Impact of Y143 HIV-1 integrase mutations on resistance to raltegravir in vitro and in vivo.
ABSTRACT Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug's activity against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance to raltegravir. Furthermore, the 50% effective concentration (EC(50)) determined for Y143R/C mutants was significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the delayed emergence of this mutation.
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ABSTRACT: Background Two randomised, placebo-controlled trials—BENCHMRK-1 and BENCHMRK-2—investigated the efficacy and safety of raltegravir, an HIV-1 integrase strand-transfer inhibitor. We report final results of BENCHMRK-1 and BENCHMRK-2 combined at 3 years (the end of the double-blind phase) and 5 years (the end of the study). Methods Integrase-inhibitor-naive patients with HIV resistant to three classes of drug and who were failing antiretroviral therapy were enrolled. Patients were randomly assigned (2:1) to raltegravir 400 mg twice daily or placebo, both with optimised background treatment. Patients and investigators were masked to treatment allocation until week 156, after which all patients were offered open-label raltegravir until week 240. The primary endpoint was previously assessed at 16 weeks. We assessed long-term efficacy with endpoints of the proportion of patients with an HIV viral load of less than 50 copies per mL and less than 400 copies per mL, and mean change in CD4 cell count, at weeks 156 and 240. Findings 1012 patients were screened for inclusion. 462 were treated with raltegravir and 237 with placebo. At week 156, 51% in the raltegravir group versus 22% in the placebo group (non-completer classed as failure) had viral loads of less than 50 copies per mL, and 54% versus 23% had viral loads of less than 400 copies per mL. Mean CD4 cell count increase (analysed by an observed failure approach) was 164 cells per μL versus 63 cells per μL. After week 156, 251 patients (54%) from the raltegravir group and 47 (20%) from the placebo group entered the open-label raltergravir phase; 221 (47%) versus 44 (19%) completed the entire study. At week 240, viral load was less than 50 copies per mL in 193 (42%) of all patients initially assigned to raltegravir and less than 400 copies per mL in 210 (45%); mean CD4 cell count increased by 183 cells per μL. Virological failure occurred in 166 raltegravir recipients (36%) during the double-blind phase and in 17 of all patients (6%) during the open-label phase. The most common drug-related adverse events at 5 years in both groups were nausea, headache, and diarrhoea, and occurred in similar proportions in each group. Laboratory test results were similar in both treatment groups and showed little change after year 2. Interpretation Raltegravir has a favourable long-term efficacy and safety profile in integrase-inhibitor-naive patients with triple-class resistant HIV in whom antiretroviral therapy is failing. Raltegravir is an alternative for treatment-experienced patients, particularly those with few treatment options. Funding Merck Sharp & Dohme.The Lancet Infectious Diseases 07/2013; 13(7):587–596. · 19.97 Impact Factor
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ABSTRACT: The possibility of replacing raltegravir or elvitegravir with dolutegravir in heavily treatment-experienced patients failing on raltegravir/elvitegravir has been evaluated in VIKING trials. All studied patients failed by the most common pathways, Y143, Q148 and N155, and dolutegravir demonstrated efficacy except for Q148 viruses. The aim of this study was to explore, in the same way, the behaviour of dolutegravir in comparison with raltegravir and elvitegravir against the atypical resistance integrase profiles, G118R and F121Y, described in HIV-1 patients failing on raltegravir therapy. The behaviour of integrases with mutations G118R and F121Y towards raltegravir, elvitegravir and dolutegravir was analysed by evaluating phenotypic susceptibility and by means of in silico techniques (investigating binding affinities and the stabilization of the inhibitors in terms of their hydrogen bond network). The phenotypic analysis of G118R and F121Y showed high resistance to raltegravir, elvitegravir and dolutegravir with a fold change >100 when the clinically derived integrase was used, and resistance was also seen when mutations were tested alone in an NL43 backbone, but more often with a lower fold change. In silico, results showed that G118R and F121Y enzymes were associated with reduced binding affinities to each of the inhibitors and with a decreased number of hydrogen bonds compared with the wild-type complexes. This study showed that G118R and F121Y mutations, rarely described in patients failing on raltegravir, induced broad cross-resistance to all currently used integrase inhibitors. These results are in accordance with our thermodynamic and geometric analysis indicating decreased stability compared with the wild-type complexes.Journal of Antimicrobial Chemotherapy 04/2014; · 5.34 Impact Factor
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ABSTRACT: HIV-1 IN is a pertinent target for the development of AIDS chemotherapy. The first IN-specific inhibitor approved for the treatment of HIV/AIDS, RAL, was designed to block the ST reaction. We characterized the structural and conformational features of RAL and its recognition by putative HIV-1 targets - the unbound IN, the vDNA, and the IN•vDNA complex - mimicking the IN states over the integration process. RAL binding to the targets was studied by performing an extensive sampling of the inhibitor conformational landscape and by using four different docking algorithms: Glide, Autodock, VINA, and SurFlex. The obtained data evidenced that: (i) a large binding pocket delineated by the active site and an extended loop in the unbound IN accommodates RAL in distinct conformational states all lacking specific interactions with the target; (ii) a well-defined cavity formed by the active site, the vDNA, and the shortened loop in the IN•vDNA complex provide a more optimized inhibitor binding site in which RAL chelates Mg(2+) cations; (iii) a specific recognition between RAL and the unpaired cytosine of the processed DNA is governed by a pair of strong H-bonds similar to those observed in DNA base pair G-C. The identified RAL pose at the cleaved vDNA shed light on a putative step of RAL inhibition mechanism. This modeling study indicates that the inhibition process may include as a first step RAL recognition by the processed vDNA bound to a transient intermediate IN state, and thus provides a potentially promising route to the design of IN inhibitors with improved affinity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.Journal of Molecular Recognition 09/2013; 26(9):383-401. · 3.01 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2010, p. 491–501
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 1
Impact of Y143 HIV-1 Integrase Mutations on Resistance to
Raltegravir In Vitro and In Vivo?
Olivier Delelis,1† Sylvain Thierry,1† Fre ´de ´ric Subra,1Franc ¸oise Simon,1Isabelle Malet,2,3,4
Chakib Alloui,5Sophie Sayon,2,3,4Vincent Calvez,2,3,4Eric Deprez,1
Anne-Genevie `ve Marcelin,2,3,4Luba Tchertanov,1
and Jean-Franc ¸ois Mouscadet1*
LBPA, CNRS, Ecole Normale Supe ´rieure de Cachan, Cachan, France1; Universite ´ Pierre et Marie Curie—Paris, UMR S-943, Paris,
France2; INSERM, U943, Paris, France3; AP-HP, Groupe Hospitalier Pitie ´ Salpe ˆtrie `re, Laboratoire de Virologie, Paris, France4;
and Service de Bacte ´riologie, Virologie-Hygie `ne, Ho ˆpital Avicennes EA 3406, AP-HP, Universite ´ Paris 13,
Bobigny, Paris, France5
Received 31 July 2009/Returned for modification 10 September 2009/Accepted 28 October 2009
Integrase (IN), the HIV-1 enzyme responsible for the integration of the viral genome into the chromosomes
of infected cells, is the target of the recently approved antiviral raltegravir (RAL). Despite this drug’s activity
against viruses resistant to other antiretrovirals, failures of raltegravir therapy were observed, in association
with the emergence of resistance due to mutations in the integrase coding region. Two pathways involving
primary mutations on residues N155 and Q148 have been characterized. It was suggested that mutations at
residue Y143 might constitute a third primary pathway for resistance. The aims of this study were to investigate
the susceptibility of HIV-1 Y143R/C mutants to raltegravir and to determine the effects of these mutations on
the IN-mediated reactions. Our observations demonstrate that Y143R/C mutants are strongly impaired for
both of these activities in vitro. However, Y143R/C activity can be kinetically restored, thereby reproducing the
effect of the secondary G140S mutation that rescues the defect associated with the Q148R/H mutants. A
molecular modeling study confirmed that Y143R/C mutations play a role similar to that determined for
Q148R/H mutations. In the viral replicative context, this defect leads to a partial block of integration
responsible for a weak replicative capacity. Nevertheless, the Y143 mutant presented a high level of resistance
to raltegravir. Furthermore, the 50% effective concentration (EC50) determined for Y143R/C mutants was
significantly higher than that obtained with G140S/Q148R mutants. Altogether our results not only show that
the mutation at position Y143 is one of the mechanisms conferring resistance to RAL but also explain the
delayed emergence of this mutation.
