for Gag assembly as mutagenesis of
this domain allows VLPs production. This region also
contains an exposed proline-rich loop required for the
recruitment to the viral particle of the host cell encoded
peptidyl-prolyl-isomerase cyclophilin A (CypA) [24].
Despite dispensable for assembly, CypA incorporation
determines HIV-1 infectivity [24,25] and plays a key role
both in viral uncoating during early steps of infection [26]
and in recognition of the incoming capsids by cellular
restriction factors including TRIM5α [27,28]. Accord-
ingly, assembly of the Gag precursor is primarily deter-
mined by CACTD while the N-terminal domain rather
contributes to assembly of mature CA.
3.3. Flexible Linkers in HIV-1 Assembly
Gag precursor is additionally characterized by the pres-
ence of flexible junctions, a common feature of retroviral
Gag proteins [29]. Junctions located at the CA/NC and
NC/p6 interface are termed SP1 and SP2 linkers respec-
tively (Figure 1). Structure prediction as well as NMR
study of a Gag fragment containing CACTD, SP1 and NC
revealed that the flanking linker domain in both CA and
NC are flexible and that the first 7 amino acids in SP1
fold into an α-helix [30]. SP1 structuration is highly in-
fluenced by the environment [31]. Upon assembly, this
domain folds into a six helix bundle as observed by elec-
Figure 1. Organization of Gag polyprotein and functional domains of mature proteins. +: positively charged amino acids;
N-Myr: Myristate; MA: matrix; CA: capsid; SP1: spacer peptide 1; NC: nucleocapsid; SP2: spacer peptide 2; Env: envelope;
ypA: cyclophilin A; MHR: Major Homology Region. C

HIV-1 Assembly, Release and Maturation 114
tron cryotomography [32]. This property was related to
changes in SP1 conformation dictated by modifications
in the local concentration of Gag molecules [31]. Such
properties are of crucial importance for HIV-1 assembly.
Indeed, SP1 plays a major role in Gag multimerization,
higher order organization of the Gag lattice and VLP
assembly [30,31,33,34]. Mutations of the CA-SP1 region,
especially modification of the first 6 residues in SP1,
prevent viral particle to assemble properly and reduce the
strength of Gag-Gag interactions underlying particle
formation [31]. Moreover, deletion of SP1 converts the
spherical particles assembled in vitro into conical parti-
cles reminiscent of mature capsids. Accordingly, SP1
was proposed to act as a molecular switch [30]. The
structural model currently proposed for SP1 in HIV-1
assembly supports that oligomerization of the C-terminal
region of Gag would trigger a signal transmitted from the
C-terminal region of Gag toward the CA domain, allow-
ing conformational changes in SP1 structure. Such modi-
fications would result in the exposure at the external part
of the complexes of assembly interfaces in the CA and
SP1 domains which are otherwise cryptic. Nevertheless,
it has to be kept in mind that this significant function of
HIV-1 spacer peptides in particle assembly is transient as
the proteolytic cleavage of SP1 linker is a prerequisite to
turn the non infectious particle into an infectious virus.
While most attention was devoted to the study of SP1
function, similar conclusions can be drawn for SP2 linker
located between NC and p6 domains of Gag. Indeed,
proper cleavage between p6 and SP2 is critical for the
formation of fully infectious particles. Mutations inhibit-
ing SP2-p6 processing result in the assembly of virions
with aberrant cores that are unable to replicate in T cells
[35]. Recently, the cleavage at the SP2-p6 site was re-
ported to be necessary for efficient genome integration
by the infecting virus [36]. Inhibition of the NC-SP2
maturation by mutations of the two proline residues in
the middle of SP2, that confer conformational constraints
to this linker, also abolished viral replication despite the
uncleaved NC-SP2 protein has been found to be more
efficient than mature NC at promoting RT-associated
functions in vitro [37]. Persistence of NC-SP2 protein
reduces genomic RNA dimer stability. Recently, the ap-
propriate maturation of the NC-SP2-p6 precursor has
been shown to be required for formation of a stable RNA
dimer within the HIV-1 particle [38]. According to this
model, the RNA dimer stabilization begins during the
primary cleavage SP1-NC of Gag and reaches stable and
uniform state allowing infectivity, once Gag processing
is complete. Altogether these observations highlight that
attention must be payed to the role of SP2 flexible linker
and uncleaved NC-SP2 proteins in assembly of fully in-
fectious HIV particles.
3.4. Nucleocapsid and Packaging of Viral RNA
The C-terminal part of Gag is central in the assembling
particle and links the retroviral RNA genome at the level
of the NC domain. NC is composed of two highly con-
served zinc fingers of the CCHC form organized in tan-
dem. Chelating of Zn2+ folds the central domain of the
protein while the N- and C-terminal moieties remain es-
sentially unfolded. Substitution of the CCHC residues,
that modify the globular folding of NC, modifies Gag
trafficking in the producer cell, alters the virion core
structure and causes a complete loss of infectivity (for
revue see [39] and [13]). The role of NC in HIV-1 as-
sembly is multiple. Primarily, its function relies on the
capacity of the zinc-finger domains to interact with the
genomic RNA and to direct the packaging of the viral
genome into the nascent particle. Both of them, with a
prominent role for the N-terminal zinc-finger structure,
are critical for the selection of the viral genomic RNA
and the assembly of the virion core [40]. At the level of
the HIV-1 full length RNA, a hundred nucleotides lo-
cated in 5’ of the untranslated region (UTR) and in 5’ of
the gag gene form the  encapsidation sequence gene
(for revue see [13]). This motif interacts with exposed
bases of the RNA loop in the packaging site of genomic
RNA while the C-terminal zinc finger motif is only part-
ly involved in NC/RNA interaction. Additional contacts
with basic residues flanking the zinc fingers along the
SP1 linker are also required for HIV-1 genome packag-
ing. Interestingly, Gag containing a leucine zipper in
place of NC domain assembles efficiently [30,41].
