Human Immunodeficiency Virus Gag and protease: partners in resistance

Article (PDF Available)inRetrovirology 9(1):63 · August 2012with46 Reads
DOI: 10.1186/1742-4690-9-63 · Source: PubMed
Human Immunodeficiency Virus (HIV) maturation plays an essential role in the viral life cycle by enabling the generation of mature infectious virus particles through proteolytic processing of the viral Gag and GagPol precursor proteins. An impaired polyprotein processing results in the production of non-infectious virus particles. Consequently, particle maturation is an excellent drug target as exemplified by inhibitors specifically targeting the viral protease (protease inhibitors; PIs) and the experimental class of maturation inhibitors that target the precursor Gag and GagPol polyproteins. Considering the different target sites of the two drug classes, direct cross-resistance may seem unlikely. However, coevolution of protease and its substrate Gag during PI exposure has been observed both in vivo and in vitro. This review addresses in detail all mutations in Gag that are selected under PI pressure. We evaluate how polymorphisms and mutations in Gag affect PI therapy, an aspect of PI resistance that is currently not included in standard genotypic PI resistance testing. In addition, we consider the consequences of Gag mutations for the development and positioning of future maturation inhibitors.


R E V I E W Open Access
Human Immunodeficiency Virus gag and
protease: partners in resistance
Axel Fun
, Annemarie MJ Wensing
, Jens Verheyen
and Monique Nijhuis
Human Immunodeficiency Virus (HIV) maturation plays an essential role in the viral life cycle by enabling the
generation of mature infectious virus particles through proteolytic processing of the viral Gag and GagPol precursor
proteins. An impaired polyprotein processing results in the production of non-infectious virus particles.
Consequently, particle maturation is an excellent drug target as exemplified by inhibitors specifically targeting the
viral protease (protease inhibitors; PIs) and the experimental class of maturation inhibitors that target the precursor
Gag and GagPol polyproteins. Considering the different target sites of the two drug classes, direct cross-resistance
may seem unlikely. However, coevolution of protease and its substrate Gag during PI exposure has been observed
both in vivo and in vitro. This review addresses in detail all mutations in Gag that are selected under PI pressure. We
evaluate how polymorphisms and mutations in Gag affect PI therapy, an aspect of PI resistance that is currently not
included in standard genotypic PI resistance testing. In addition, we consider the consequences of Gag mutations
for the development and positioning of future maturation inhibitors.
Keywords: HIV, Particle maturation, Protease inhibitors, Maturation inhibitors, Gag mutations, Resistance,
HIV maturation
HIV is released from the host cell membrane as a non-
infectious particle that is called the immature virion.
After budding and release, the virion undergoes a dra-
matic structural rearrangement that results in fully infec-
tious virus. Transition of the amorphous, non-infectious
virion into the mature, infectious virion that is charac-
terized by an electron-dense conical core is called mat-
uration (Figure 1). This transition is triggered by the
proteolytic cleavage of the Gag (Pr55
) and GagPol
) precursor polyproteins by the viral enzyme
protease (PR). Gag is cleaved into the structural proteins
matrix (MA, p17), capsid (CA, p24) and nucleocapsid
(NC, p7), p6 and two small spacer peptides (p1 and p2).
Pol, which is translated as the GagPol polyprotein after
a -1 nucleotide frameshift event, that occurs with a
frequency of 5-10% [1], encodes the viral enzymes PR,
reverse transcriptase (RT) and integrase (IN). Analysis of
different Gag substrates revealed that HIV PR recognizes
the asymmetric, 3-dimensional conformation of the Gag
substrate, rather than a particular peptide sequence [2].
The peptides that form the different cleavage sites (CS)
have a superimposable secondary structure, yielding the
so-called substrate envelope which fits within the sub-
strate binding pocket of the viral PR. However, each sub-
strate has a unique structure, and there are subtle
differences in the way the amino acids protrude from
the substrate envelope. It is thought that these small dif-
ferences in substrate structure impact affinity for the
viral protease and contribute to the highly regulated and
ordered stepwise process of viral maturation in which all
the individual cleavages occur at different rates [3-6]
(Figure 1). First, the scissile bond between p2 and NC
(MA-CA-p2#NC-p1-p6) is cleaved, followed by separ-
ation of MA from CA-p2 (MA#CA-p2). Subsequently
p6 is cleaved from NC-p1 (NC-p1#p6). Finally, the two
small spacer peptides are removed in the rate-limiting
cleavage steps NC#p1 and CA#p2, of which CA#p2 is
thought to be the final cleavage (Figure 1). This ordered
cleavage is mainly regulated by those amino acids in the
substrate that are in direct contact with the viral PR
* Correspondence:
Department of Virology, Medical Microbiology, University Medical Center
Utrecht, HP G04.614, Heidelberglaan 100, Utrecht 3584 CX, The Netherlands
Full list of author information is available at the end of the article
© 2012 Fun et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Fun et al. Retrovirology 2012, 9:63
(p4-p3position, Figure 1) [6-8]. Although most studies
have focused on the impact of these substrate residues
that are in direct contact with the viral PR, it has been
demonstrated that the more distantly located p4and p5
residues can also affect processing efficiency [9-13].
HIV protease
HIV protease is a member of the family of aspartic pro-
teases and is a symmetrically assembled homodimer
consisting of two identical subunits of 99 amino acids.
Both subunits contribute catalytic residues to the active
site (an aspartic acid at codon 25) [14,15]. The substrate
binding pocket is at the center of the dimer and inter-
acts with the different substrate sequences in the Gag
and GagPol polyproteins. The mechanism that activates
the viral PR, which is embedded in the GagPol poly-
protein itself, is not yet fully understood. It is known
however, that the viral PR is responsible for its own
Figure 1 A schematic representation of HIV particle maturation. At the top, the viral GagPol polyprotein is depicted. On the left, the 5
sequential proteolytic processing steps of Gag are shown. In the middle, the 5 Gag cleavage sites (CS) and their nucleotide and corresponding
amino acid sequences are shown. The numbers above the residues correspond to their position in the Gag polyprotein. At the top of the middle
panel, the location of the p5-p5positions is indicated. On the right are schematic representations and electron-microscopy images of an HIV
particle. Top: the immature, non-infectious particle with its granulated core. Bottom: the fully mature and infectious virion with its characteristic
electron-dense conical core. The pacman figure represents the viral protease enzyme.