Integration of the HIV-1 viral genome into that of the host
cell is a key step of viral replication, since the integrated ge-
nome, the provirus, is the genomic DNA form that allows the
expression of the viral gene and the subsequent formation of
progeny viruses. This step is catalyzed by the viral protein
integrase (IN), encoded by the pol gene and responsible for the
integration of double-stranded DNA generated by reverse
transcription of the RNA genome (for a review, see reference
11). To complete this process, IN catalyzes two successive
reactions. The first is the 3?-processing (3?-P) reaction, during
which terminal GpT dinucleotides are cleaved from each 3?
end of long terminal repeats (LTR), producing CpA 3?-hy-
droxyl ends. This reaction takes place within a nucleoprotein
complex known as the preintegration complex (PIC), which
translocates through the nuclear pore into the nucleus, where
the second reaction or strand transfer (ST) occurs. During this
second step, IN transfers both newly exposed 3? extremities of
viral DNA into the target genome in a concerted manner by
one-step transesterification reactions, resulting in full-site in-
tegration (2). Due to this key function in the viral cycle, IN is
an attractive target for antiretroviral drugs (ARVs). IN inhib-
itors constitute a new class of antiretroviral agents blocking
HIV-1 activity (19). To date, only integrase strand transfer
inhibitors (INSTIs) have shown potent antiviral activity in vivo.
Raltegravir (RAL), which was approved in October 2007, is the
first drug of the INSTI group to reach therapeutic use after
having demonstrated a rapid, potent, and sustained antiretro-
viral effect in patients with advanced HIV-1 infection (17, 30).
RAL is well tolerated and, due to its mechanism of action, is
active against viruses resistant to other classes of antiretro-
viral drugs, such as nucleoside reverse transcriptase inhibi-
tors (NRTI), nonnucleoside reverse transcriptase inhibitors
(NNRTI), protease inhibitors (PI), and entry inhibitors (17).
However, as observed for other ARVs, specific resistance mu-
tations located in the IN coding region of replicating viruses,
associated with a reduced susceptibility to RAL, have been
identified in patients failing to respond to treatment with RAL
(28). Although resistance to RAL in vivo has been linked to 14
mutations (35), each to a varying degree, virological failure is
clearly linked to two main independent genetic pathways in-
volving primary mutations on residues N155 (N155H) and
Q148 (Q148K/R/H). Secondary mutations that increase the
fitness of the resistant viruses have been identified in both
pathways. In particular, the secondary G140S mutation, ob-
served in tandem with the Q148H mutation, rescues a replica-
* Corresponding author. Mailing address: LBPA, CNRS UMR8113,
Ecole Normale Supe ´rieure de Cachan, 61 Avenue du Pre ´sident Wil-
son, 94235 Cachan, France. Phone: (33) 1 47 40 76 75. Fax: (33) 1 47
40 76 84. E-mail: firstname.lastname@example.org.
† These authors contributed equally to the work.
?Published ahead of print on 9 November 2009.
tive defect due to the presence of the primary mutation Q148H
(12). Several other mutations at other positions have also been
described, such as L68I/V, D232N, G163K/R, E138A, E157Q,
L74M, and V151I (For a review, see reference 5). Neverthe-
less, with the hindsight of several months of treatment, only a
few mutations that might constitute another pathway of resis-
tance have emerged. These mutations involve residues E92,
E157, and Y143. While the first two mutations are subject to
controversy as primary mutations for RAL resistance (35), the
third one seems to significantly decrease susceptibility to the
inhibitor. Interestingly, Y143R/C/H occurs less frequently and
appears to occur later than the first two pathways (9). We
identified Y143R/C mutations in the IN mutation patterns
associated with patients that failed to respond to RAL treat-
ment. We investigated the impact of the substitution at the
Y143 residue in their sensitivity to RAL, the overall catalytic
activity of the recombinant protein, and the replication effi-
ciency of viruses with the Y143R/C mutation. Y143R/C muta-
tions conferred a high resistance to RAL in vitro as well as in
vivo. The overall catalytic activity of IN in vitro and in vivo was
highly impaired, and this occurred mainly at the integration
MATERIALS AND METHODS
Patients. Four patients who failed to respond to RAL treatment were retro-
spectively studied. The genotypic analysis of IN resistance and the follow-up of
HIV-1 viral load were carried out following French national guidelines (39). All
patients received at least one NRTI with one boosted PI with or without enfu-
virtide in their optimized regimen. The optimized regimen associated with RAL
was selected according to previous antiretroviral exposure and genotypic resis-
tance testing interpreted using the last French ANRS algorithm (www
.hivfrenchresistance.org). RNA was extracted from 500 ?l of plasma, and a
fragment encompassing the entire IN coding region of the pol gene was amplified
and sequenced as described previously (27).
Cells and viruses. MT4 cells were cultured in RPMI 1640 containing 10% fetal
calf serum. 293T and HeLa-P4 cells were cultured in Dulbecco’s modified Eagle
medium supplemented with 10% fetal calf serum, 100 U penicillin/ml (Invitro-
gen), and 100 ?g streptomycin/ml (Invitrogen). HIV-1 IN (Y143R/C) mutants
were generated by site-directed mutagenesis as previously described (12). Briefly,
the fragment encoding IN of the replication-competent pNL4.3 virus was di-
gested with AgeI and EcoRI and inserted into the Bluescript vector, and IN
mutants were obtained by mutagenesis (QuikChange mutagenesis kit; Strat-
agene). The constructs were checked by sequencing, and the fragment was then
inserted into pNL4.3. HIV-1 virus stocks of all mutants were prepared by trans-
fecting 293T cells. Transfection assays were carried out by the calcium phosphate
method. Forty-eight hours posttransfection, viral supernatants were filtered
through a 0.45-?m-pore-size filter and immediately frozen at ?80°C. The HIV-1
p24gagantigen content in viral supernatants was determined by enzyme-linked
immunosorbent assay (Perkin-Elmer Life Sciences).
HIV infectivity assay. The viral titer was determined in HeLa-P4 cells.
HeLa-P4 cells are HeLa CD4 LTR-LacZ cells in which lacZ expression is
induced by the HIV transactivator protein Tat, allowing the precise quantifica-
tion of HIV-1 infectivity. Cells were infected in triplicate in 96-well plates with
wild-type (WT) or Y143R/C viruses (equivalent of 3 ng of p24gagantigen or with
higher viral concentrations, as described in Results). The viral inoculum was left
for 48 h. The viral titer was determined 48 h after infection by quantifying
?-galactosidase activity in HeLa-P4 lysates in a colorimetric assay based on the
cleavage of chlorophenol red-?-D-galactopyranoside (CPRG) by ?-galactosidase
as described previously (12). For 50% effective concentration (EC50) determi-
nation, cells were infected with viruses and grown in the presence of increasing
concentrations of RAL. The EC50was defined as the drug concentration result-
ing in ?-galactosidase levels that are 50% lower than those in untreated infected
cells. Cell survival was also estimated with a standard MTT (3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazolium bromide) assay (12).
Viral infections. MT4 cells were concentrated to 2.106/ml and infected with
viruses (50 ng of p24 antigen par 106cells). Viral inoculum remains throughout
the course of the experiment. If required, cells were treated with 100 nM RAL
inhibitor (Merck & Co.) 1 h before infection. RAL was maintained during the
course of the experiment. At various time points after infection, 1 million to 2
million cells were harvested and dry cell pellets were frozen at ?80°C until use.