Therefore, the RNA binding property of NC has been
proposed to promote Gag concentration and organization
at the budding site, the RNA genome playing the role of
a scaffold in Gag lattice formation. Despite NC displays
a strong preference for HIV-1 genomic RNA, contacts of
the zing-fingers with non specific cellular RNAs or
DNAs can also occur and promote Gag multimerization.
In addition, the contribution of NC in HIV-1 assembly
also relies on the presence of a stretch of basic residues
located in the NTD and of charged amino acids located
throughout NC that contribute to Gag multimerization
[42]. Finally, the NC domain of Gag associated with ge-
nomic RNA is a key player in the recruitment of host
cofactors of HIV-1 assembly into the viral particle. This
family of proteins includes Staufen1 [43], nucleolin, [44],
ABCE1 [45] and Bro1 [46], all of these proteins being
reported as cofactors for HIV-1 assembly.
3.5. Gag p6 Domain and Viral Release
To the exception of a very recent study indicating that
mutations in the C-terminal p6 domain in the Gag poly-
protein interfere with CA-SP1 processing and generate

HIV-1 Assembly, Release and Maturation115
noninfectious particles [47], p6 is generally admitted to
play no apparent role in retroviral assembly to the excep-
tion of its capacity to recruit the cellular machinery re-
quired for viral budding and release. At the cell mem-
brane, a number of host cell components are recruited to
allow the final release of the nascent particle. The most
striking example is the hijacking of the Endosomal Sort-
ing Complexes Required for Transport (ESCRT) (for
revue see [48]). The primary function of the ESCRT
complexes is to create internal vesicles in multivesicular
bodies (MVBs)/late endosomes and to participate in the
recognition, sorting and degradation of membrane mole-
cules in lysosomes. During this process, ESCRT com-
plexes dictate the incorporation of ubiquitinylated car-
goes into budding exosomes and the pinching off of en-
dosomal vesicles into MVBs. During retroviral assembly
and budding, these complexes assist processes that are
topologically related to reactions occurring during in-
ward budding of cellular vesicles into MVBs lumen. En-
gagement of this pathway relies on its recruitment
through initial interaction of a P(T/S)AP late domain in
p6 domain at the C-terminus of Gag with Tsg101 par-
ticipating in ESCRT-I complex. Then the LYPXL motif
in p6 interacts with the V-domain of Alix [ALG-2 (apo-
ptosis-linked gene 2)-interacting protein X] [49]. This
interaction is regulated by structural constraints in p6
[50]. Despite not fully understood, Gag ubiquitinylation
might participate in this process as suggested by the ca-
pacity of proteasome inhibitors to reduce HIV-1 release
[51,52]. The nature of the ubiquitin ligase participating in
Gag ubiquitination is not fully elucidated. However, the
PPXY motif in Gag binds members of the Nedd4 family
of E3 ubiquitin ligases. Moreover, Nedd4-2 ubiquitinates
Gag and may, in this way, allow the recognition by ubiq-
uitin-binding domains within the ESCRT machinery [48].
Identifying the ubiquitin ligases required for HIV-1 bud-
ding as well as decrypting the exact role played by these
enzymes and by Gag ubiquitinylation in retroviral release
would provide new insight in the mechanisms of retrovi-
ral budding.
Of note, advances in the field of HIV-1 budding have
been achieved in the last years with the identification of
BST2/tetherin, a cellular protein found to counteract the
release of assembled particles [53,54]. This GPI-an-
chored molecule retains virions on the surface of infected
cells and within endosomes following virions internaliza-
tion [53,54]. In wild type conditions, the colocalization
of Tetherin with nascent particles is counteracted by
HIV-1 Vpu [53,55]. When produced in the absence of
Vpu, viral particles accumulate in a mature form at the
plasma membrane and in intracellular compartments of
tetherin-expressing cells [56]. Accordingly, the host cell
machinery either assists or counteracts the final virus-cell
membrane scission through contacts with retroviral pro-
teins.
4. Cellular Compartments Supporting Gag
Assembly
4.1. Membrane Microdomains and Viral
Assembly
The production of a HIV-1 immature particle results
from targeting of Gag and GagPol precursors, viral RNA
and Env glycoproteins to a unique site of assembly (Fig-
ure 2). The place where HIV-1 assembly takes place is
Figure 2. (A) Morphological consequences of Gag process-
ing visualized by electron microscopy imaging; (B) Sche-
matic representation of HIV-1 assembly, budding and
maturation. Gag precursors are addressed to the plasma
membrane (a) and insert into the lipid bilayer through in-
teraction of MA with phosphoinositides (b). Gag oligomer-
izes into microdomains through contacts occurring thr-
oughout the polyprotein domain. Contacts between the NC
domain of Gag and full length RNA serve as a scaffold to
Gag assembly and direct incorporation of the viral genome
into the nascent particle. At this time, the retroviral enve-
lope is incorporated into the viral bud (c). The viral particle
is released as an immature non infectious virion (d). Once
released, Gag polyprotein in the immature virion is proc-
essed by the protease. The matrix remains associated to the
lipid bilayer while the mature capsid protein refolds into a
cone shape structure containing the RNA dimer (e).

