ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2010, p. 4016–4019
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 9
Baseline Genotypic and Phenotypic Susceptibilities of HIV-1
Group O to Enfuvirtide?
Agne `s Depatureaux,1† Charlotte Charpentier,1† Gilles Collin,2Marie Leoz,1Diane Descamps,2
Aure ´lia Vessie `re,3Florence Damond,2Dominique Rousset,3
Franc ¸oise Brun-Ve ´zinet,2and Jean-Christophe Plantier1*
Laboratoire associe ´ au Centre National de Re ´fe ´rence du VIH, EA2656, CHU Charles Nicolle, Rouen,1and
Assistance Publique—Ho ˆpitaux de Paris (AP-HP), Ho ˆpital Bichat-Claude Bernard, Laboratoire de
Virologie, Paris,2France, and Centre Pasteur du Cameroun, Yaounde ´, Cameroon3
Received 22 January 2010/Returned for modification 8 March 2010/Accepted 2 June 2010
We assessed the natural genotypic and phenotypic susceptibilities to enfuvirtide of 171 HIV group O
(HIV-O) samples and 29 strains, respectively. The N42D resistance-associated mutation in the gp41 region was
detected in 98% of cases. The phenotypic assay showed a wide range of baseline susceptibilities, with 50%
inhibitory concentrations (IC50s) from 4 to 5,000 nM, a range similar to that reported for HIV-1 group M.
Thus, despite the natural genotypic resistance conferred by the N42D signature mutation, HIV-O variants
appear to be phenotypically susceptible. Enfuvirtide could therefore potentially be used in antiretroviral
treatments for HIV-O-infected patients.
Human immunodeficiency virus (HIV) is characterized by
high levels of genetic diversity within HIV type 1 (HIV-1) and
HIV type 2 (HIV-2). HIV-1 is subdivided into four groups: M
(major), O (outlier), N (non-M, non-O), and P. HIV-1 group
O (HIV-O) is endemic in Western Central Africa, including
Cameroon in particular, where the prevalence of this group is
estimated at about 1% of all HIV infections (about 10,000
patients) (2, 26). The prevalence of HIV-O remains low, with
limited spread, outside Western Central Africa. In France, the
first HIV-O-infected patient was described in 1992 (1), and the
viral strain was characterized in 1994 (3). As part of our sur-
veillance activities on divergent variants of HIV, we set up, in
2005, a network (RES-O) of physicians and biologists for the
surveillance and monitoring of HIV-O infections in France.
This network has identified 123 patients (6; also our unpub-
lished data), most of whom were originally from Cameroon or
had a partner originating from Cameroon.
HIV-O displays strong genetic divergence from HIV-1
group M and a high degree of intragroup diversity (22, 28).
This divergence has clinical implications, complicating the pro-
cesses of diagnosis (18) and virological monitoring (11, 19),
and necessitating the use of adapted techniques and treatment
strategies. Indeed, HIV-O is considered to be naturally resistant
to nonnucleoside reverse transcriptase inhibitors (NNRTI), due
to the presence of the Y181C mutation (6). Few studies have
assessed the virological response to treatment regimens contain-
ing nucleoside reverse transcriptase inhibitors (NRTI) and pro-
tease inhibitors (PI) (5, 21), and this response has not been eval-
uated for the most recently developed drugs.
Enfuvirtide (T20), an HIV-1 fusion inhibitor, is a peptide
based on the sequence of the heptad repeat 2 (HR2) region of
the gp41 transmembrane glycoprotein derived from the sub-
type B HIV-1LAIsequence. It was not clear whether T20 would
be active against HIV-O, because this peptide was designed to
work against HIV-1 group M. Furthermore, the reported
natural presence in HIV-O of an N42D mutation (4, 20),
associated with resistance in HIV-1 group M according to
the interpretation of the algorithm defined by the ANRS (http:
.kuleuven.be/cev/), whether in isolation or associated with
other mutations, would be expected to compromise the activity
of T20 in HIV-O variants (14, 15, 16, 23). However, prelimi-
nary data showed that T20 was active against six HIV-O pri-
mary isolates in vitro (4) and in one HIV-O-infected patient in
vivo (20). This apparent discordance with the expected findings
and the limited number of cases of HIV-O infection evaluated
in these studies precluded the development of a reliable rep-
resentation of the high level of intragroup diversity.
In this study, we assessed the genotypic susceptibility to T20
of a large panel of HIV-O samples (n, 171) representative of
the diversity of group O. Phenotypic susceptibility to T20 was
evaluated for 29 of these samples, and the results obtained
were compared with the genotypic interpretation. None of the
patients studied had ever been treated with T20, and all were
identified by the French RES-O network (n, 91) and through
collaboration with the Centre Pasteur du Cameroun in
Yaounde ´ (n, 80).