DNA extraction and real-time PCR. Total cell DNA was extracted with a
QIAamp blood DNA minikit (Qiagen, Courtaboeuf, France). Quantifications of
total HIV-1 DNA, 2-LTR circles, and integrated HIV-1 DNA were performed by
real-time PCR on a LightCycler instrument (Roche Diagnostics) using the fit
point method provided by the LightCycler quantification software, version 3.5
(Roche Diagnostics) as previously described (3). Cell equivalents were calculated
based on amplification of the ?-globin gene with commercially available mate-
rials (control kit DNA; Roche Diagnostics). 2-LTR circles and total and inte-
grated HIV-1 DNA levels were determined and expressed as copy numbers per
Characterization of IN protein activity in vitro. The Y143R and Y143C mu-
tations were obtained by site-directed mutagenesis of pET-15b containing the
WT sequence. The wild-type and mutant HIV-1 INs used for DNA-binding and
3?-P assays were produced in Escherichia coli BL21(RIL) and purified under
nondenaturing conditions as previously described (25). Activity assays were car-
ried out as described in reference 25. Oligonucleotides (ODNs) were radiola-
beled with T4 polynucleotide kinase (Biolabs) and [?-32P]ATP (3,000 Ci/mmol)
(Amersham) and purified on a Sephadex G-10 column (GE Healthcare). Dou-
ble-stranded ODNs were obtained by mixing equimolar amounts of complemen-
tary strands in the presence of 100 mM NaCl. 3?-P and strand transfer assays
were carried out at 37°C in a buffer containing 10 mM HEPES (pH 7.2)–1 mM
dithiothreitol (DTT) (7.5 mM) in the presence of 3.25 nM and 12.5 nM DNA
substrates, respectively. Products were separated by electrophoresis in denatur-
ing 18% acrylamide/urea gels. Gels were analyzed with a Molecular Dynamics
Storm phosphorimager (GE Healthcare) and quantified using the Image Quant
4.1 software program. Fifty percent inhibitory concentration (IC50) calculation
and t tests were performed using Prism 5.0 software (GraphPad Software, San
Steady-state fluorescence anisotropy-based assay. Steady-state fluorescence
anisotropy values (r) were recorded on a Beacon 2000 instrument (Panvera,
Madison, WI) in a cell maintained at 25°C or 37°C under thermostatic control.
The principle underlying the anisotropy-based assay has been published else-
where for DNA binding (1, 36) and 3?-P (10, 18), respectively. Briefly, IN binding
to fluorescein-labeled DNA (double-stranded 21-mer oligonucleotide [ODN]
mimicking the U5 viral DNA end) increases the r value, making it possible to
calculate the fractional saturation function: [DNA?IN]/[DNA]0. The DNA bind-
ing step was recorded at 25°C, using ODNs fluorescein labeled at the 3?-terminal
GT nucleotide. The percentage of complexes was then calculated as
r ? rfree
rsat? rfree? 100(1)
where rfreeand rsatare the anisotropy values characterizing the free and bound
oligonucleotides, respectively. Following DNA binding, the sample was then
incubated at 37°C to record 3?-P activity. As the fluorophore is linked to the
released dinucleotide, 3?-P activity significantly decreases the r value with respect
to that for nonprocessed DNA. Activity can be calculated in fixed-time experi-
ments by disrupting all the IN?DNA complexes with SDS (0.25%, final). The
fraction of dinucleotides released is given by
where rNPand rdinuare the anisotropy values for pure solutions of nonprocessed
double-stranded ODN and dinucleotide, respectively. The formation of IN/DNA
complexes and the subsequent 3?-processing reaction were carried out by incu-
bating fluorescein-labeled ODNs (4 nM) with IN in 20 mM HEPES (pH 7.2), 1
mM dithiothreitol, 30 mM NaCl, and 10 mM MgCl2. Standard 3?-P and ST
activity tests based on gel electrophoresis were performed as previously de-
Theoretical studies. All calculations were carried out on a PC running RedHat
Enterprise Linux 5 software. Modeling, analysis, and graphic generation were
performed with SYBYL (version 8.0) software (Tripos Inc., 2008).
Molecular modeling. Three-dimensional (3D) models of raltegravir resistance
mutants were built by individual amino acid substitutions, using previously gen-
erated models of the catalytic core of wild-type IN, and were optimized as
previously described (31). The stereochemical quality of the models was assessed
with the ProTable Procheck software program (24), which showed that more
than 97% of the nonglycine residues in all models had dihedral angles in the most
favored and allowed regions of the Ramachandran plot, consistent with high
492 DELELIS ET AL.ANTIMICROB. AGENTS CHEMOTHER.
The molecular Connolly (7) and MOLCAD (20) surfaces were generated, and
the lipophilic and hydrogen-bonding properties were displayed using an opaque
Lipophilic potential (LP). the overall hydrophobicity (lipophilicity) of a mol-
ecule was calculated/predicted by its partition coefficient (logP), represented as
fragmental increments, fi:
On the basis of the atomic partial lipophilic values, fi, a distance-dependent
function has been defined for the lipophilicity potential (LP) of proteins (20):
i ? 1
i ? 1
where di? distance of a certain point in space from atom i with
LPHMwas implemented in MOLCAD with two of fisets based on Crippen
atomic partial lipophilicities (16) used for characterizing hydrophobicity of the
wild type and the mutants.
HB sites. Hydrogen-bonding (HB) sites on the molecular surface were local-
ized by the original MOLCAD method.
Patients and HIV-1 viral load. Sequence analysis of clinical
isolates obtained during RAL treatment has led to the identi-
fication of various mutations at specific positions only observed
in the IN coding region (28). Indeed, two main genetic path-
ways have been characterized involving residues N155 (muta-
tion N155H) and Q148 (mutations Q148H/K/R) (23). Recent
data suggest the existence of a third pathway, involving the
Y143 residue, correlated with virological resistance to RAL
(35). Here, four HIV-1-infected patients failing to respond to
400 mg RAL, administered twice daily, and for whom a mu-
tation in position 143 was found in the viral IN sequence at the
time of failure were studied retrospectively. These patients
with isolates with the Y143 mutation represented an approx-
imate ratio of 4% among patients failing to respond to RAL,
while clinical isolates displaying the N155H or G140S/
Q148R/H mutations represented, respectively, 61 and 35%
of the resistant profiles observed in our clinical center.
Isolates from patients 1 to 3 had a Y143R mutation, and
virus from patient 4 had two mutations, Y143Y/C and
N155N/H (Tables 1 and 2). Their baseline characteristics are
given in Table 1. At the start of RAL therapy, the median CD4
count was 164.5 cells/mm3(range, 3 to 383 cells/mm3) and the
median plasma HIV-1 RNA level was 4.7 log10copies/ml
(range, 3.7 to 4.8 log10copies/ml). All patients harbored highly
mutated viruses with resistance to NRTI, NNRTI, and PI; their
genotypic sensitivity score (number of active ARV in the back-
ground regimen associated with RAL) was between 1 and 2.
The complete nucleotide sequences of IN were determined for
clinical isolates from each patient at various time points before
and during RAL treatment. We found no IN resistance muta-
tions in the bulk of the PCR products amplified from plasma
HIV-1 RNA before the introduction of RAL for any of the
patients. At the time of failure (median, 41 weeks; range, 16 to
48 weeks), the only change in sequence analyzed was a muta-
tion in position 143 in the IN sequence. These findings con-
firmed that a specific mutation at Y143 was correlated with a
failure to respond to RAL treatment. Mutation of this residue
may constitute a third mechanism of resistance against RAL.
Thus, we investigated Y143 and its role in resistance to RAL by
producing viruses that have the Y143R or Y143C mutation.