Fun et al. Retrovirology 2012, 9:63 Page 2 of 14
release from the precursor polyprotein (autoprocessing).
Since PR is active only as a dimer, it is thought that
autoprocessing is initiated by dimerization of two prote-
ase domains that are still embedded in the GagPol pre-
cursor. The initial cleavage is a transient, intramolecular
event and the low occupancy of the embedded dimer
configuration can explain its low enzymatic activity
compared to the fully matured PR enzyme [16,17].
Protease inhibitors
Detailed structural knowledge of HIV PR and its sub-
strate led to the development of specific protease inhibi-
tors (PIs). To date, nine different PIs have been
approved for clinical use: saquinavir (SQV), ritonavir
(RTV), indinavir (IDV), nelfinavir (NFV), (fos)amprena-
vir (FPV/APV), lopinavir (LPV), atazanavir (ATV), tipra-
navir (TPV) and darunavir (DRV). All PIs, with the
exception of tipranavir, are competitive peptidomimetic
inhibitors, mimicking the natural substrate of the viral
PR. The peptidomimetic inhibitors contain a hydro-
xyethylene core which prohibits cleavage by the viral PR
[18-25]. Instead of a hydroxyethylene core, tipranavir
contains a dihydropyrone ring as a central scaffold [26].
In general, all these compounds have been designed to
bind to the substrate binding region of the mature viral
PR dimer with high affinity, but they tend to occupy
more space than the natural substrate. For tipranavir
and darunavir, it has been demonstrated that they have a
dual mechanism of inhibition as they also impede
dimerization of the viral PR [27]. This may contribute to
their antiviral potency and high genetic barrier towards
resistance, although the impact of the anti-dimerization
activity has not been elucidated yet.
Inhibition of the initial GagPol processing steps which
involve self-cleavage of the embedded HIV PR from the
GagPol polyprotein (autoprocessing) would prevent viral
maturation at the earliest stages and therefore be an
ideal drug target. However, all PIs have been developed
to bind the active site of the mature PR dimer, and it
was shown that the embedded HIV PR dimer is 10,000
fold less sensitive to RTV than the mature PR dimer
[16]. More recently, two different groups demonstrated
independently and using different assays, that of the
nine approved PIs, DRV and SQV are the most potent
inhibitors of autoprocessing. However, both inhibitors
are still three orders of magnitude less active against the
embedded dimer compared to the mature PR dimer
Currently, first line highly active antiretroviral therapy
(HAART) regimens usually consist of a combination of
two nucleoside reverse transcriptase inhibitors (NRTIs)
with either a non-nucleoside reverse transcriptase inhibi-
tor (NNRTI), an integrase inhibitor, or a PI. Effective
HAART has reduced HIV-related morbidity and
mortality and greatly improved therapeutic success rates
[30]. However, in the early days of PI therapy, high pill
burden and related toxicity, low bioavailability and a low
barrier to the emergence of resistance severely impaired
effective treatment of HIV infected individuals. Resist-
ance to PIs was usually associated with the selection of
multiple mutations in the viral PR resulting in broad
class cross-resistance. Since then, several strategies
have been developed to improve clinical outcome and
increase the barrier to development of PI resistance.
Co-administration with ritonavir, an inhibitor of the
cytochrome P450 3A4 isoenzyme, which is involved in
the metabolism of all PIs, considerably improved the
bioavailability and half-life of PIs [31] resulting in higher
PI plasma concentrations with a reduced pill burden
and related drug toxicity [32]. Another improvement
was the development of second generation PIs that have
an intrinsic higher genetic barrier to development of re-
sistance, which are (fos)amprenavir, lopinavir, atazanavir,
tipranavir and darunavir [20,23-26,33-36]. In patients on
second generation PI based HAART and who have not
received prior PI therapy, selection of primary resistance
mutations in the viral PR is rare, even in case of therapy
Evolution of PI resistance usually has a biphasic signa-
ture in which mutations develop initially in or near the
substrate binding pocket of the viral PR. In fact, it has
been shown that resistance mutations mainly develop
where the PIs protrude beyond the substrate binding
pocket at residues that are in direct contact with the in-
hibitor, but not with the natural substrate [37-39]. These
mutations lower the affinity for the drug more than for
the natural substrate, which decreases the susceptibility
to the drug, resulting in a resistant virus. However, by
changing the substrate binding region of the enzyme,
the affinity for the natural substrate (Gag) is also slightly
altered, often resulting in reduced viral replication
[40-42]. In a second step, compensatory or secondary
mutations can be selected that restore viral replication
and/or enhance drug resistance. These mutations are
found in the viral PR itself as well as in the Gag sub-
strate and in particular, in the NC/p1 and p1/p6 cleavage
sites [9,12,43-49]. It has also been shown that several
Gag substrate mutations are primary drug resistance
mutations that confer PI resistance in the absence of PR
mutations [10,50].
In this review we describe the results of a comprehen-
sive search of the available literature investigating nat-
ural variation in Gag and coevolution of Gag and
protease during protease inhibitor exposure. We provide
a detailed overview of Gag mutations that are observed
during PI exposure, both in vivo and in vitro and how
they affect PI therapy and resistance. Furthermore, we
evaluated the impact of these Gag mutations on the
Fun et al. Retrovirology 2012, 9:63 Page 3 of 14
efficacy of the novel antiretroviral class of CA/p2 matur-
ation inhibitors.