Genotypic assays involved amplification of the whole
ectodomain of gp41, residues 1 to 179, encompassing the HR1
and HR2 regions and the start of the transmembrane region.
Viral RNA was extracted from 142 cell-free plasma samples
and 29 cell-free viral supernatant samples (obtained by cocul-
ture [see below]) and was subjected to reverse transcription-
PCR with the primers and cycling conditions described else-
where (17, 26). We developed a phenotypic test adapted to the
gp41 region, using a peripheral blood mononuclear cell
* Corresponding author. Mailing address: Laboratoire de Virologie,
Institut de Biologie Clinique, Ho ˆpital Charles Nicolle, Centre Hospi-
talier Universitaire de Rouen, 1 rue de Germont, 76031, Rouen,
France. Phone: 33 2 32 88 14 62. Fax: 33 2 32 88 04 30. E-mail:
† A.D. and C.C. contributed equally to this work.
?Published ahead of print on 4 June 2010.
(PBMC) assay, as previously described (8). Briefly, 29 strains
were isolated by coculturing PBMCs from HIV-O-infected pa-
tients with PBMCs from healthy donors. We used 1 ml of the
viral supernatant, corresponding to a viral density of 1 ? 106
copies/ml and one 50% tissue culture infective dose (TCID50)
per ml of sample, to infect cells in the presence of various
concentrations of T20 (range, 4.5 nM to 12,000 nM). Viral
replication was then evaluated by an HIV-O-specific real-time
PCR assay (10). We interpreted 50% inhibitory concentrations
(IC50) using the values obtained for two laboratory reference
strains: (i) HIV-1BRU, which is susceptible to T20, and (ii)
HIV-2ROD, which is considered to be naturally resistant to T20
We found that 168 of the 171 HIV-O samples (98%) carried
the N42D mutation within the HR1 domain of gp41. One
sample carried the N42S mutation; one carried the natural
residue; and the remaining sample was found to correspond to
an N42D N43K double mutant virus, detected in a patient
followed up at the Centre Pasteur du Cameroun in Yaounde ´.
Phenotypic susceptibility was successfully determined for all
29 HIV-O viral supernatants tested. The IC50obtained were
widely scattered over a range extending from 4 nM to 5,000
nM, with a median value of 149 nM (Fig. 1). The sample
characterized genotypically as a double mutant (N42D N43K)
gave a low value (IC50, 6.4 nM), similar to that for the HIV-1
group M control. Even for the HIV-O samples with the highest
IC50in this series, the IC50obtained were lower than those for
We then analyzed the sequences of the HR1 and HR2 re-
gions of these 29 HIV-O strains in order to identify possible
determinants linked to high or low IC50. A comparison be-
tween the HIV-O sequences and the sequences of the HIV-1
group M reference strain HIV-1BRUshowed a high level of
polymorphism in HIV-O viruses, associated with a high level of
intragroup diversity, particularly for the HR2 region (Fig. 2).
This high level of polymorphism was confirmed for the 142
sequences obtained from plasma samples (data not shown).
Furthermore, comparisons of sequences of viruses for which
the IC50exceeded 1,000 nM with sequences of viruses for
which the IC50were below 1,000 nM (Fig. 2) showed two
positions (residues 27 and 119) to be significantly different
between these two groups of sequences (alpha, 5% by Pear-
son’s chi-square test). The T27A mutation was more frequent
in the group of HIV-O sequences with IC50of ?1,000 nM
(54% versus 6% [P, 0.008]), whereas the E119Q mutation was
less frequent among HIV-O sequences with IC50of ?1,000 nM
(0% versus 44% [P, 0.026]).
Finally, we assessed the possible impact of genetic diversity
on phenotypic susceptibility, since we had previously demon-
strated the clade dependence of the Y181C mutation associ-
ated with resistance to NNRTI (7). A phylogenetic tree includ-
ing all 29 HIV-O gp41 sequences used for the phenotypic
analysis was generated. No relationship was found between
IC50and a specific genetic cluster or a specific clade (data not
We have reported the genotypic susceptibilities of 171
HIV-O viruses to T20, an HIV-1 fusion inhibitor, and the
phenotypic susceptibilities of 29 of these viruses. In our study,
98% of HIV-O viruses naturally harbored the N42D mutation
in the gp41 region, associated with a wide range of baseline
levels of phenotypic susceptibility to T20. The lowest IC50in
our study were similar to those previously reported (4, 20),
probably due to the limited number of samples tested. Our
results, obtained on a larger panel, indicate that IC50are more
scattered than previously thought, with a range potentially
spanning 3 log orders of magnitude, as reported for HIV-1
group M viruses, which had IC50from 1 to 480 ng/ml in the
study by Sista et al. (23), 3 to 1,002 ng/ml in the study by
Labrosse et al. (12), and 4 to 8,192 ng/ml in the study by Melby
et al. (13). The similar results obtained for HIV-1 groups M
and O led us to conclude that this specific antiretroviral drug
displayed a broad baseline susceptibility. In our study, the
highest IC50, approaching that for HIV-2ROD, which is consid-
ered naturally resistant to T20 (27), require careful interpre-
tation. One important consequence of this wide range of values
is that it is difficult to state phenotypic resistance based solely
on the finding of a high IC50. Thus, for a more accurate defi-
nition of resistance, a comparison of IC50is required, with the
definition of a significant fold change between the IC50for the
baseline sample and those for samples obtained during treat-
ment in the context of virological failure with a T20-containing
regimen, as described for HIV-1 group M.