HIV-1 Assembly, Release and Maturation 116
still a matter of debate (reviewed in [8]). The current
knowledge indicates that it may occur in distinct com-
partments considering the different cell types susceptible
to support HIV-1 replication. In most cell types, includ-
ing T lymphocytes, HIV-1 assembles predominantly at
the plasma membrane. In contrast, retroviral particles
were proposed to assemble and to bud into intracellular
late endosomal vacuoles/multivesicular bodies (MVB) in
macrophages. However, the presence of viruses in intra-
cytoplasmic vacuoles was later reported to illustrate the
presence of virions into extracellular spaces limited by
membrane invaginations with the appearance of a bona
fide intracellular compartment. In this context, a major
argument is that the manipulation of the endocytic
compartment inhibits Gag localization in endosomes
without effect neither on Gag expression at the plasma
membrane or on extracellular HIV-1 release [57]. To
date, this question remains controversial and it is not
clear whether HIV-1 has acquired different mechanisms
of budding in different cell types including in macro-
phages. The possibility that Gag precursor could be ad-
dressed first to MVBs before being relocalized to the
plasma membrane has been evoked. This model is quite
similar to that proposed for HTLV-1 assembly and bud-
ding [58].
At the plasma membrane, the viral assembly is likely
to be supported by lipid enriched domains, the nature of
which is widely discussed [59]. This model relies first on
the observation that virions are markedly enriched in
specific phospholipids (cholesterol, PI(4,5)P2 phospho-
inositide) relative to the plasma membrane [60]. More-
over, the incorporation of cellular proteins generally as-
sociated with membrane microdomains (namely Thy-1,
CD55, CD59 GPI-anchored proteins, tetraspanins) to-
gether with the selective exclusion of proteins from these
areas (CD45) are additional arguments supporting that
HIV-1 assembles and buds in specific membrane micro-
domains [61]. Both the lipid rafts and the tetraspanin
enriched domains (TEMs), despite distinct, were pro-
posed as preferential platforms for retroviral assembly as
Gag colocalizes in vivo with each domain [8]. The role of
lipids rafts as assembly platforms is supported by bio-
chemical evidences demonstrating that Gag associates
with detergent-resistant membrane and by the drastic
inhibition of HIV-1 release observed in cells depleted for
membrane cholesterol, an essential component for raft
integrity. Finally, coimmunoprecipitation of Gag with
some CD81 tetraspanin pointed the possible role of tet-
raspanin enriched domains in retroviral assembly. The
question whether Gag is targeted preferentially to
pre-existing microdomains or whether these areas are
structured upon Gag targeting at the plasma membrane
remains to be answered.
In the context of the uncleaved Gag, the N-terminal
matrix domain interacts with the lipid bilayer through the
insertion of a myristic acid moiety covalently attached to
the Gly1 residue at the N-terminus of MA [62,63]. This
motif facilitates interactions of MA with phosphoinositi-
des, mainly with phosphatidylinositol-(4,5)-bisphosphate
[PI(4,5)P2], present in the cytoplasmic leaflet. Lipid
binding and localization of the Gag precursor to the
plasma membrane is additionally reinforced by electro-
static interactions of acidic phospholipids with a stretch
of basic residues located at positions 17 - 30 in MA do-
main of Gag the presence [8]. NMR studies revealed that
the inositol head group and the 2’ acyl chain of PI(4,5)P2
fit into a hydrophobic cleft in MA, promotes exposure of
the sequestered myristate group, thereby promoting the
stable association of the viral matrix with membranes
and protein oligomerization [64,65]. Upon proteolytic
processing, MA refolding decreases its capacity to bind
to membranes [66].
4.2. Molecular Interactions Required for
Trafficking of Gag and Env
The hijacking of cellular transport systems is generally
required to overcome the limited capacity of most vi-
rus-encoded components to diffuse freely in the host cell
cytoplasm to the site of assembly. Regarding HIV-1,
early experiments based on the use of chemical inhibitors
including brefeldin and monensin evidenced that Env but
not Gag transport is ensured by the vesicular pathway
(see [8]). A series of data reporting the capacity of wort-
mannin, an inhibitor of myosin light chain kinase, or of
actin depolymerisation agents mycalolide B and cyto-
chalasin D to reduce the transport of nascent HIV-1 pro-
tein in the host cell and to disrupts Gag compartmentali-
zation within the polarized raft-like domains of T cells
has suggested that the cell cytosketon assists viral parti-
cle assembly and is required for spread of HIV-1 (for
review see [67]). The data supporting the active transport
of Gag to the assembly site and the contribution of the
cell cytoskeleton in viral assembly were reinforced by
the description of direct interaction between Gag and
actin and between Gag and the kinesin motor KIF-4. Fi-
nally, the association of components of actin microfila-
ments with highly purified HIV-1 particles provides ad-
ditional support to the role of cytoskeleton in HIV-1 as-
sembly [1]. Additional interaction between Gag and
hVps18, Mon2 proteins and with AP1 and AP3 clathrin
adaptors have been reported to assist virion assembly
[68-70], suggesting that interaction between HIV and
cellular proteins specialized in vesicle formation and
endosome biogenesis are required during the late stages
of the viral life cycle.
An important question to be answered is the exact

HIV-1 Assembly, Release and Maturation117
place where the viral components self associate, and the
locus where Gag, Env and the RNA genome encounter to
assemble into a correctly folded immature particle. This
question was partly solved for Rous sarcoma virus (RSV).
In this model, the Gag protein undergoes a transient nu-
clear trafficking prior to plasma membrane transport and
binds viral RNAs to form ribonuclear protein complexes
that are exported from the nucleus for packaging into
virus particles (for review see [71]). Regarding HIV-1, in
the absence of Gag, genomic RNA accumulates at the
nuclear envelope. The retroviral nucleic acids are redi-
rected to the plasma membrane when Gag is expressed.
Despite, mutations in MA relocalizing Gag to the nucleus
impair HIV-1 replication [72], the role of HIV-1 Gag in
triggering export of ribonucleic complexes from the nu-
cleus remains undemonstrated conversely to RSV.