Natural variation of Gag cleavage sites
Only a limited number of studies evaluated the natural
variation within Gag and its cleavage sites and most data
are from studies focusing on subtype B [51-58]. The lim-
ited data that are available suggest that the variation in
non-B subtypes is greater than in subtype B [52,53,56].
All these studies show that the degree of conservation
differs dramatically between individual amino acid posi-
tions as well as between the different cleavage sites as a
whole (Table 1). Cleavage site p2/NC is the most vari-
able of the 5 Gag cleavage sites, followed by p1/p6, NC/
p1, CA/p2 and finally MA/CA, which is the most con-
served CS in subtype B isolates. Amino acids 369-371 in
p2 are included in this table as they are important for
CA/p2 maturation inhibitor susceptibility, which will be
described later in this review.
Selection of Gag cleavage site mutations during protease
inhibitor exposure
During PI exposure, substitutions in all Gag CS have been
described. Mutations in MA/CA (codon 128), NC/p1
(codons 431, 436 and 437) and p1/p6 (codons 449, 452
and 453) are observed most frequently in vitro and
in vivo and have been shown to confer PI resistance
(Table 2). The effect of these different CS mutations is
described in detail below.
MA/CA mutations
Several mutations at MA codon 128 (V128T/A/del) were
associated with exposure to PIs in vivo (FPV/ATV/r) [56].
In addition, substitution V128I was observed more fre-
quently in subtype G isolates from PI experienced
patients compared to PI naïve patients [65]. Mutation
V128I was also associated with virological rebound in
patients on a boosted DRV containing regimen and was
positively correlated with presence of PR mutation V32I
[66]. It also has been selected in vitro with GS-8374, an
experimental high genetic barrier PI [64].
NC/p1 mutations
NC/p1 CS mutation A431V is the most frequently occur-
ring Gag CS mutation during PI exposure. It has been
observed in vivo during PI therapy with RTV
[46,51,79,80], IDV [45,51], NFV [77], SQV [51,79], LPV
[81] and was also associated with PI exposure in unspeci-
fied therapy or cross-sectional analyses [10,55,57,78,82]. It
is often observed in combination with one or more of
Table 1 Natural variation of Gag cleavage sites in subtype B isolates
MA // CA
HXB2 aa V S Q N Y // P I V Q N
position 128 129 130 131 132 // 133 134 135 136 137
variability (%) 3.5 - 4.3 - 3.5 // - - 0.9 0.9 -
CA // p2
HXB2 aa K A R V L // A E A M A Q V T
position 359 360 361 362 363 // 364 365 366 367 368 369 370 371
variability (%) 0.2 0.2 1.8 11.5 1.2 // - - - - 1.2 4.1 24.8 16.4
p2 // NC
HXB2 aa S A T I M // M Q R G N
position 373 374 375 376 377 // 378 379 380 381 382
variability (%) 36.3 32.6 42.7 23.6 1.8 // 5.5 - 40.9 5.5 2.1
NC // p1
HXB2 aa E R Q A N // F L G K I
position 428 429 430 431 432 // 433 434 435 436 437
variability (%) 2.3 3.5 - 0.5 - // - 0.1 - 6.3 5.5
p1 // p6
HXB2 aa R P G N F // L Q S R P
position 444 445 446 447 448 // 449 450 451 452 453
variability (%) - 0.1 - - - // 9.1 - 22.8 - 8.4
Data describing the natural variation at individual codons in the five Gag cleavage sites were pooled and the frequency of polymorphisms as compared to the
HXB2 reference sequence was calculated. Only data describing subtype B isolates were included in the analysis. Codons 369-371 in p2 are included because of
their important role in CA/p2 maturation inhibitor susceptibility. A dash indicates 100% conservation.
Fun et al. Retrovirology 2012, 9:63 Page 4 of 14
Table 2 All Gag mutations associated with PI exposure and/or resistance and maturation inhibitor resistance
Gag mutation Associated with PI exposure Associated with
PI resistance
Associated with
maturation inhibitor
in vivo in vitro
E12K yes [59,60]
G62R yes [61]
L75R yes [59,60]
R76K yes [62,63] yes [62]
Y79F yes [62,63] yes [62]
T81A yes [62,63] yes [62]
K112E yes [64]
V128I/T/A/del yes [56,65,66] yes [64] yes [64]
Y132F yes [46,67]
M200I yes [64]
H219Q/P yes [59,60,64]
CS CA/p2
A360V yes [46]
V362I yes [68] yes [69,70]
L363M/F/C/N/Y yes [61] yes [69,71-74]
S368C/N yes [51,54] yes [69]
Q369H yes [54] yes [72,74]
V370A/M/del yes [54] yes [69,70,72,74-76]
T371del yes [77] yes [72,74,76]
CS p2/NC
S373P/Q/T yes [51,54,78]
A374P/S yes [78]
T375N/S yes [46,78]
I376V yes [46,51]
G381S yes [46]
I389T yes [77]
V390A/D yes [59,60]
I401T/V yes [77] yes [64]
R409K yes [59-61,64]
CS NC/p1
E428G yes [66]
Q430R yes [44]
A431V yes [10,45,46,51,55,57,77-83] yes [10,44,84,85] yes [13,55,81]
K436E/R yes [10,55,82] yes [10] yes [10,13,55]
I437T/V yes [10,45,46,51,55,78,82,86] yes [10,61] yes [10,13,55]
Fun et al. Retrovirology 2012, 9:63 Page 5 of 14
the following PI resistance mutations in the viral PR:
L24I, M46I/L, I50L, L76V, V82A/T/F and I84V. In vitro,
mutation A431V was selected during exposure to RTV
[10,84], LPV [85] and experimental PI BILA 2185 BS
[44]. Mutation A431V confers resistance to all PIs except
DRV [10,55] and can be considered a primary PI resist-
ance mutation as it confers PI resistance in the absence
of mutations in the viral PR [10]. The level of resistance
caused by this mutation is comparable to that of single
PI resistance mutations in PR (M46I and V82A) [81].