We found no association between IC50and a specific HIV-O
phylogenetic cluster, as might have been expected following
our recent description of the clade A dependence of the
Y181C mutation responsible for NNRTI resistance (7). Inter-
estingly, significant changes to residues 27 and 119 of gp41
have been associated with changes in IC50but not with sus-
ceptibility, since all samples were nonetheless considered to be
susceptible to T20. It is difficult to determine the individual
roles of these specific mutations, because they have not been
described in previous studies, and the accurate assessment of
their roles is likely to be difficult, because the considerable
intragroup genetic diversity within HIV-O viruses must be
taken into account. These two mutations do not affect residues
forming the leucine zipper, an essential part of the final struc-
ture of gp41, but they may help to stabilize the helix or may act
as accessory mutations in the restoration of fitness, as sug-
gested by previous studies (24, 25).
FIG. 1. Baseline susceptibilities to enfuvirtide (T20) of 29 HIV-1
group O viral strains. The 50% inhibitory concentrations (IC50) are
expressed as nanomolar concentrations for each of the 29 HIV-1 group
O viral supernatants. The results were obtained in two independent
experiments, with quadruplicate determinations for each point. The
IC50of the reference laboratory virus HIV-1BRUis indicated by an
open square, and that of the reference laboratory virus HIV-2RODis
indicated by an open circle.
VOL. 54, 2010 HIV-1 GROUP O AND ENFUVIRTIDE4017
The apparent discrepancy between genotypic and pheno-
typic results raises questions about the real impact of the N42D
mutation on the mechanism of resistance of HIV-O variants to
enfuvirtide. This mutation may not be the only determinant of
resistance. Compensatory mutations may also be present,
though difficult to identify, due to the high degree of polymor-
phism of these variants. This discordance between phenotypic
results and the interpretation of genotypic resistance on the
basis of HIV-1 group M algorithms highlights the need to
optimize current interpretations for HIV-O. Finally, our find-
ings demonstrate that T20 may potentially be useful for the
treatment of HIV-O-infected patients whose treatment op-
tions are already limited. The prevalence of HIV-O is low
outside the Western Central Africa region, and the short-term
FIG. 2. Alignment of the sequences of the 29 HIV-1 group O strains used in the phenotypic analysis. The sequences correspond to the
ectodomain of gp41; HIV-O sequences are compared with HIV-1BRU, used as a reference, and are ranked in ascending order of associated IC50.
The IC50threshold of 1,000 nM is indicated by a horizontal line. Resistance-associated positions are boxed, and the residues at positions 27 and
119 are boxed and indicated by arrows. A schematic linear structure of the gp41 ectodomain is shown in the center. FP, fusion peptide; HR1 and
HR2, heptad repeats 1 and 2; TM, transmembrane region. The positions of the HR1 and HR2 domains were defined according to reference 9.
4018 DEPATUREAUX ET AL.ANTIMICROB. AGENTS CHEMOTHER.
use of T20 in Cameroon seems to be compromised by logistical Download full-text
obstacles. Nevertheless, this study shows that T20 was active in
vitro against a large panel of diverse HIV-O viruses, increasing
the number of antiretroviral drugs that may be considered
active against this particular group of HIV-1 viruses.
Nucleotide sequence accession numbers. The HIV-O se-
quences determined in this study have been submitted to
GenBank under accession numbers GU935937 to GU936106
We thank the Institut de Veille Sanitaire (InVS), the Agence Na-
tionale de Recherche sur le SIDA et les He ´patites Virales (ANRS),
and Rouen University Hospital for financial support.
We thank all the physicians and biologists involved in the RES-O
network for the surveillance of HIV-O infections in France.
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VOL. 54, 2010HIV-1 GROUP O AND ENFUVIRTIDE 4019