Resistance to RAL of Y143R/C viruses. To determine whether
the Y143R and Y43C mutations conferred resistance to RAL,
these mutations were introduced into a WT pNL4.3 viral back-
bone. First, mutated viruses were produced following transfec-
tion of these genomic clones into 293T cells. Levels of p24
similar to those obtained with the WT virus were observed for
each mutant, showing that none of these mutations signifi-
cantly impaired late viral replication steps (Fig. 1A). Then, the
TABLE 1. Patient and virus baseline characteristicsa
No. of CD4
treatment at day 0
1B 4.8 383M41L, E44D, D67N,
Y188L L10I, I15V, K20R, V32I,
L33F, I47V, I54 M,
L63P, A71V, G73S,
V82A, L90 M
V10I, E36I, M46I, I50V,
Q58E, I62V, L63P,
A71V, L74P, I84V,
L10F, I15V, K20 M,
V32I, L33F, M36I,
M46I, I54V, L76V,
L10F, L24I, M46I, I54V,
I62V, L63P, A71V,
ZDV, 3TC, ABC,
2B 3.7 323 K103N, Y181C,
3B 4.76 M184V, M41L,
L100I, K103NABC, ETR,
4B 4.73T215I- TDF, DRV/r,
aAbbreviations; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor; PI, protease; ZDV, zidovudine; 3TC,
lamivudine; ABC, abacavir; ATV/r, atazanavir/ritonavir; FPV/r, fosamprenavir/ritonavir; T20, enfuvirtide; RAL, raltegravir; TDF, tenofovir; TPV/r, tipranavir/ritonavir;
ETR, etravirine; DRV/r, darunavir/ritonavir.
bLevels of HIV RNA in plasma were determined by using the Cobas AmpliPreps/Cobas TaqMan HIV-1 test.
cGSS, genotypic sensitivity score, according to genotypic resistance testing interpreted using the French ANRS algorithm.
VOL. 54, 2010RESISTANCE TO RAL OF Y143 HIV-1 INTEGRASE MUTANTS493
impact of these mutations on the early replication steps, i.e.,
from entry to integration, was determined at 48 h postinfection
on HeLa-P4 reporter cells (Fig. 1B). Cells were infected with
similar amounts of viral particles (3 ng of p24), and ?-galac-
tosidase levels were evaluated 48 h postinfection. Under these
conditions, about 40% of the cells were infected with the wild-
type virus. To rule out a possible bias due to the colorimetric
measurement method, the background corresponding to the
CPRG signal obtained for an abortive viral infection in the
presence of 50 ?M zidovudine (ZDV) was systematically de-
termined. Both the Y143R and Y143C mutations significantly
decreased viral infectivity (15% and 10% of the WT level for
Y143C and Y143R, respectively), suggesting that mutant IN
activity was affected (Fig. 1B). We then quantified RAL resis-
tance in Y143R/C mutants following infection with higher virus
concentrations (30 ng p24 antigen). To avoid a bias due to a
possible correlation between the amount of infectious viruses
present and the effectiveness of RAL, we determined first
whether a change in the infectious virus concentration affects
the apparent EC50. In agreement with previous studies (33), no
significant change was observed, with an EC50equal to 7 nM
after various infectious virus concentrations to more than 1
order of magnitude, thereby confirming the high potency of
RAL as an inhibitor of HIV-1 integration (Fig. 1C). No cyto-
toxicity due either to RAL or to viral replication was observed
in this model by a standard MTT assay up to 72 h postinfection
as already described (12). For both Y143R and Y143C viruses,
the EC50was not reached within the range of concentrations
tested (EC50? 100 nM), thus demonstrating a strong resis-
tance to RAL (Fig. 1C).
Y143R/C viruses are defective for viral DNA integration.
Although Y143R/C viruses are highly resistant to RAL, their
infectivity was deeply altered, suggesting a defect during the
early replicative steps. To determine which replicative step was
impaired, MT4 cells were infected with either WT or resistant
viruses in the presence or absence of 100 nM RAL, and the
various viral DNA species were quantified by real-time PCR
24 h postinfection. The amounts of total viral DNA were sim-
ilar for all viruses, indicating that the mutations did not affect
the viral entry and reverse transcription steps (data not
shown). Moreover, the amount of viral DNA was not de-
creased by the addition of 100 nM RAL, confirming that this
ARV does not affect the reverse transcription step.
Efficiency of viral integration was evaluated by Alu-LTR
real-time nested PCR amplification of integrated proviruses
(3). For the wild-type virus, 10% of the total DNA was inte-
grated, in agreement with previous reports (3). In contrast, for
the Y143R and Y143C mutants, the amount of integrated
DNA was equal to 2.1% and 1.1%, respectively. Thus, the
integration for the Y143 mutants was much less than that for
WT viruses, thereby indicating that a block in integration may
account for the replication defect (Fig. 2A). In the presence of
100 nM RAL, only 27% of the total DNA of the WT virus was
integrated in comparison with results for the control without
RAL, thereby confirming the efficacy of INSTI in blocking the
integration step (Fig. 2A). This decrease in the amount of
integrated viral DNA was not observed for the Y143R/C mu-
tants, since the ratio between integrated DNA and uninte-
grated DNA was not affected by the addition of RAL despite
the weaker integration efficiency. The ratio of integrated DNA
in the absence of RAL over integrated DNA in the presence of
RAL was not significantly different for the Y143R and Y143C
mutants, whereas it was equal to 3.6 for the WT. Thus, the
inhibitor was not efficient at blocking viral integration of resis-
A defect in viral integration affects the formation of epi-
somic 2-LTR circles, for which accumulation is a hallmark of
integration impairment. This impairment is observed if ST
inhibitors are added during infection or in the case of infection
by IN catalytic mutants (19, 32, 37). Infection with WT virus in
the presence of RAL led to an increase in 2-LTR circles, as
expected (Fig. 2B). The number of 2-LTR circles resulting
from resistant viruses in the absence of RAL was also slightly
higher than that observed for WT viruses, thus confirming a
partial block of integration for the mutants (Fig. 2B). Finally,
no significant difference in the amount of 2-LTR circles was
observed for resistant Y143R viruses in the presence or ab-
sence of RAL, thus confirming that the inhibitor was not able
to block viral integration of this mutant. Intriguingly, a slight
increase of 2-LTR circles was observed for the Y143C mutant
in the presence of RAL. This may hint that this mutant re-
mains partially sensitive to the inhibitor. However, this hypoth-
esis is not supported by the absence of integration inhibition
(Fig. 2A). Nevertheless, the increase is small compared to that
observed with the wild-type virus. Together, these findings
demonstrated that (i) the Y143R/C mutation affects viral in-
TABLE 2. Integrase mutations in isolates from patients at failure of therapy
Baseline amt of
Baseline integrase mutations
Amt of HIV RNA
Integrase mutations at failure
Time at failure
1 4.8 D10E, V32I, S39C, V72I, K111R, G123S,
A124N, T125A, K127K, G193E,
3.6 D10E, V32I, S39C, V72I, K111R,
G123S, A124N, T125A,
K127K, Y143R, G193E,
G123S, R127K, Y143R, A205S,
I208L, K211Q, E212A, N232D
V72I, I113V, G123S, A124N/S,
T125A, R127K, Y143R, T206S,
I220L, Y227F, N232D
H51D, K111T, S119T, G123S,
A124T, R127K, Y143Y/C,
2 3.7G123S, R127K, A205S, I208L, K211Q,
V72I, I113V, G123S, A124N/S, T125A,
R127K, T206S, I220L, Y227F, N232D
3 4.7 4.816
4 4.7 H51D, K111T, S119T, G123S, A124T,
494DELELIS ET AL.ANTIMICROB. AGENTS CHEMOTHER.
tegration efficiency and (ii) this mutation provides the virus
with potent resistance to RAL originating in its inability to
block viral integration.