Substitutions at amino acids 436 and 437 in the
NC/p1 CS have been observed during PI therapy with
RTV [46,51], IDV [45], SQV [51] and were associated
with PI exposure in unspecified therapy or cross-
sectional analyses [10,55,78,82,86]. Mutations at Gag
position 436 are associated with PR mutation V82A and
mutation I437V with PR mutations: I54V, V82F/T/S and
I84V. They have been selected in vitro with experimental
high genetic barrier PIs (RO033-4649; 436E + 437T,
437T and 437V [10] and (GRL-02031; 437T [61]). These
mutations confer PI resistance and mutation I437V and
the double mutation K436R + I437T also confer PI re-
sistance in the absence of PR mutations [10,13,55]. Mu-
tation I437V alone results in low-level PI resistance,
but the double mutation K436E + I437T has a greater
impact on PI susceptibility and confers slightly more
resistance than mutation A431V [13].
p1/p6 mutations
Mutations in the p1/p6 CS and especially substitutions
at codons 449, 452 and 453 are also often observed
during PI therapy [9,45,46,55-57,77,79-82,86].
Mutations L449F/V/P have been associated with PI
therapy in a number of cross-sectional studies (Table 2)
and have been directly related to treatment with RTV
[80], IDV [45],NFV [67], FPV, ATV [56], SQV [46,56]
and APV [9]. Mutation L449F often occurs in combin-
ation with PR mutations D30N/N88D, I50V and I84V
and mutation L449V is observed with PR mutations
L449F has been selected in vitro using LPV [85], APV
[49], and experimental PIs BILA 1906 BS, BILA 2185 BS
[44] and GW640385 [87]. Alone, mutation L449F has no
effect on PI susceptibility, but in combination with
mutations in the viral PR it affects inhibitor resistance.
Combined with D30N/N88D, it decreases susceptibility
to IDV, SQV, APV and TPV. In combination with V82A
or V82A/L90M, mutation L449F decreases susceptibility
to all PIs (DRV was not tested). Interestingly, when com-
bined with PR mutation I50V, it induces hypersuscept-
ibility to IDV, LPV and especially RTV [87].
Amino acid substitutions at position 452 have been
associated with exposure to RTV, SQV [79], DRV [66]
and in two cross-sectional studies [55,82]. In vivo, muta-
tions at this position associate with PR mutations D30N/
N88D, I50V and I84V [55]. In vitro, mutation P452K has
been selected with experimental PI GW640385 [87]. In
combination with PR substitutions I84V or I84V/L90M,
R452 mutations decrease susceptibility to all PIs except
TPV (DRV was not tested) [55].
Mutations at position 453 have been associated with
PI exposure in vivo to APV [9], LPV [81], NFV [57,67],
RTV, IDV and SQV [57]. It is often seen together with
PR mutations D30N, I50V, I84V, N88D and L90M
[9,55,57]. In vitro, mutation P453L has only been
described being selected with IDV and P453T with ex-
perimental PI GW640385 [87]. P453L does not confer
resistance on its own, but enhances PI resistance in
Table 2 All Gag mutations associated with PI exposure and/or resistance and maturation inhibitor resistance
CS p1/p6-gag
L449F/P/V yes [9,45,46,55-57,67,77,79,80,82,86] yes [44,49,85,87] yes [55]
S451T/G/R yes [55,66]
R452S/K yes [55,66,79,82] yes [87] yes [55]
P453A/L/T yes [9,55,57,77,81,82][67] yes [84,87] yes [9,55]
E468K yes [59]
Q474L yes [77]
A487S yes [77]
P497L yes [77]
V484G/I/P/S yes [88]
CS TFP/p6pol
D437N yes [56,64]
Fun et al. Retrovirology 2012, 9:63 Page 6 of 14
combination with PR mutation I50V [9,55] and I84V to
NFV, APV and SQV and to all PIs in combination with
L90M (DRV not tested) [55].
Gag CS mediated PI resistance
The observed association between PI exposure and Gag
CS mutations signifies the close relationship of the viral
PR and the Gag CSs and their contribution to escape PI
pressure. The effect of Gag CS mutations on PI suscepti-
bility has been studied in detail. Structural and func-
tional analyses of the processing efficiencies of wild type
or mutant substrates showed improved processing and/
or higher predicted binding affinities for the mutant sub-
strates [9,11,37,89,90]. Enhanced processing of Gag is
believed to shift the equilibrium of protease inhibitor/
Gag substrate with the viral PR in favour of the Gag sub-
strate and thereby confers resistance. This proposed
mechanism of Gag CS mediated PI resistance is sup-
ported by studies that show altering just one CS (NC/p1
in these studies) also affects the processing efficiency at
other CSs and thereby the entire substrate processing
cascade [13,91]. Nevertheless, the difference in affinity of
the viral PR for either the substrate or the PIs seems to
contradict this explanation. Whereas the PRs affinity
constant for the PIs is thought to be in the low nanomo-
lar range, the affinity constant for the natural substrates
is in the millimolar range [92]. How a small shift in
equilibrium induced by CS mutations can negate this
million fold difference in binding affinity is not fully
understood. One possible explanation that is offered
comes from the stoichiometry of viral PR relative to its
substrate, as it is present at the site of assembly and mat-
uration [93]. The ratio between PR and natural substrate
compared to PR and PI might be very different at the
site of HIV maturation than in the cell-free environment
used for in vitro enzymatic analysis which could strongly
affect the actual in vivo kinetics [12].