The recent development of live imaging techniques
provided some the field of HIV-1 proteins trafficking.
First, these approaches established that assembly of a
single particle almost complete between 5 to 9 min [73].
Then, investigation of trafficking in living cells evi-
denced that the genomic RNA encounters monomers or
low-order multimers of Gag present in the cell cytoplasm
and form a subvirion complex required for anchoring of
HIV-1 genome at the plasma membrane. Gag-RNA con-
tacts are stabilized by interactions in CACTD. Once ad-
dressed to the plasma membrane, this subunit nucleates
further accumulation of Gag and the complexes assemble
into high-order multimers [7].
The trafficking of gp160 Env polyprotein is extremely
complex (reviewed in [12]). It is synthesized by ri-
bosomes located in the rought RE, transported to the cell
membranes by the secretory pathway and finally recycled
by the endosomal/lysosomal compartment before final
expression at the cellular membrane. During transporta-
tion, the envelope polyprotein precursor undergoes gly-
cosylation and proteolytic processing into mature gp120
and gp41. Direct interaction of viral proteins with the
cellular lipid cargo protein TIP47 is required for HIV-1
assembly. Indeed, the inhibition of interactions between
TIP47 and the matrix domain in Gag or between the in-
tracytoplasmic domain of gp41 and TIP47 prevents the
colocalization and interaction of Gag and Env in macro-
phages, thereby inhibiting Env incorporation into bud-
ding virions. This observation supports the notion that
TIP47 is required for Gag and Env to encounter at the
retroviral assembly site and for the assembly of infec-
tious particles.
5. Maturation of HIV-1 Virions
HIV-1 particles are initially released from the producing
cell as immature non infectious virions containing un-
cleaved precursors that are turned into mature fully in-
fectious viruses following proteolytic processing and
reorganization of the structural proteins [2]. The entire
processing of Gag and GagPol precursors is finely coor-
dinated and regulated. It requires the activity of the ret-
roviral protease (PR), a 99 amino acids protein that re-
lates to the family of cellular aspartyl proteases. At initial
step, an unknown signal, probably based on the local
concentration of GagPol precursors favors dimerization
of the PR in GagPol polyprotein and autocleavage of the
active enzyme. Activation of the viral protease and ma-
turation of structural precursors need to be timely regu-
lated to ensure the optimal maturation of the viral particle.
Indeed, premature PR activation or excessive activity and
inactive PR equally abolish the assembly of infectious
particles [74]. Processing of HIV-1 proteins by the retro-
viral protease involves no strict consensus amino acid
motif. The recognition of the cleavage sites is rather de-
termined by the local conformation of the target.
Gag cleavage by the retroviral protease occurs sequen-
tially at five specific sites (Figure 3). During this step-
wise reaction, the SP1-NC junction is cleaved with
greater efficiency. Then, the joint SP2-p6 and the MA-
CA regions are cleaved, allowing the release of free MA
and p6 together with the p25 (CA-SP1) and NC-SP2 in-
termediates. The separation of spacer peptides is the final
maturation step that turns the p25 intermediate into the
mature p24 capsid protein and allows the release of the
mature NC. The cleavage of HIV-1 structural proteins is
accompanied by a morphologic conversion of the viral
particle. When studied by electron microscopy imaging,
the most critical change relates to the conversion of the
electron dense ring of assembled Gag polyproteins be-
neath the lipid bilayer into a conical shaped capsid re-
sulting from assembly of mature CA (Figure 2(A)). The
assembly of this protein core, together with its stability
and its capacity to disassemble at the appropriate time
regulate viral infectivity by allowing early step of infec-
tion to occur in a new target cell. The mature core is of
fullerene-type geometry. It consists of a lattice of 250
CA hexamers separated by a 9.6-nm hexamer-hexamer
Figure 3. Schematic representation of timely processing of

HIV-1 Assembly, Release and Maturation 118
Gag polyproteins.
spacing and is closed at the narrow end by 5 pentamers
and by 7 pentamers at the broad end [2,22]. At the mo-
lecular level, the release of the cleaved CA is accompa-
nied by a significant refolding of the protein. The CTD
rearranges into its mature position and engages intra-
hexamer interaction with the NTD of an adjacent mole-
cule. The 13 N-terminal residues refold into a β-hairpin
that engages the formation of a salt bridge between
Proline 1 and Aspartate 51 residues in the NTD of CA.
This salt bridge is essential for proper core assembly and
viral infectivity. NTD refolding was proposed to create a
new dimeric interface required for appropriate assembly
of the conical core. This function was recently contra-
dicted [75], and the contribution of the β-hairpin was
rather proposed to determine and stabilize the orientation
of CA proteins in the mature lattice [76].
Finally, the release of mature CA is accompanied by
production of processed MA and NC proteins. Release
of fully mature NC is required for infectivity as it favors
the condensation of the ribonucleoprotein complex and
stabilizes dimers of genomic RNA [77]. Processing also
lowers membrane-binding affinity of MA, reduces mul-
timerization and results in the sequestration of the my-
ristate moiety. Such modification may be required for
appropriate membrane dissociation of viral preintegra-
tion complexes of incoming particles. Finally, the pro-
teolytic processing of Gag also regulates Env-mediated
cell-to-cell fusion, perhaps by altering the interaction
between MA and the cytoplasmic tail of gp41 [78,79].