Y143R/C mutations provide greater resistance than G140S/
Q148H. Although greater resistance may explain the emer-
gence of the Y143 mutants, their weak replicative capacity is an
obstacle to it. To address the possibility that Y143 mutants
have a significant advantage over the other resistant viruses, we
compared the viral replication of Y143R/C and G140S/Q148H
resistant viruses in the single-round infection assay in the pres-
ence of increasing concentration of RAL. As shown in Fig. 3,
Y143R/C viruses remained poorly susceptible to RAL for con-
centrations up to 5 ?M, demonstrating a dramatic resistance to
the drug. In contrast, G140S/Q148H mutants were affected for
concentrations above 100 nM, with an apparent EC50of the
drug reached at 2 ?M. Furthermore, Y143R/C viruses were
FIG. 2. Impairment of viral integration for Y143R and Y143C mu-
tants. MT4 cells (106) were infected with 50 ng of p24 antigen, and
(A) Percentage of integrated viral DNA 24 h postinfection for each
mutant in the presence (dark-gray bars) or absence (light-gray bars) of
RAL. The percentage of integrated viral DNA was determined by divid-
ing the integrated viral DNA copy number by total viral DNA 24 h
postinfection. (B) Percentage of 2-LTR circles for each mutant in the
presence (black bar) or absence (white bar) of RAL. “#” indicates a
significant difference with the wild-type virus in a t test with a P value ?
FIG. 1. Infectivity and resistance of the mutants. (A) Quantifica-
tion of p24 protein 48 h after transfection of 5 ?g of the DNA corre-
sponding to each virus. (B) Viral infectivity of each mutant was deter-
mined using the CPRG assay. HeLa-P4 indicator cells (105) were
infected with 3 ng/10,000 cells of p24 antigen for 48 h, corresponding
to about 40% of the cells being infected for the wild-type virus. The
early stages of infection were assessed by measuring the ?-galactosi-
dase activity in cell extracts by the CPRG method compared to the
signal obtained for uninfected cells (n.i., no infection). The back-
ground, corresponding to the CPRG signal obtained for an abortive
viral infection, was determined in the presence of 50 ?M AZT.
(C) HeLa-P4 cells were infected in triplicate, using amounts of p24
antigen that give the same infectivity without RAL. Data from a rep-
resentative experiment are shown. The EC50was determined as the
concentration of RAL inhibiting ?-galactosidase production by 50% in
comparison with results for untreated infected cells. For all panels,
confidence interval analysis was used with n ? 3 and with a p value ?
0.05 considered statistically significant.
FIG. 3. Comparison of resistance levels of Y143R/C and G140S/
Q148H mutants. HeLa-P4 cells were infected in triplicate with an
amount of p24 antigen (30 ng) yielding the same infectivity for mutated
viruses as that obtained with 3 ng of wild-type virus in the absence of
RAL (i.e., multiplicity of infection ? 0.4). The EC50was determined as
the concentration of RAL inhibiting ?-galactosidase production by
50% in comparison to results for the untreated infected cells. Confi-
dence interval analysis was used with n ? 3 and with a P value ? 0.05
considered statistically significant.
VOL. 54, 2010RESISTANCE TO RAL OF Y143 HIV-1 INTEGRASE MUTANTS495
significantly more resistant to RAL than the other single mu-
tants, Q148H and N155H viruses (12). In conclusion, Y143R/C
mutations provide additional resistance compared to that of
In vitro activity of Y143R/C IN mutants. The defect in the
integration process observed in the viral context could be due
to a defect in the 3?-P or ST reaction. To address this question,
we investigated the impact of Y143 mutations on the two
catalytic activities of the enzyme (3?-P and ST reaction). We
first used a steady-state fluorescence anisotropy assay. The
determination of the anisotropy of the fluorescence emitted by
a fluorophore covalently linked to an ODN mimicking the viral
DNA allows monitoring of both the binding of IN to the ODN
substrate and the subsequent 3?-P reaction (18). The binding of
IN to the ODN increases the steady-state anisotropy value (r),
allowing the calculation of the fractional saturation function
(Fig. 4A). When the fluorophore is linked to the 3? extremity,
the release of the dinucleotide product resulting from the 3?
processing significantly decreases r. First, we used this assay to
check that Y143R/C mutants did not affect IN-DNA complex
formation (Fig. 4A). We then monitored 3? processing as a
function of IN concentration in the steady-state anisotropy
assay, allowing the reaction to be followed from an initial
precisely quantified amount of IN/DNA complexes (18). 3?-P
activity as a function of the IN concentration gave a charac-
teristic bell-shaped curve, with activity reaching a plateau at a
concentration of about 200 nM similarly to that of the wild-
type protein (Fig. 4B). However, the 3?-P efficiency of the
Y143R and Y143C mutants was significantly reduced in com-
parison with that of the WT, since the overall yields were only
30% and 22% that of WT enzyme for Y143C and Y143R
mutants, respectively. Moreover, increasing the mutant con-
centration did not allow the activity of the wild-type protein to
be reached. This result is consistent with previous results show-
ing that mutant protein DNA binding properties were not
affected but rather displayed a catalysis-related defect.
Taken together, these data indicate that the Y143R and
Y143C mutations affect 3?-P activities of the proteins; these
effects occur mostly at the catalytic step without affecting the
overall affinity for the viral substrate, a situation reminiscent of
that with the Q148 mutation (12). We have previously reported
that the RAL-resistant G140S/Q148H double mutant is a cat-
alytic mutant, the activity of which is recovered by allowing the
reaction to proceed for an extended time at 37°C. The yield
from the 3?-P reaction in Y143R/C mutants also continuously
increased over time, reaching up to 80% of the yield obtained
with the WT (Fig. 4C). Thus, Y143R/C mutants may behave
similarly to the previously described G140S/Q148H mutant.
To determine whether viral resistance to RAL was related to
how it affects IN, we quantified the efficacy of RAL against the
enzyme in vitro. To quantify both activities, we used a gel assay
that allows the monitoring of both 3?-P and ST (4, 25). We first
quantified the 3?-P activity in the presence of increasing RAL
concentrations (Fig. 5A). Results obtained for the 3?-P reac-
FIG. 4. Comparative study of the DNA-binding and 3?-processing activity of wild-type and Y143R/C INs. (A) DNA binding of wild-type and
Y143R/C mutants. The DNA binding step was assessed as described in Materials and Methods (see equation 1). Increasing concentrations of IN
and DNA (4 nM) were incubated together for 15 min before steady-state anisotropy as recorded. (B) 3?-Processing activity after 3 h of incubation
at 37°C as a function of the IN concentration monitored by steady-state fluorescence anisotropy using a 21-mer DNA substrate (4 nM) with MgCl2
(10 mM) as a metallic cofactor in 20 mM HEPES (pH 7.2), 1 mM DTT, and 30 mM NaCl. IN and DNA were incubated together for 15 min before
steady-state anisotropy as recorded. 3?-P activities were quantified as described in Materials and Methods (see equation 2). The same symbols were
used in panels A and B: black triangle, WT IN; white square, Y143R IN; and black circle, Y143C IN. (C) 3?-Processing kinetics for the WT and
Y143R/C mutants. 3?-P activity for the Y143R/mutants was normalized against WT activity during the course of the experiment. Time is indicated
in hours. Symbols used are as follows: white bar, Y143R IN; black bar, Y143C IN.
496DELELIS ET AL.ANTIMICROB. AGENTS CHEMOTHER.
tion with radiolabeled ODNs were consistent with those ob-
tained by steady-state anisotropy (reduction of the 3?-P activity
of the Y143C and Y143R mutants in comparison with that of
WT IN) (Fig. 5A, right panel). RAL did not significantly in-
hibit the 3?-P reaction in vitro, as expected for an INSTI com-
pound (14, 21, 29). This assay, which used short blunt ODNs,
also revealed that the ST reaction was impaired for both mu-
tants. To allow a precise quantification of RAL inhibition of
the ST reaction, the experiment was performed with pre-
cleaved ODNs mimicking the processed substrate, which have
been reported to enhance ST activity (26). Again, we found
that the ST reaction in Y143R/C mutants was less efficient than
that in the WT, with the Y143C mutant being the most affected
(Fig. 5B). The ST reaction yield was 60% and 21% of that of
the WT for the Y143R and Y143C mutants, respectively (Fig.