Selection of Gag non-cleavage site mutations during
protease inhibitor exposure
Besides HIV-1 CS mutations, accumulation of non-CS
mutations during PI therapy has been observed in all
Gag proteins (MA, CA, NC, p6) as well as in spacer
peptide p2 (Table 2). Additionally, several non-CS muta-
tions in Gag have been identified in vitro to contribute
to PI resistance in the presence of PR mutations
[34,59,64], but can also mediate reduced PI susceptibility
in the absence of PR mutations [62,94]. The impact of
single non-CS Gag mutations on PI susceptibility has
not been investigated, but combinations of non-CS Gag
substitutions have been shown to affect both viral repli-
cation capacity and PI susceptibility [59,60,62,77]. The
underlying mechanism of non-CS mediated resistance is
not well understood. Since Gag non-CS mutations did
not accumulate in functional related regions but were
found throughout the whole Gag precursor protein,
multiple mechanisms are likely to be involved and differ-
ent per gene-segment.
Substitutions in MA have been associated with viro-
logical failure against boosted LPV containing therapy
(R76K, Y79F and T81A) [62,63]. They have been
selected in vitro with APV (E12K and L75R [59,60]) and
experimental PIs GRL-02031(G62R [61]) and GS-8374
(K112E) [64]). The mechanism of MA-mediated resist-
ance remains elusive, but it is speculated that MA muta-
tions change the multimerization of viral Gag. Another
mechanism was suggested in a study demonstrating that
changes in the tertiary protein structure of the Gag pre-
cursor proteins, caused by three mutations in the matrix
protein, conferred drug resistance [62]. The three resist-
ance associated residues (R76K, Y79F and T81A) are
located in an alpha helical structure within MA, and the
mutations result in loss of certain hydrogen bonds and
thus more flexibility around the helix. It is hypothesized
that the greater flexibility increases either the affinity
or the availability/accessibility of the MA-CA CS with
respect to the PR.
Mutations in CA have not been associated with viro-
logical failure to PI therapy, but substitutions M200I
(with GS-8374 [64]) and H219Q/P (with GS-8374 [64]
and APV [59,60]) have been selected during PI exposure
in vitro. The effect of mutation M200I is unknown, but
substitutions at codon H219 interfere with binding of
cyclophilin A and possibly reduce the requirement for
cyclophilin A for efficient replication and thereby
increase viral replication [59].
Mutations in p2 at codons 369-371 appear to accumu-
late during PI therapy [54,77], but they have not been
associated with virological failure to PI therapy. They
have also not been observed during in vitro selections
with PIs or have demonstrated to contribute to PI resist-
ance. The p2 mutations that accumulate during PI ther-
apy are located in a proposed alpha helical structure
spanning the CA/p2 CS [54]. Therefore, the selection by
PIs of these mutations might be explained by changes in
the stability or the conformation of the alpha helical
structure influencing the accessibility of the CA/p2 CS
by the viral PR.
Amino acid substitutions in NC have been associated with
NFV failure (I389T and I401V [77]). Other mutations in
Fun et al. Retrovirology 2012, 9:63 Page 7 of 14
NC have been selected in vitro with APV (V390A/D and
R409K [59,60]) and GS-8374(I401T and R409K [64]).
None of these mutations demonstrated to confer PI re-
sistance and their contribution to PI therapy failure and
PI resistance remains unclear.
Mutations in p1 outside its cleavage sites have not been
described to be associated to PI exposure and resistance,
in vivo or in vitro.
Only a handful of mutations in p6 have been described
in relation to PI exposure in vivo (NFV; Q474L, A487S
and P497L [77]) and in vitro (APV; E468K [59]). E468K
in combination with other mutations improves viral rep-
lication in the presence of PIs, but the mechanism has
not been elucidated [59].
One paper describes (partial) duplication of the
P(S/T)APP motif in relation to PI therapy response.
P(S/T)APP is a proline rich domain in p6-gag (Gag aa
455-459) that recruits Tsg101, a cellular factor involved
in HIV budding. The authors found a significant associ-
ation of partial or complete P(S/T)APP duplication with
a decrease in virological response to APV at week
12 in highly pre-treated but APV naïve patients [95]. In
addition, P(S/T)APP duplications were significantly asso-
ciated with the presence of a mutation at V82 in PR.
The hypothesized mechanism is an increase in viral
packaging efficiency and budding, leading to an enhanced
viral fitness. One other study describes an accumulation
of P(S/T)APP insertions/duplications during HAART,
but it does not comment on the type of antiretroviral
therapy [96]. In contrast, three other studies did not ob-
serve a correlation between P(S/T)APP duplications and
antiretroviral therapy [97-99], and one study even found
a non-significant trend that HIV-1 patients harbouring
P(S/T)APP insertions were less likely to experience
virological failure [100].
Impact of Gag mutations on PI therapy
Only a few studies investigated the impact of Gag muta-
tions on viral response during subsequent PI therapy.
We summarize the available data of CS substitutions at
MA codon 128, substitutions in p2/NC (codon 373),
NC/p1 (codons 428, 431 and 437), p1/p6 (codons, 449,
451, 452 and 453) and non-CS p6Gag substitutions at
codon 484.
Mutations at MA/CA CS 128 (3.5% natural variability
in subtype B isolates (Table 1)) were negatively asso-
ciated with virological response in ANRS 127, a trial in-
volving naïve patients receiving one of two dual-boosted
PI combinations (FPV/ATV/r or SQV/ATV/r) [56].
Mutation V128I was also observed in >10% of virological
rebounders in an analysis of the combined POWER 1, 2
and 3 trials that evaluated virological response to DRV/r
plus optimized background therapy in PI-experienced
patients [66].
Mutation S373Q (codon 373 is highly polymorphic,
36% variability in subtype B isolates (Table 1)) in the p2/
NC CS, which was associated with the emergence of
specific PR mutations during SQV therapy (K20R/I/M
and L89M/I) did not have an effect on the virological re-
sponse. In contrast, mutation S373P negatively impacted
virological response to SQV [78].
The frequently observed NC/p1 CS mutation A431V
(highly conserved, 0.5% variability in subtype B isolates
(Table 1)) was not associated with a poorer virological
outcome in several studies [10,78] and remarkably, was
correlated with a better outcome to DRV/r therapy [101].