6. New Inhibitors of HIV-1 Replication
At the end of 2010, approximately 33.3 million people
were estimated to be infected worldwide with HIV-1
(UNAIDS Global report 2010; http://www.unaids.org/
globalreport/). Significant progress has been made in the
treatment of HIV-1-infected people since the introduc-
tion of highly active antiretroviral therapy (HAART)
based on the combined use of nucleoside/nucleotide re-
verse transcriptase inhibitors (NRTIs), non-nucleoside
reverse transcriptase inhibitors (NNRTIs), and/or prote-
ase inhibitors (PIs) (Figure 4). However, serious adverse
effects together with the emergence of multi-drug-resis-
tant viral strains transmitted to more than 25% of newly
infected individuals are major concerns in the use of
these drugs. Emergence of variants resistant to approved
drugs suggests the need for not only novel compounds,
but also compounds active against novel targets. A de-
tailed understanding of HIV-1 assembly, release and
maturation has made it possible to design or discover
small molecules and peptides that interfere with assem-
bly release and maturation.
6.1. Assembly and Release Inhibitors
The identification of active peptides as candidates for
intervention at the virus assembly level is one promising
strategy. As exposed above, the assemblies of Gag pro-
teins into immature viral particles followed by prote-
olytic disassembly of the Gag shell to mature capsids are
crucial steps for the formation of infectious HIV-1 parti-
cles. In this process the function CA in context of Gag or
in assembling the conical core of viral particles is of cen-
tral importance. Inhibitors that bind CA could therefore
disrupt virus assembly at various steps. The identification
by phage display screening of a 12-mer peptide capable
to inhibit both mature and immature particles in vitro
provided a proof of concept for this new class of inhibi-
tors. This peptide termed capsid assembly inhibitor (CAI)
binds residues 169 - 191 in CA and interacts with the last
α-helix of the protein [80]. A high-resolution X-ray
structure of CAI in complex with CTD of HIV-1 capsid
protein (CACTD) has revealed that the peptide binds to a
hydrophobic groove formed by helices 1, 2 and 4 [81].
However, the CAI peptide itself fails to inhibit HIV-1
assembly in cell culture due to its inability to penetrate
cells. A structure-based rational design approach known
as hydrocarbon stapling has then been developed to sta-
bilize the alpha-helical structure of CAI and convert it to
a cell-penetrating peptide. The resulting molecules,
named NYAD-1 and NYAD-13 efficiently disrupt HIV-1
assembly in cell cultures [82]. However, because they
have a relatively low affinity for CA, these peptides are
unlikely to progress to the clinic [80,82,83]. Anyway,
NYAD peptides should be considered as a proof of con-
cept for cell penetrating peptide which inhibits both as-
sembly of immature particles and core formation [80,82].
Information gained in the study of these molecules has
been used as a starting point for the design of pepti-
domimetics and small molecule drugs targeting HIV-1
assembly and have warranted high-throughput virtual
docking screens for compounds binding in the pocket
formed by helices 1, 2 and 4 in CA. After selection, 2
compounds out of a series of 8 hits have been found to
retain an antiviral activity. Both are active at low mi-
cromolar concentrations in cell culture to disrupt ma-
ture assembly but not immature assembly in vitro [84].
While all the above mentioned compounds act through
binding to CACTD, candidate molecules screened by in
silico modeling and NMR titration spectroscopy of
molecules interacting with the N-terminal moiety of
CA have been identified [85]. Two molecules, CAP-1
and CAP-2, have been reported to inhibit the assembly
and/or the stability of the mature capsid lattice. CAP-1
interacts with the CANTD with a Kd of 1 mM and
CAP-2 with a Kd of 52 μM. The binding sites

HIV-1 Assembly, Release and Maturation 119
Figure 4. HIV-1 life cycle and current inhibitors of HIV-1 replication. Current anti-HIV-1 therapies inhibit reverse tran-
scriptase and the HIV-1-encoded protease. Alternative strategies under development target the integrase, binding of the viral
envelope with its receptors and fusion of the viral particle with the host cell membrane. The most recent approaches are de-
signed to inhibit viral maturation in a protease independent way and to perturb assembly of the mature capsid.
determined by NMR perturbation experiments map to a
single sequence located at the apex of a helical bundle
composed of helices 1, 2, 3, 4, and 7 which have been
demonstrated to be involved in an intersubunit CANTD-
CACTD interaction unique to the mature lattice. The
structure of complexes formed by CANTD and CAP-1
evidenced that upon CAP-1 binding, CANTD undergoes
conformational rearrangements with a displacement of
F32 residue from its buried position in the protein core
[86]. Both CAP-1 and CAP-2 block in vitro assembly of
CA into helical tubes; CAP-2 has been proven to be cy-
totoxic, but CAP-1 was nontoxic and resulted in greater
than 95% inhibition of virus replication in cell culture
[85]. Morphological analysis indicates that virions pro-

HIV-1 Assembly, Release and Maturation 120
duced in the presence of CAP-1 are more heterogeneous
in size than those produced in its absence and display
aberrant core morphology. Based on the measured
CAP-1-CANTD affinity, it is estimated that as few as ~25
molecules of CAP-1 per particle are sufficient to inhibit
the formation of a functional core particle. As CAI and
its derivatives, CAP-1 disturbs interaction with CANTD-
CACTD interface. The extensive knowledge gained in
studying these inhibitors and their molecular targets has
paved the way for subsequent efforts to screen for small
molecules inhibitors of HIV-1 assembly and/or matura-
tion.
Screens using a cell-based assay that supports a com-
plete viral replication cycle, identified a series of com-
pounds that interfere with assembly and uncoating.
Among these compounds PF-74 presents an antiviral
activity with an EC50 of 0.6 μM. This molecule, unlike
CAP-1 does not induce conformational change but rather
binds in a pocket defined by helices 3, 4, 5 and 7 into the
CANTD-CACTD interface. Interestingly, PF-74 was also
capable to act early during infection by altering post-
entry uncoating. All these studies validate assembly and
more precisely both the NTD and the CTD of HIV-1
capsid protein as potential antiviral targets.