5B, right panel). Despite this weak activity, the quantification
of RAL inhibition demonstrated that both mutants were highly
resistant to RAL (IC50above 300 nM) in comparison with the
WT (Fig. 6).
These findings demonstrated that the mutation at the Y143
position was detrimental to overall IN activity, explaining the
integration defect observed in cell culture. The nature of the
amino acid at the Y143 position (Y143R or Y143C) observed
in our clinical study slightly influenced the 3?-P reaction (the
difference was not statistically significant in the gel assay). How-
ever, the substitution of a C residue (instead of R) was more
detrimental to the ST reaction. Nevertheless, even though Y143
mutants are less active than the WT, the mutations observed in
patients and studied in this report led to strong RAL resistance.
Molecular modeling. To gain insight into the origin of the
resistance induced by the Y143R/C mutations and for compar-
ison against results with the Q148R mutation, we analyzed the
FIG. 5. In vitro RAL resistance of the wild-type and Y143R/C mutants. (A) 3?-P activity (after 3 h of incubation at 37°C) at a concentration
of 200 nM IN. A representative gel showing RAL resistance with respect to the IN mutation. Drug concentrations are indicated above each lane.
The32P-labeled oligonucleotide, the substrate used in this reaction, is indicated by an arrow (21-mer). The product of the 3?-P reaction is indicated
by an arrow (19-mer). Products of the ST reaction are indicated (ST products). Activity of each protein at 200 nM is indicated on the right of the
gel, normalized against WT activity. (B) Strand transfer reaction of the Y143R/C mutants. The ST reaction was performed using a32P-labeled
oligonucleotide mimicking the preprocessed substrate. Drug concentrations are indicated above each lane. Percentage of the ST activity of each
mutant was normalized against WT activity. 3?-P and ST activities were quantified as described in Materials and Methods. Experiments were
performed three times. An unpaired t test was used to derive P values.
0,1110 100 1000
% Strand-transfer activity
FIG. 6. Effect of the mutations on RAL resistance in strand trans-
fer assay. Strand-transfer reactions were carried out for 3 h in the
presence of increasing RAL concentrations.
VOL. 54, 2010RESISTANCE TO RAL OF Y143 HIV-1 INTEGRASE MUTANTS497
498 DELELIS ET AL.ANTIMICROB. AGENTS CHEMOTHER.
structural and molecular effects induced by these alternative
RAL resistance mutations. First we observed that the 3D mod-
els of the Y143R/C and Q148R mutants were structurally
equivalent and could be superimposed on the WT protein
perfectly. In particular, the Y143R/C and Q148R mutations
result in the conservation of the catalytic loop, particularly its
?-shaped hairpin. Y143 and Q148 are adjacent residues in the
catalytic loop structure at the neck of its hairpin. This proxim-
ity in an invariant structure, as well as the high mobility of the
loop, suggests comparable roles of these mutations in resis-
tance to RAL.
We previously demonstrated that the Q148R/H/K mutation
alters the specificity of DNA recognition by IN (31). We thus
compared the hydrophobicity and hydrogen-bonding patterns
of the WT and mutant proteins, both factors playing key roles
in the binding of substrates or inhibitors. To compare the
contributions of residues Y143R/C and Q148R in modulating
the target binding properties and their roles in the complex
formation with substrate (DNA) or RAL, we generated the
molecular Connolly and MOLCAD surfaces for the WT and
mutant INs and analyzed the lipophilic potential (LP) and
hydrogen bonding (HB) properties. Lipophilicities of the
Y143R/C and Q148R mutants were similar, whereas LP on
the IN surface in mutants was considerably lower than that in
the WT enzyme (Fig. 7, top). Therefore, a reduced capability
to stabilize inhibitors through nonspecific hydrophobic inter-
action in the vicinity of these residues is expected. The analysis
of hydrogen bonding sites on the IN surface also highlighted
significant changes to the donor/acceptor properties of the
mutants in comparison with those of the WT enzyme (Fig. 7,
bottom). In particular, both mutations Q148R and Y143R/C
contributed to form a pure hydrogen donor site instead of the
mixed donor/acceptor binding sites observed in the context of
the wild-type enzyme (Fig. 8A).
Next, two theoretical models of the tetrameric HIV-1 IN
with bound LTR DNAs were used to analyze the interactions
between the LTR terminal nucleotides and the integrase res-
idues involved in resistance to raltegravir (6, 38). According to
the theoretical model suggested by Chen et al. (6), the un-
paired 5?-AC base pair of the viral DNA is precisely positioned
between these two residues, allowing 3?-processing contacts
between Q148/Y143 and the 5?-AC overhang (Fig. 8B, left). In
a model suggested by Wielens et al. (38), the 3?-processed viral
DNA is situated in a position allowing short-range contacts
between both the Q148 and Y143 residues and the A-T base
pair in the third position on the viral LTR (Fig. 8B, right).
Each model corresponds to one of the two possibilities pre-
dicted for the way wild-type residues recognize the single ad-
enine at position ?2 (6) or the A-T base pairs at position 1
(38). Thus, Y143R could be considered to be a functional and
structural alternative to the Q148R mutation.
To date, two different resistance pathways are associated
with failure to respond to RAL therapy; these involve the
primary mutation N155H or Q148R/H/K (8, 23, 28, 35). A
retrospective study of four patients who failed to respond to
RAL therapy revealed Y143R/C mutations, supporting the
suggestion that these mutations are also responsible for pri-
mary resistance to RAL. Here we studied the impact of
Y143R/C mutations on viral replication and on the activity of
Unlike what we previously found for the G140S/Q148H mu-
tant virus, which replicate efficiently in the single-round repli-
cation assay (12), the replicative capacity of Y143 mutant virus
evaluated in a similar assay was dramatically lower than that of
the wild-type virus. This result is consistent with previous ob-
servations that show that Y143N mutant replication is signifi-
cantly delayed in comparison with that of the wild-type virus
owing to a specific defect in early replication (34). Quantifica-
tion of viral DNA species clearly indicated that the Y143 mu-
tations led to a significant replicative defect, resulting in low
fitness in the absence of the integrase inhibitor. In contrast, the
integration process of both Y143R/C mutants was not affected
by RAL, highlighting strong resistance conferred by Y143 sub-
FIG. 7. Lipophilic potential (LP) (top) and hydrogen bonding site (HB) (bottom) surfaces of wild-type and raltegravir-resistant mutants. LP
and HB calculations performed on the Connolly solvent-accessible surface of the INs were determined using the MOLCADE subroutine from
SYBYL 8.0 (Tripos Inc., St Louis, MO). The color ramp for LP ranges from brown spots (highest lipophilic potential area of the molecule) to blue
spots (highest hydrophilic area). The color ramp for HB ranges from red (hydrogen donors; low electronegativity) to blue (hydrogen acceptors;
high electronegativity). The RAL-resistant and catalytic residues are shown as sticks and in magenta, respectively. The areas corresponding to the
RAL resistance mutations on the IN surface are delimited by dashed circles, yellow for Q148R and Y143R/C and blue for N155H.
FIG. 8. HIV-1 IN interactions with viral DNA based on theoretical
models. (A) Donor (red) and acceptor (blue) surfaces of the WT (left)
and mutated residues (right) Q148R and Y143R. Arginine is pre-
sented as two tautomeric forms. (B) HIV-1 IN interactions with viral
DNA according to theoretical models by Chen et al. (6) (left) and by
Wielens et al. (38) (right).
VOL. 54, 2010RESISTANCE TO RAL OF Y143 HIV-1 INTEGRASE MUTANTS 499
stitutions; this may thus confer a higher fitness to the mutant
virus in the presence of the inhibitor. In the presence of 5 ?M
RAL, Y143 mutants were still capable of replicating whereas
G140S/Q148R resistant virus was fully susceptible to RAL.
This comparison shows that Y143 mutants have a replicative
advantage in the presence of the drug over the double mutant,
possibly explaining the eventual emergence of this mutant de-
spite its poor replicative capacity.