Analysis of the NARVAL trial [102] revealed that
mutation I437V (5.5% natural variability in subtype B
isolates (Table 1)) was significantly associated with a
reduced virological response to different PI therapies
(RTV, IDV, NFV, SQV and APV) [10]. It was also
associated with virological failure in patients on DRV
containing therapy in absence of multiple primary DRV
resistance mutations in the viral PR [103]. Conversely,
mutations at this position in the pol open reading
frame (CS TFP/p6pol) positively impacted virological
response to double boosted PI therapy (FPV/ATV/r or
SQV/ATV/r) [56].
The study on ANRS 127 also revealed a negative asso-
ciation of CS p1/p6 mutation L449P (9.1% natural vari-
ability in subtype B isolates (Table 1)) on virological
response to (FPV/ATV/r and SQV/ATV/r) [56]. In
addition, mutations E428G, S451T and R452S (2.3, 22.8
and 0% natural variability in subtype B isolates (Table 1))
were linked with a reduced response to DRV/r in the
POWER trials [66]. In contrast mutations S451G/N/R
were associated to a better virological outcome in
patients receiving first-line LPV/r monotherapy [88].
This study also showed a negative effect on virological
response to LPV/r monotherapy of non-CS Gag muta-
tions at codon 484 (V484G/I/P/S).
Mutations at codon 453 (8.4% natural variability in
subtype B isolates (Table 1)) were not associated with
virological response [57,78].
Maturation inhibitors
Similar to PIs, the novel class of maturation inhibitors
prevents viral replication by inhibiting particle matur-
ation, but instead of targeting the viral PR, they target
the Gag and GagPol precursor proteins directly. Within
this experimental class of antiretrovirals, the CA assem-
bly inhibitors and CA/p2 inhibitors are the most
advanced in their development. CA assembly inhibitors
are thought to bind CA and inhibit particle maturation
Fun et al. Retrovirology 2012, 9:63 Page 8 of 14
by interfering with CA-CA interactions required for the
formation of the conical-shaped capsid core. The CA
subunits consist of two domains, the N-terminal and
C-terminal domains (NTD and CTD). The mature
capsid is constructed as a lattice of hexamers and
NTD-NTD, NTD-CTD as well as CTD-CTD interac-
tions are required to build the hexamer lattice [104,105].
Binding of the CA assembly inhibitor disrupts the mo-
lecular interface between the functional N-terminal and
C-terminal structures of CA, which are located adja-
cently in the hexamer lattice, thereby preventing core
assembly. Both small molecules and peptide derivatives
are being investigated as potential CA assembly inhibitors
and examples of CA assembly inhibitors that were or are
in development are: CAP-1 [106,107], CAI [108], NYAD-I
[109], BI-257, BI-627 and BI-720 from Boehringer-
Ingelheim [110], PF3450074 from Pfizer [111,112] and
CAC1, CAC1M and H8 [113].
As the name suggests, CA/p2 inhibitors impede par-
ticle maturation by specifically blocking the cleavage of
CA from p2, which is one of the final and rate-limiting
steps in the Gag processing cascade (Figure 1). Unpro-
cessed CA/p2 (p25) interferes with core assembly and
results in the formation of non-infectious particles
[91,114]. Most data on CA/p2 inhibitors are derived
from work on bevirimat (BVM, Panacos PA-457, Myriad
MPC-4326), which was the most advanced maturation
inhibitor in its development (phase II clinical trials).
Western blotting and in vitro resistance selection studies
identified CA/p2 as the target region of bevirimat
[71,114], which was later confirmed by cross-linking
studies [115]. It has also been shown that bevirimat has
a stabilizing effect on the immature Gag lattice which
indicates that bevirimat already binds during assembly
and must be incorporated to inhibit maturation [116].
This observation offers an explanation why CA/p2 inhi-
bitors are unable to inhibit the processing of monomeric
Gag in solution. CA/p2 inhibitors include bevirimat,
PA1050040 which is a second generation maturation
inhibitor from Panacos [117] based on bevirimat, two
maturation inhibitors from Myriad Pharmaceuticals,
Vivecon (MPC-9055) [118,119] and MPI-461359 [120],
and PF-46396 [121] from Pfizer.
The initial in vitro selection studies with bevirimat
identified resistance mutations in the CA/p2 CS at Gag
positions 358, 363, 364 and 366 [71]. A more recent
study identified additional resistance mutations at Gag
codons 362, 368 and 370 [69]. Phase 2b clinical studies
demonstrated that baseline polymorphisms (substitution
and/or deletions) slightly downstream of the CA/p2
cleavage site (Gag p2 aa 369, 370 and 371, known as
the QVT-motif) also confer resistance [72,122]. All cur-
rently known bevirimat resistance mutations are located
in or near the CA/p2 CS (Gag 359-368) (Table 2
[69-72,122,123]). The genetic barrier of CA/p2 matur-
ation inhibitors appears to be low. Most single resist-
ance mutations confer high levels of resistance. This is
confirmed by the in vitro studies where, in contrast to
protease and integrase inhibitors, during in vitro selec-
tions with bevirimat, no accumulation of mutations is
Impact of Gag mutations on CA/p2 maturation inhibitor
Several amino acid positions in Gag that are known to
affect CA/p2 maturation inhibitor susceptibility are
highly polymorphic, including codons 362, 370 and 371
(Table 1). In the treatment-naïve population, approxi-
mately 30% of patients infected with subtype B harbored
an isolate with at least one mutation associated with a
reduced susceptibility to bevirimat [54], and this appears
to be much higher in non-B subtypes with a prevalence
ranging from over 70% to as high as 93% [124,125].