To complete the viral life cycle, following assembly,
HIV-1 particles are subsequently released from the plas-
ma membrane of infected cells. Release of the virus re-
quires the Gag protein and the active participation of
multiple host proteins [88]. As mentioned above, the p6
region of Gag known as late domain, contains a highly
conserved P(T/S)AP tetrapeptide motif which binds to
the ESCRT-I component Tsg101 and LYPXL which in-
teracts with Alix ([48]). Inhibition of Tsg101 synthesis or
overexpression of the N-terminal Gag binding domain of
Tsg101 (TSG-5’) severely impairs virus production by
arresting the release of viral particles from the plasma
membrane of host cells. In the same way, overexpression
of the Gag-binding domain of Alix (V-domain) also
blocks HIV-1 release [89]. Even if the overexpression of
TSG5’ or Alix V-domain inhibits virus budding in tissue
culture conditions, it seems hard to imagine they can be
used for therapeutic application. As high resolution
structural information is available for p6-Tsg101 and
p6-Alix [90] several research teams have been able to
achieve a rational design of budding inhibitors. A
PTAP-based peptide which presents an increased affinity
for Tsg101 has been designed [91]. Using a bacterial
reverse two hybrid system a library of 3.2 106 cyclic pep-
tides have been screened to identify hits that interfere
with p6-Tsg101 interaction. Five peptides can disrupt
this interaction and inhibit HIV-1 release [92]. Collec-
tively, all these results support the notion that small the-
rapeutic molecules that inhibit specific oligomerization
or multimerization of Gag or CA or contacts between
Gag and host proteins represent a significant challenge
for the development of new classes of antiretrovirals.
6.2. Maturation Inhibitors
6.2.1. Chemical Agents Targeting SP1 Junction and
Antiviral Properties
As the proteolytic processing of Gag is conditioned by
the functionality of the viral protease and by the accessi-
bility of the recognition site in Gag, strategies aimed at
interfering either with PR enzymatic activity or to reduce
access to protein substrate have been considered in the
development of anti-HIV-1 drugs. The later concept is
represented by a new class of HIV-1 antivirals referred to
as maturation inhibitors that have been developed and
tested in phase IIb clinical trials. This class of com-
pounds developed in the recent years is characterized by
its ability to inhibit proteolytic maturation of Gag inde-
pendently of an inhibition of protease activity. The lead
drug of this pharmacologic class is betulinic acid (BA), a
triterpene compound isolated from the clove-like plant
Sygizum claviflorum. This molecule has been initially
identified as an anti-cancer agent with significant activity
against melanoma. In the last years it has been identified
as an anti-HIV-1 molecule [93]. Modification of side
chains at positions C3 and C28 in the betulinic acid
scaffold has been used to derive a library of compounds
that has been evaluated in vitro for its inhibitory proper-
ties [94] (Figure 5). Among these molecules, an acyl de-
rivative of the C3 hydroxyl, 3-O-(3’,3’-dimethylsuccinyl)
betulinic acid, later renamed bevirimat (BVM) or PA-
457 or DSB, displays a mean IC50 value of approximately
10 nM against primary HIV-1 isolates [95] and retains a
therapeutic index greater than 20,000 in H9 lymphocytes
[94]. The viral steps inhibited by these molecules have
been investigated in depth. No activity against reverse
transcriptase or integration has been observed for the
lead compounds [94,96]. Marginally, triterpenes have
been reported to inhibit HIV-1 protease dimerization, a
prerequisite for enzymatic activation [97]. However,
BVM does not exhibit a significant inhibitory effect on
protease cleavage when a synthetic peptide or recombi-
nant Gag are used as substrates [95]. Two possible mode
of action have been identified as primary mechanisms for
betulinic acid-derived molecules. Compounds with modi-
fications at the carboxylic group in position C28 interfere
with HIV-1 entry into target cells [98]. Such effect has
been reported for the RPR103611 and IC9564 derivatives
[98]. These compounds both inhibited cell-cell fusion in-
duced by a variety of viral isolates, without interfering
with the binding of viral particles to the target cell. How-
ever their respective mechanisms of action may differ

Current Distortion Evaluation in Traction 4Q Constant Switching Frequency Converters 121
Figure 5. Inhibitors of HIV-1 maturation and consequences on viral core morphology. (a) Structure of betulinic acid and
some derivatives. C-3 and C-28 atoms are marked; (b) Morphology of HIV-1 particles produced from untreated cells or in
the presence of bevirimat (BVM). The arrow indicates a viral particle with a partial immature morphology. The scale is in-
dicated by a bar.
in some point since the prolonged use of RPR103611
favored the acquisition by the virus of mutations in gp41
[99] and resistance to IC9564 was associated to muta-
tions in gp120 [100]. Although not elucidated, the me-
chanism of action of C28 derivatives of betulinic acid
resembles that of T20 inhibitor and may rely on the inhi-
bition of a late step of the fusion process, occurring after
lipid mixing [101]. Conversely to C28-modified BA de-
rivatives, molecules composed of a triterpenic scaffold
with modification of hydroxylic group at position C3 do

HIV-1 Assembly, Release and Maturation 122
not inhibit viral entry but rather interfere with late stages
of HIV-1 assembly. This inhibitory effect has been ini-
tially identified for the lead compounds BA and BVM.
These compounds, when added to a HIV-1 producing
cell culture, induce a dose-dependent change in the viral
protein profile both in cells and in viral particles [95].
The amount of processed p24 protein decreases in favor
of the immature p25 protein reflecting unprocessed
CA-SP1 junctions. This phenotype is reminiscent of that
observed for HIV-1 bearing mutations in CA or at SP1
junction that abolish CA-SP1 processing [30]. Consistent
with the capacity of BA-derived inhibitors to interfere
with viral maturation, the capacity of viral particles to be
released in the extracellular medium remains unaffected.