In vitro, we observed a significant catalytic defect for both 3?
processing and strand transfer activities of Y143R/C proteins.
This confirms that integration impairment observed when
these mutations were introduced into the viral background
originated from a defect in IN activity. If assayed in vitro,
mutated INs showed a reduced susceptibility to RAL, which
accounts for the resistance of the virus. Thus, Y143R/C ap-
pears to be sufficient at conferring high resistance to the en-
zyme both in vitro and in the viral context.
Interestingly, 3?-P activity was slightly more affected for
Y143R mutants, but not ST activity, which was more efficient
for this mutant than for the Y143C mutant. This observation
could provide a possible explanation for the prevalence of this
specific mutant, since both mutants displayed similar resis-
tances to RAL in vitro and in vivo. From this viewpoint, it is
interesting to note that the Y143C change requires only a
single nucleotide mutation (UAU to UGU or UAC to UGC)
whereas Y143R involves a minima two successive mutations
(UAU to CGU or UAC to CGC), and thus, Y143C may be a
transient form of the Y143R pathway.
Y143 mutants do compare, both in vitro and in vivo, with the
Q148H mutant that emerges only in the context of the double
mutation G140S/Q148H (12, 35). We previously demonstrated
that the Q148H mutant is a thermodynamic mutant, whereas
the G140S/Q148H mutant is a kinetics mutant (12). In contrast
to the Q148H mutant, which was highly impaired in its overall
activity, the G140S/Q148H double mutant was capable of
reaching wild-type levels of activity provided that incubation
time was increased, indicating a functional rescue of the
Q148H defect by the G140S substitution (12). Increasing the
incubation time of the reaction in Y143 mutants also restored
the enzyme activity to levels observed for the wild-type en-
zyme, albeit less rapidly than with the G140S/Q148H mutant.
This thus suggests that Y143 mutants are functionally equiva-
lent to the G140S/Q148H mutants.
The modeling analysis of the molecular effects induced by
these alternative RAL resistance mutations supported this hy-
pothesis in that both the G140S/Q148H and Y143R mutations
might prompt alternative substrate recognition, particularly
the post-3?-processing contact with the 5?-AC overhang. Al-
though this hypothesis remains a theoretical model owing to
the lack of structural data, it is supported by biochemical re-
sults showing that both the Y143 and Q148 residues are in-
volved in specifically binding the extremity of viral DNA (13,
15, 22). The resistance of recombinant IN to RAL in vitro and
molecular modeling study suggested that the presence of R or
H at position 148 prevents RAL binding while mutated IN is
still capable of interacting with DNA (12, 31). Thus, the re-
placement of neighboring Y by R may provide a similar effect,
thereby providing an alternative pathway for RAL resistance.
In conclusion, this study shows that mutations at position
Y143 constitute one pathway conferring high-level resistance
to RAL both in vivo and in vitro. Despite impairment of IN
activities observed in vitro, these mutated viruses are able to
replicate efficiently in vivo as was observed before for the two
other pathways of resistance to RAL (positions 155 and 148).
From this viewpoint, it is possible that the polymorphisms
present at the origin in the sequences of patients have a pos-
itive effect on the activity of the integrase containing the Y143
mutations, which went unnoticed for the single mutations in an
HXB2 background that we studied. However, first the ge-
netic backgrounds of the four patients were quite different,
and second, several of these polymorphisms (T125A, H51D,
N232D, and T206S) are commonly found, in particular T125A
and T206S, associated with the CRF02_AG strains, and so far
the studies of these strains have failed to show any difference
in the resistance patterns induced by RAL (5). Thus, the mech-
anism underlying this in vivo replicative efficiency of a virus
encoding a catalytically crippled integrase remains to be elu-
The research leading to these results received funding from Sidac-
tion, Agence Nationale de Recherches sur le SIDA (ANRS), and the
European Community’s Seventh Framework Programme (FP7/2007-
2013) under the project Collaborative HIV and Anti-HIV Drug Re-
sistance Network (CHAIN), grant agreement no. 223131. S. Thierry is
the recipient of a fellowship from ANRS.
1. Agapkina, J., M. Smolov, S. Barbe, E. Zubin, T. Zatsepin, E. Deprez, B. M.
Le, J. F. Mouscadet, and M. Gottikh. 2006. Probing of HIV-1 integrase-
DNA interactions using novel analogs of viral DNA. J. Biol. Chem. 281:
2. Brown, P. O. 1990. Integration of retroviral DNA. Curr. Top. Microbiol.
3. Brussel, A., O. Delelis, and P. Sonigo. 2005. Alu-LTR real-time nested PCR
assay for quantifying integrated HIV-1 DNA. Methods Mol. Biol. 304:139–
4. Bushman, F. D., and R. Craigie. 1991. Activities of human immunodeficiency
virus (HIV) integration protein in vitro: specific cleavage and integration of
HIV DNA. Proc. Natl. Acad. Sci. U. S. A. 88:1339–1343.
5. Ceccherini-Silberstein, F., I. Malet, R. D’Arrigo, A. Antinori, A. G. Marcelin,
and C. F. Perno. 2009. Characterization and structural analysis of HIV-1
integrase conservation. AIDS Rev. 11:17–29.
6. Chen, A., I. T. Weber, R. W. Harrison, and J. Leis. 2006. Identification of
amino acids in HIV-1 and avian sarcoma virus integrase subsites required for
specific recognition of the long terminal repeat ends. J. Biol. Chem. 281:
7. Connolly, M. L. 1983. Solvent-accessible surfaces of proteins and nucleic-
acids. Science 221:709–713.
8. Cooper, D. A., R. T. Steigbigel, J. M. Gatell, J. K. Rockstroh, C. Katlama, P.
Yeni, A. Lazzarin, B. Clotet, P. N. Kumar, J. E. Eron, M. Schechter, M.
Markowitz, M. R. Loutfy, J. L. Lennox, J. Zhao, J. Chen, D. M. Ryan, R. R.
Rhodes, J. A. Killar, L. R. Gilde, K. M. Strohmaier, A. R. Meibohm, M. D.
Miller, D. J. Hazuda, M. L. Nessly, M. J. DiNubile, R. D. Isaacs, H. Teppler,
B. Y. Nguyen, and B. S. Teams. 2008. Subgroup and resistance analyses of
raltegravir for resistant HIV-1 infection. N. Engl. J. Med. 359:355–365.
9. Croxtall, J. D., and S. J. Keam. 2009. Raltegravir: a review of its use in the
management of HIV infection in treatment-experienced patients. Drugs
10. Delelis, O., K. Carayon, E. Guiot, H. Leh, P. Tauc, J. C. Brochon, J. F.
Mouscadet, and E. Deprez. 2008. Insight into the integrase-DNA recognition
mechanism. A specific DNA-binding mode revealed by an enzymatically
labeled integrase. J. Biol. Chem. 283:27838–27849.
11. Delelis, O., K. Carayon, A. Saib, E. Deprez, and J. F. Mouscadet. 2008.
Integrase and integration: biochemical activities of HIV-1 integrase. Retro-
12. Delelis, O., I. Malet, L. Na, L. Tchertanov, V. Calvez, A. G. Marcelin, F.
Subra, E. Deprez, and J. F. Mouscadet. 2009. The G140S mutation in HIV
integrases from raltegravir-resistant patients rescues catalytic defect due to
the resistance Q148H mutation. Nucleic Acids Res. 37:1193–1201.
13. Engelman, A., and R. Craigie. 1992. Identification of conserved amino acid
residues critical for human immunodeficiency virus type 1 integrase function
in vitro. J. Virol. 66:6361–6369.
500DELELIS ET AL. ANTIMICROB. AGENTS CHEMOTHER.
14. Espeseth, A. S., P. Felock, A. Wolfe, M. Witmer, J. Grobler, N. Anthony, M.
Egbertson, J. Y. Melamed, S. Young, T. Hamill, J. L. Cole, and D. J. Hazuda.