Although PIs and maturation inhibitors have a differ-
ent target site, this review clearly indicates that PI expos-
ure can result in selection of mutations in Gag,
including the CA/p2 cleavage site which affects CA/p2
maturation inhibitor susceptibility (Table 2). Several
studies showed an accumulation of bevirimat resistance
mutations during PI treatment in bevirimat naïve
patients [54,124,126]. These mutations were mainly
observed in the QVT-motif. In subtype B isolates with
PI resistance, the prevalence of bevirimat resistance
mutations increased to 45%, a statistically significant in-
crease. Accumulation of mutations at 4 individual posi-
tions in the CA/p2 region was also statistically
significant and involved amino acid substitutions S368C,
Q369H, V370A and S373P (Table 2 [54]). In addition,
mutations associated with bevirimat resistance were
significantly more detected in HIV isolates with 3PI
resistance mutations than in those with less than three
PI mutations [54,124].
The data presented in this review show that the
CA/p2 region is variable and affected by PI exposure.
A reduced maturation inhibitor activity can be ex-
pected in one-third of the treatment-naïve HIV-1 sub-
type B isolates and significantly more in PI resistant
HIV. Moreover, one could speculate that even in those
individuals who do not have mutations in the CA/p2
region, mutations in the viral PR may affect subsequent
resistance development to the CA/p2 maturation in-
hibitor. One study demonstrated that an impaired Gag
processing efficiency caused by PI resistance mutations,
delayed the development of bevirimat resistance and
reduced the level of bevirimat resistance conferred by
bevirimat resistance mutations [127]. Conversely, we
showed that an increased Gag processing efficiency can
result in enhanced levels of bevirimat resistance [69].
Fun et al. Retrovirology 2012, 9:63 Page 9 of 14
Mutations at codons 362 and 368 give rise to low level
bevirimat resistance (2-6 fold) in the presence of wild
type PR, whereas in the presence of a drug resistant
HIV PR with increased Gag processing bevirimat resist-
ance increases to >150 fold [69]. This intricate relation
between HIV PR and Gag cleavage is supported by a
study by Doyon et al., who also demonstrated that a
CA/p2 mutation at position 362 has differential effects
on CA/p2 processing depending on the genotype of the
protease present in the virus [68].
In conclusion, these studies indicate that during PI ex-
posure, mutations in the target region of the CA/p2
inhibitors may be selected, reducing the baseline suscep-
tibility to the maturation inhibitor. Furthermore, the
level of Gag processing of a PI resistant isolate may im-
pact the development of bevirimat resistance.
Clinical perspective
This review highlights the complex interactions between
the viral protease and its Gag substrates and how muta-
tions in Gag can affect PI and maturation inhibitor sus-
ceptibility. The data summarized in this review clearly
show that mutations in Gag accumulate during PI
therapy and that these mutations can contribute to PI
susceptibility. Even though contemporary therapy suc-
cess rates are very high and development of primary
resistance to PI containing HAART is rare, the relative
high incidence of unexplained failure without major PI
resistance mutations in PR supports including Gag in
the resistance analysis.
Especially, the addition of the C-terminal region of
Gag (NC/p1 and p1/p6gag CSs) to routine testing could
substantially improve our knowledge on genetic vari-
ability and the predictive value of genotypic resistance
testing. However, this coins a paradox as the actual
contribution of Gag mutations to virological failure is
largely unknown, and this question can only be
answered by including Gag in genotypic testing in
clinical trials and cohorts.
HIV maturation inhibitors target the Gag proteins
directly, and therefore genotypic analysis of Gag is
invaluable for the development and clinical implemen-
tation of these inhibitors. This review illustrates that
naturally occurring Gag polymorphisms dramatically
affect the susceptibility to maturation inhibitors in clinical
studies. Furthermore, accumulation of mutations at these
polymorphic positions in Gag is observed during PI ther-
apy failure, strongly affecting the sequential utilization of
maturation inhibitors. New and more potent maturation
inhibitors should therefore overcome the resistance asso-
ciated with these highly variable positions in Gag and
exhibit synergy with protease inhibitors. They should
capitalize on the reduced processing often caused by PI
resistance mutations in such a way that there is added
value from the use of a maturation inhibitor in salvage
therapy for PI experienced patients.
HIV: Human Immunodeficiency Virus; PR: Protease; MA: Matrix; CA: Capsid;
NC: Nucleocapsid; RT: Reverse transcriptase; IN: Integrase; CS: Cleavage sites;
PI: Protease inhibitor; HAART: Highly active antiretroviral therapy;
NRTI: Nucleoside reverse transcriptase inhibitor; NNRTI: Non-nucleoside
reverse transcriptase inhibitor; NTD: N-terminal domain; CTD: C-terminal
Domain; BVM: Bevirimat.
Competing interests
The authors declare they have no competing interests.
AF and MN wrote the manuscript. AMJW modified and contributed parts of
the manuscript in her role as clinical virologist. JV reviewed the literature and
contributed parts of the manuscript. All authors read and approved the final
The Netherlands Organization for Scientific Research (NWO VIDI grant
91796349). NWO had no role in the study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Author details
Department of Virology, Medical Microbiology, University Medical Center
Utrecht, HP G04.614, Heidelberglaan 100, Utrecht 3584 CX, The Netherlands.
Institute of Virology, University of Cologne, Cologne, Germany.
Received: 8 May 2012 Accepted: 17 July 2012
Published: 6 August 2012
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Cite this article as: Fun et al.:Human Immunodeficiency Virus gag and
protease: partners in resistance. Retrovirology 2012 9:63.