Used in de novo infection experiments, these viruses are
competent for binding and fusion events, but are unable
to reverse transcribe genomic RNA into proviral DNA
[102]. From the morphological point of view, these par-
ticles show aberrant core condensation when studied in
electron microscopy. The inner capsid instead of cone-
shaped is acentric, spherical and cohabits with patches of
uncleaved Gag beneath the lipid bilayer [94]. Cryo-EM
analysis has recently revealed that they contain an in-
complete Gag hexameric shell underlying the viral enve-
lope lacking the NC domain [103]. The presence of
dense material at the center of the particle probably re-
flects the release of CA/CA-SP1 molecule in quantities
sufficient to assemble small capsid with altered size and
shape or alternatively that of ribonucleic complexes
mainly comprising NC and genomic RNA (Figure 5(b)).
Thus, it is conceivable that the small amounts of incom-
pletely processed Gag molecules generated by treatment
with inhibitors are sufficient to interfere with the ordered
maturation of Gag, and inhibit HIV-1 infectivity in a
trans-dominant manner as proposed [104]. This defect is
usually associated with incomplete maturation in re-
sponse to SP1 mutations. Isolation experiments have
established that the corresponding core structure is un-
stable [105].
The region binding BA and BVM is not formally de-
termined. However, a number of studies provided sup-
port on the role of the CA-SP1 junction in Gag. Indeed,
BVM, despite inhibiting VLPs in Sf9 insect cells infected
with a recombinant baculovirus expressing HIV-1 or
SIVmac251 Gag precursors with IC50 of 10 µM and 20 µM
respectively, was unable to block assembly of MLV
Pr72Gag [106]. In contrast, BVM efficiently blocks as-
sembly of a MLV-HIV-1 chimera containing SP1-NC-p6
domains from HIV-1 and MA-CA domains from MLV
with an IC50 of 30 µM. Altogether these observations
lead to the conclusion that the main target on Gag is SP1,
and suggested that other upstream Gag region(s) might
contribute to BVM reactivity in that particular model.
The fact that BVM and BA lack activity against HIV-2
and SIV provides support to the hypothesis that
BA-derived molecules act at the level of CA-SP1 matu-
ration [102]. Indeed, these viruses display relatively low
genetic homology with HIV-1 in this particular domain
(Figure 6). Modeling analysis and sequence alignment
indicate that HIV-1 and SIVmac CA-SP1 differ regarding
their hydrophobicity supporting that this intrinsic prop-
erty may dictate sensitivity to BVM. In addition, muta-
tions at the CA-SP1 junction at positions 362 and 363 in
Gag render SIV sensitive to BVM [105]. Mutations ac-
quired by BVM-resistant viruses are expected not to
modify the structure of this particular domain and to in-
crease its hydrophobicity.
Going deeper into the mode of action of BA and BA-
derived molecules, the capacity of these molecules to
inhibit Gag processing by blocking access of the retrovi-
ral protease to the Gag precursor or alternatively through
fixing the Gag shell into as configuration unfavorable to
its proteolytic processing has been questioned. The re-
quirement for a higher order oligomeric Gag structure for
BVM activity has been established. Indeed, the inhibitor
is inactive on recombinant Gag in solution unable to as-
semble. To the opposite, BVM inhibits CA-SP1 process-
ing of in vitro preassembled Gag [95]. Moreover, the
mode of action of BVM apparently requires the binding
to Gag. This mechanism is supported by its selective in-
corporation into HIV-1 immature particles at a 1:1 sto-
echiometry [107]. The observation that mutations in
CA-SP1 conferring resistance to this molecule abolish
incorporation finally suggested that a specific binding of
the inhibitor to the Gag shell is required. Accordingly,
the model of action of BVM and derived molecules cur-
rently accepted indicates that 1) BVM binds Gag when
present in a specific conformation that is unique into the
immature lattice; 2) the bioactive molecule inhibits in-
teractions within the Gag hexamer; 3) the SP1 linker
domain participates in the interaction between Gag and
BVM. A model was proposed in which a trimer of BVM,
stabilized by intermolecular interaction of the hydropho-
bic betulinic acid core, associates with the CA-SP1 junc-
tion present as trimers within the Gag lattice [107]. This
model would explain why BVM is inefficient at inhibit-
ing protease-dependent processing of soluble Gag.
6.2.2. Clin i c al E val uation of Bevirimat and Betulin i c
Acid-Derivatives
The marked capacity of BVM and BA derivatives to po-
tently inhibit HIV-1 replication in vitro together with
toxicological studies showing a very good tolerance of
this series of molecules in rat and marmoset models, a
good absorption after oral administration together with
the lack of serious adverse effects [108] prompted further

HIV-1 Assembly, Release and Maturation 123
Figure 6. Representation of CA-SP1 sequences in HIV-1, HIV-2 and SIV and of mutations acquire d in this region by HIV-1
viruses in response to bevirimat treatment (BVM res). The deletion of amino acid at position 8 in SP1 of low susceptible (Low
susc.) viruses is indicated by *.