2000. HIV-1 integrase inhibitors that compete with the target DNA substrate
define a unique strand transfer conformation for integrase. Proc. Natl. Acad.
Sci. U. S. A. 97:11244–11249.
15. Esposito, D., and R. Craigie. 1998. Sequence specificity of viral end DNA
binding by HIV-1 integrase reveals critical regions for protein-DNA inter-
action. EMBO J. 17:5832–5843.
16. Ghose, A. K., and G. M. Crippen. 1986. Atomic physicochemical parameters
for 3-dimensional structure-directed quantitative structure-activity-relation-
ships. 1. Partition-coefficients as a measure of hydrophobicity. J. Comput.
17. Grinsztejn, B., B. Y. Nguyen, C. Katlama, J. M. Gatell, A. Lazzarin, D.
Vittecoq, C. J. Gonzalez, J. Chen, C. M. Harvey, and R. D. Isaacs. 2007.
Safety and efficacy of the HIV-1 integrase inhibitor raltegravir (MK-0518) in
treatment-experienced patients with multidrug-resistant virus: a phase II
randomised controlled trial. Lancet 369:1261–1269.
18. Guiot, E., K. Carayon, O. Delelis, F. Simon, P. Tauc, E. Zubin, M. Gottikh,
J. F. Mouscadet, J. C. Brochon, and E. Deprez. 2006. Relationship between
the oligomeric status of HIV-1 integrase on DNA and enzymatic activity.
J. Biol. Chem. 281:22707–22719.
19. Hazuda, D. J., P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J. A. Grobler,
A. Espeseth, L. Gabryelski, W. Schleif, C. Blau, and M. D. Miller. 2000.
Inhibitors of strand transfer that prevent integration and inhibit HIV-1
replication in cells. Science 287:646–650.
20. Heiden, W., G. Moeckel, and J. Brickmann. 1993. A new approach to anal-
ysis and display of local lipophilicity hydrophilicity mapped on molecular-
Surfaces. J. Comput. Aided Mol. Des. 7:503–514.
21. Johnson, A. A., C. Marchand, S. S. Patil, R. Costi, S. R. Di, T. R. Burke, Jr.,
and Y. Pommier. 2007. Probing HIV-1 integrase inhibitor binding sites with
position-specific integrase-DNA cross-linking assays. Mol. Pharmacol. 71:
22. Khan, E., J. P. Mack, R. A. Katz, J. Kulkosky, and A. M. Skalka. 1991.
Retroviral integrase domains: DNA binding and the recognition of LTR
sequences. Nucleic Acids Res. 19:851–860.
23. Kobayashi, M., K. Nakahara, T. Seki, S. Miki, S. Kawauchi, A. Suyama, C.
Wakasa-Morimoto, M. Kodama, T. Endoh, E. Oosugi, Y. Matsushita, H.
Murai, T. Fujishita, T. Yoshinaga, E. Garvey, S. Foster, M. Underwood, B.
Johns, A. Sato, and T. Fujiwara. 2008. Selection of diverse and clinically
relevant integrase inhibitor-resistant human immunodeficiency virus type 1
mutants. Antiviral Res. 80:213–222.
24. Laskowski, R. A., M. W. Macarthur, D. S. Moss, and J. M. Thornton. 1993.
Procheck—a program to check the stereochemical quality of protein struc-
tures. J. Appl. Crystallogr. 26:283–291.
25. Leh, H., P. Brodin, J. Bischerour, E. Deprez, P. Tauc, J. C. Brochon, E.
LeCam, D. Coulaud, C. Auclair, and J. F. Mouscadet. 2000. Determinants of
Mg2?-dependent activities of recombinant human immunodeficiency virus
type 1 integrase. Biochemistry 39:9285–9294.
26. Li, M., and R. Craigie. 2005. Processing of viral DNA ends channels the
HIV-1 integration reaction to concerted integration. J. Biol. Chem. 280:
27. Malet, I., O. Delelis, C. Soulie, M. Wirden, L. Tchertanov, P. Mottaz, G.
Peytavin, C. Katlama, J. F. Mouscadet, V. Calvez, and A. G. Marcelin. 2009.
Quasispecies variant dynamics during emergence of resistance to raltegravir
in HIV-1-infected patients. J. Antimicrob. Chemother. 63:795–804.
28. Malet, I., O. Delelis, M. A. Valantin, B. Montes, C. Soulie, M. Wirden, L.
Tchertanov, G. Peytavin, J. Reynes, J. F. Mouscadet, C. Katlama, V. Calvez,
and A. G. Marcelin. 2008. Mutations associated with failure of raltegravir
treatment affect integrase sensitivity to the inhibitor in vitro. Antimicrob.
Agents Chemother. 52:1351–1358.
29. Marinello, J., C. Marchand, B. T. Mott, A. Bain, C. J. Thomas, and Y.
Pommier. 2008. Comparison of raltegravir and elvitegravir on HIV-1 inte-
grase catalytic reactions and on a series of drug-resistant integrase mutants.
30. Markowitz, M., B. Y. Nguyen, E. Gotuzzo, F. Mendo, W. Ratanasuwan, C.
Kovacs, G. Prada, J. O. Morales-Ramirez, C. S. Crumpacker, R. D. Isaacs,
L. R. Gilde, H. Wan, M. D. Miller, L. A. Wenning, and H. Teppler. 2007.
Rapid and durable antiretroviral effect of the HIV-1 integrase inhibitor
raltegravir as part of combination therapy in treatment-naive patients with
HIV-1 infection: results of a 48-week controlled study. J. Acquir. Immune
Defic. Syndr. 46:125–133.
31. Mouscadet, J. F., R. Arora, J. Andre, J. C. Lambry, O. Delelis, I. Malet, A. G.
Marcelin, V. Calvez, and L. Tchertanov. 2009. HIV-1 IN alternative molec-
ular recognition of DNA induced by raltegravir resistance mutations. J. Mol.
32. Pauza, C. D., P. Trivedi, T. S. McKechnie, D. D. Richman, and F. M.
Graziano. 1994. 2-LTR circular viral DNA as a marker for human immu-
nodeficiency virus type 1 infection in vivo. Virology 205:470–478.
33. Quercia, R., E. Dam, D. Perez-Bercoff, and F. Clavel. 2009. Selective-advan-
tage profile of HIV-1 integrase mutants explains in vivo evolution of ralte-
gravir resistance genotypes. J. Virol. 83:10245–10249.
34. Shin, C. G., B. Taddeo, W. A. Haseltine, and C. M. Farnet. 1994. Genetic
analysis of the human immunodeficiency virus type 1 integrase protein.
J. Virol. 68:1633–1642.
35. Sichtig, N., S. Sierra, R. Kaiser, M. Daumer, S. Reuter, E. Schulter, A.
Altmann, G. Fatkenheuer, U. Dittmer, H. Pfister, and S. Esser. 2009. Evo-
lution of raltegravir resistance during therapy. J. Antimicrob. Chemother.
36. Smolov, M., M. Gottikh, V. Tashlitskii, S. Korolev, I. Demidyuk, J. C.
Brochon, J. F. Mouscadet, and E. Deprez. 2006. Kinetic study of the HIV-1
DNA 3?-end processing. FEBS J. 273:1137–1151.
37. Svarovskaia, E. S., R. Barr, X. Zhang, G. C. Pais, C. Marchand, Y. Pommier,
T. R. Burke, Jr., and V. K. Pathak. 2004. Azido-containing diketo acid
derivatives inhibit human immunodeficiency virus type 1 integrase in vivo
and influence the frequency of deletions at two-long-terminal-repeat-circle
junctions. J. Virol. 78:3210–3222.
38. Wielens, J., I. T. Crosby, and D. K. Chalmers. 2005. A three-dimensional
model of the human immunodeficiency virus type 1 integration complex.
J. Comput. Aided Mol. Des. 19:301–317.
39. Yeni, P. 2008. Prise en charge me ´dicale des personnes infecte ´es par le
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