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Fun et al. Retrovirology 2012, 9:63 Page 14 of 14
    • "Our genotypic analysis was limited to the pol region. There are indications of polymorphic mutations associated with PI resistance in the gag cleavage site and in env [43, 44], which have not been measured in our study. Their potential contribution to PI failure is not captured in conventional resistance testing and is understudied. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: As antiretroviral therapy (ART) programs in sub-Saharan Africa mature, increasing numbers of persons with human immunodeficiency virus (HIV) infection will experience treatment failure, and require second- or third-line ART. Data on second-line failure and development of protease inhibitor (PI) resistance in sub-Saharan Africa are scarce. Methods: HIV-1-infected adults were included if they received >180 days of PI-based second-line ART. We assessed risk factors for having a detectable viral load (VL, ≥400 cps/mL) using Cox models. If VL was ≥1000 cps/mL, genotyping was performed. Results: Of 227 included participants, 14.6%, 15.2% and 11.1% had VLs ≥400 cps/mL at 12, 24, and 36 months, respectively. Risk factors for a detectable VL were as follows: exposure to nonstandard nonnucleoside reverse-transcriptase inhibitor (NNRTI)-based (hazard ratio, 7.10; 95% confidence interval, 3.40-14.83; P < .001) or PI-based (7.59; 3.02-19.07; P = .001) first-line regimen compared with zidovudine/lamivudine/NNRTI, PI resistance at switch (6.69; 2.49-17.98; P < .001), and suboptimal adherence (3.05; 1.71-5.42; P = .025). Among participants with VLs ≥1000 cps/mL, 22 of 32 (69%) harbored drug resistance mutation(s), and 7 of 32 (22%) harbored PI resistance. Conclusions: Although VL suppression rates were high, PI resistance was detected in 22% of participants with VLs ≥1000 cps/mL. To ensure long-term ART success, intensified support for adherence, VL and drug resistance testing, and third-line drugs will be necessary.
    Full-text · Article · Jul 2016
    • "Alternatively, if this relaxation occurred immediately after the recombination that gave rise to CRF06_cpx, it could have been caused by the fact that the CRF06_cpx PR gene was derived from subtype G while CRF06_cpx gag gene was derived from subtype A1. These two HIV proteins co-evolve due to the fact that the gag is the substrate of PR cleavage (Fun et al., 2012), and a gene shuffle could have required PR to recognise new cleavage sites in gag, which would imply the relaxation of the purifying selection that maintained this co-evolution and the high mutation rates observed in PR regardless its codon position. Our estimate of the time to the most recent common ancestor (tMRCA) for the African CRF06_cpx was 1979CRF06_cpx was (1973CRF06_cpx was – 1983) in PR and 1981PR and (1978PR and –1983) in gp41, which agrees with the date provided by the only phylogeographic analysis of CRF06_cpx published to date (Delatorre and Bello, 2013), 1979 (1970–1985). "
    [Show abstract] [Hide abstract] ABSTRACT: Background: HIV-1 circulating recombinant forms (CRFs) represent viral recombinant lineages that play a significant role in the global epidemic. Two of them dominate the epidemic in Burkina Faso: CRF06_cpx (first described in this country) and CRF02_AG. We reconstructed the phylodynamics of both recombinant viruses in Burkina Faso and throughout West Africa. Methods: We analysed CRF06_cpx and CRF02_AG sequences (protease/gp41) from early samples collected in Burkina Faso in 1986 together with other GenBank sequences (1984-2013) in 4 datasets: African CRF06_cpx (210/60); down-sampled CRF06_cpx (146/45); Burkina Faso CRF02_AG (130/39) and West/Central African CRF02_AG (691/298). For each dataset, we analysed both protease and gp41 jointly using the BEAST multilocus analysis and conducted phylogeographic analysis to reconstruct the early migration routes between countries. Results: The time to the most recent common ancestor (tMRCA) of CRF06_cpx was 1979 (1973-1983) for protease and 1981 (1978-1983) for gp41. The gp41 analysis inferred the origin of CRF06_cpx (or at least its parental subtype G lineage) in the Democratic Republic of Congo but migrated to Burkina Faso soon after (1982). Both genes showed that CRF06_cpx radiated to the rest of West Africa predominantly after around 1990. These results were robust to the oversampling of Burkina Faso sequences as they were confirmed in the down-sampled dataset. The tMRCA of the Burkina Faso CRF02_AG lineage was 1979 (1977-1983) for protease and 1980 (1978-1981) for gp41. However, we reconstructed its presence in West Africa much earlier (mid-1960s), with an initial origin in Cameroon and/or Nigeria, and its phylogeographic analysis revealed much interconnection within the region with a lack of country-specific phylogenetic patterns, which prevents tracking its exact migration routes. Conclusions: Burkina Faso presents a relatively young HIV epidemic, with the diversification of the current in-country CRF02_AG and CRF06_cpx lineages taking place around 1980. This country represents the main source of CRF06_cpx in West Africa. The CRF02_AG epidemic started at least a decade earlier and showed much interchange between West African countries (especially involving coastal countries) suggesting a great population mobility and an extensive viral spread in the region.
    Full-text · Article · Apr 2016
    • "This can be explained by RAMs emerging upon single dose NVP exposure that are linked on the same viral genome. Similarly, coevolution of protease and its substrate Gag during protease inhibitor (PI) exposure can affect PI-based therapy [74,75]. Haplotype reconstruction of NGS data may help to address this, but this remains challenging for short read data [76]. "
    [Show abstract] [Hide abstract] ABSTRACT: Genetic analyses play a central role in infectious disease research. Massively parallelized "mechanical cloning" and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have, however, remained largely limited to small genomic regions. The growing need to monitor multiple genome regions for drug resistance testing, as well as the obvious benefit for studying evolutionary and epidemic processes makes complete genome sequencing an important goal in viral research. In addition, a major drawback for NGS applications to RNA viruses is the need for large quantities of input DNA. Here, we use a generic overlapping amplicon-based near full-genome amplification protocol to compare low-input enzymatic fragmentation (Nextera™) with conventional mechanical shearing for Roche 454 sequencing. We find that the fragmentation method has only a modest impact on the characterization of the population composition and that for reliable results, the variation introduced at all steps of the procedure-from nucleic acid extraction to sequencing-should be taken into account, a finding that is also relevant for NGS technologies that are now more commonly used. Furthermore, by applying our protocol to deep sequence a number of pre-therapy plasma and PBMC samples, we illustrate the potential benefits of a near complete genome sequencing approach in routine genotyping.
    Full-text · Article · Jan 2016
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