evaluation of BVM in vitro and in clinical trials. How-
ever, soon after discovery, BA-derived molecules have
been reported to generate the acquisition of muta-
tion-associated resistances. In vitro, serial passage of
HIV-1NL4.3 in the presence of DSB favored the emer-
gence of resistant strains. Individual mutations responsi-
ble for DSB resistance have been mapped both to resi-
dues immediately flanking the CA-SP1 cleavage site and
to the C-terminus of CA. No other change either in the
protease or in the other domain of Gag correlates with
DSB resistance. Six individual mutations have been first
identified: H226Y, L231F, and L231M in CA and A1V,
A3T, A3V in SP1. [95,105,109]. L231M and A1V muta-
tions decrease BVM susceptibility by 37.6-fold and more
than 77.5-fold, respectively [110]. In phase 1/2 and 2a
clinical trials, 50 to 60% of HIV-1 patients experience
resistance to BVM [110]. From the genotypic point of
view, the in vivo situation appears quite different of that
established in vitro since patient isolates do not contain
the corresponding mutations [111]. Instead, a predictive
value for BVM resistance has been attributed to poly-
morphisms in a QVT motif at positions 6, 7, and 8 in SP1
[110-113]. In patients, the presence of the T8 deletion
often coincided with the presence of a polymorphism at
T7, making the role of the T8 deletion in resistance more
difficult to assess. More recently, SP1 residue 7 of the
Gag protein has been proposed to be the primary deter-
minant for SP1 polymorphism associated drug resistance
to BVM for subtype C isolates [114]. Nevertheless, the
T371 deletion plays a less critical role than V7A in resis-
tance to BVM [112]. This deletion is observed in
PI-experienced patients and is proposed to be co-selected
with treatment with protease inhibitors. Polymorphism
study has finally revealed that in vivo, the CA pro-
tein-V230I mutation is also a major mutation conferring
resistance to BVM [112]. From these studies it appears
that the knowledge on molecular mechanisms of inhibi-
tion by BVM needs to be significantly improved before
BA-derived molecules are further optimized.
Of note, the capacity of BVM and BA-derived mole-
cules to inhibit HIV-1 maturation is shared by another
inhibitor of HIV-1 maturation isolated in the same time.
PF-46396, a pyridone-based compound has been discov-
ered as a hit from high-throughput full HIV-1 replication
screen of inhibitors [115]. This small molecule has been
further identified as an inhibitor of viral maturation. As
previously observed for BVM and BA-derived com-
pounds, PF-46396 interferes specifically with the cleav-
age of the CA-SP1 without effect on PR activity [116].
Selection pressure exerted by PF-46396 yields the acqui-
sition of a I201V mutation in the C-terminal region of
HIV-1, a mutation that also confers resistance to BVM.
Moreover, an HIV-1 mutant with the BVM-induced
SP1A1V mutation displays resistance to PF-46396. Ac-
cordingly, both molecules have been proposed to share
the same mechanism of action despite their distant struc-
tural relationships.
6.2.3. Perspectives in the Study of Maturation
Inhibitors
The future optimization of triterpene compounds has
been evoked. Indeed, chemical modifications at both C-3
and C-28 result in bi-functional BA derivatives. [[N-
[3β-O-3’,3’-Dimethylsuccinyl-lup-20(29)-en-28-oyl]-7-
aminoheptyl]-carbamoyl]methane (A12-2) is one of the
most potent BA derivatives that inhibits HIV-1 replica-
tion at both maturation and entry steps [117]. These mo-
lecules are certainly of interest to target simultaneously
early and late steps of HIV-1 replication. In addition,
modification BA with insertion of a shorter C-28 side

HIV-1 Assembly, Release and Maturation 124
chain, an equivalent of CONH-(CH2)nR with n being 4 or
5, results in optimal anti-HIV-2 activity [118]. This
compound acts by targeting the V3 loop of gp120. Ac-
cordingly, these compounds could benefit to HIV-2 in-
fected patients.
In addition, a large panel of areas remains to be inves-
tigated regarding the mode of action of BA-derived
compounds and the resistance acquired in response to
treatment. The first area regards the defects generated at
the level of virus assembly by this series of molecules.
This includes possible consequences of genomic RNA
packaging. First, structural studies will be of help to de-
fine the capacity of SP1 junction to oligomerize in the
presence of BA-derived molecules. Indeed, during as-
sembly SP1 domain that folds into an amphipathic helix,
assemble via hydrophobic and hydrophilic contacts [119].
Investigating the morphology of VLPs produced in the
absence of viral protease from mammalian cells main-
tained in the presence of these molecules. Second, SP1
domain participates in contacts with SL3 RNA loop as
shown RMN study of CA148-231-SP1-NC1-55/SL3 com-
plexes [120]. HIV-1 RNA dimerization and Gag matura-
tion are intimately linked [38]. Formation of a uniform
RNA dimer is acquired after complete virion maturation
since protease deficient viruses fail to stably dimerize
genomic RNA [74]. Recently, evidence was provided for
the contribution of Gag cleavage products in stabilizing
genomic RNA dimers. Indeed, the initial RNA dimer is
stabilized during the primary SP1-NC cleavage and
complete RNA dimerization depends of ensuing prote-
olytic processing. Accordingly, perturbations of Gag
processing might affect viral infectivity through altera-
tion of RNA dimer stabilization. The contribution of such
mechanisms in inhibitory effects of BA and derived
compounds needs to be investigated.
In summary, despite the intensive investigation de-
voted to the study of HIV-1 assembly, release and matu-
ration during the past 25 years, the precise molecular
mechanisms required to assemble and to maturate HIV-1
virions are far from elucidated. The recent advances in
the field have demonstrated that the identification of
these crucial steps will offer new ways to develop bioac-
tive molecules capable of inhibiting the replication cycle
of the virus. The many examples mentioned in this re-
view of molecules that have an antiviral effect by target-
ing viral assembly, release or maturation, show that ef-
forts to elucidate the antiviral mechanisms of this family
of anti-viral compounds will not only lead to the devel-
opment of new bioactive molecules used in infected pa-
tients but will also help to better understand the biology
of HIV-1.
7. Acknowledgements
We are grateful to members of the laboratory for helpful
discussions. Research in our laboratory is supported by
the French AIDS Agency ANRS, by the CNRS-UM1-
UM2 and Sidaction. We apologize to all authors whose
work could not be included in this review due to space
limitations.
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