JOURNAL OF VIROLOGY, July 2008, p. 6678–6688
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 13
Selection of T1249-Resistant Human Immunodeficiency Virus
Type 1 Variants?
Dirk Eggink,1Christopher E. Baldwin,1Yiqun Deng,2Johannes P. M. Langedijk,3Min Lu,2
Rogier W. Sanders,1and Ben Berkhout1*
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam,
Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands1; Department of
Biochemistry, Weill Medical College of Cornell University, New York, New York2; and
Pepscan Therapeutics, Lelystad, The Netherlands3
Received 18 February 2008/Accepted 16 April 2008
Human immunodeficiency virus type 1 (HIV-1) entry is an attractive target for therapeutic intervention. Two
drugs that inhibit this process have been approved: the fusion inhibitor T20 (enfuvirtide [Fuzeon]) and, more
recently, the CCR5 blocker maraviroc (Selzentry). T1249 is a second-generation fusion inhibitor with improved
antiviral potency compared to the first-generation peptide T20. We selected T1249-resistant HIV-1 variants in
vitro by serial virus passage in the presence of increasing T1249 doses after passage with wild-type and
T20-resistant variants. Sequence analysis revealed the acquisition of substitutions within the HR1 region of the
gp41 ectodomain. The virus acquired mutations of residue V38 to either E or R in 10 of 19 cultures. Both E and
R at position 38 were confirmed to cause resistance to T1249, as well as cross-resistance to T20 and C34, but
not to the third-generation fusion inhibitor T2635. We also observed substitutions at residues 79 and 90 (Q79E
and K90E), which provide modest resistance to T1249 and, interestingly, T2635. Thus, the gp41 amino acid
position implicated in T20 resistance (V38 replaced by A, G, or W) is also responsible for T1249 resistance (V38
replaced by E, R, or K). These results indicate that T20 and T1249 exhibit very similar inhibition modes that
call for similar but not identical resistance mutations. All T1249-resistant viruses with changes at position 38
are cross resistant to T20, but not vice versa. Furthermore, substitutions at position 38 do not provide
resistance to the third-generation inhibitor T2635, while substitution at positions 79 and 90 do, suggesting
different resistance mechanisms.
Human immunodeficiency virus type 1 (HIV-1) entry is me-
diated by the envelope (Env) glycoproteins gp120 and gp41.
Env is arranged on the virus particle as trimeric spikes, com-
prising three gp120 molecules and three gp41 molecules, an-
chored within the viral membrane via the gp41 transmembrane
domain. The first step in HIV-1 entry is the binding of gp120
to CD4 followed by binding to a coreceptor on the T-cell
surface that triggers conformational changes in Env, resulting
in the insertion of the hydrophobic N-terminal fusion peptide
of gp41 into the target cell membrane (reviewed in reference
18). Subsequent changes within gp41 involve two leucine zip-
per-like motifs, heptad repeat 1 (HR1) and heptad repeat 2
(HR2), assembling into a highly stable six-helix bundle struc-
ture which juxtaposes the viral and cellular membranes for the
fusion event (9, 49, 52). The change in free energy associated
with this structural transition is predicted to be sufficient to
cause lipid mixing and membrane fusion (25, 39). Peptide
fusion inhibitors that bind to one of the HR motifs can block
this conformational switch and thus inhibit viral entry (4, 10,
T20, the first approved fusion inhibitor for HIV-1, is a 36-
amino-acid peptide that mimics HR2 and acts by binding to
HR1, thus preventing the HR1-HR2 interaction (Fig. 1) (29,
32, 53, 54). In vitro passaging of HIV-1 in the presence of
increasing T20 concentrations resulted in the selection of re-
sistant virus variants with mutations within a stretch of three
HR1 amino acids, glycine-isoleucine-valine (the GIV motif,
HXB2 amino acid positions 36 to 38 of gp41) (16, 22, 43, 44,
55, 56). Resistance mutations have also been identified within
the viral quasispecies of patients on T20 therapy, specifically at
positions 36 to 45 (1, 5, 12, 40, 42, 51).
The fusion inhibitor C34 also corresponds to part of HR2
(Fig. 1; see Fig. 5b) (35). The inhibitory activity of C34 depends
on its ability to bind to a prominent hydrophobic pocket in
HR1, which may make it less susceptible to the evolution of
drug-resistant viruses (36, 43). In vitro passaging of HIV-1 in
the presence of increasing C34 concentrations resulted in the
selection of resistant virus with the V38E substitution (2). An
second-generation fusion inhibitor, T1249, which has greater
inhibitory potency than C34 and is also effective against T20-
resistant variants in vitro and in vivo, was developed (20, 21).
T1249 is composed of sequences from HIV-1, HIV-2, and
simian immunodeficiency virus (20, 47). The C-terminal region
of T1249 is almost identical to T20, but the peptide differs in
the N-terminal sequence, which extends a further three resi-
dues (total length, 39 amino acids) and bears little similarity to
T20 in its amino acid composition (Fig. 1). In vitro resistance
has been described in a study using a randomized mutagenesis
strategy in HR1 (13). Changes at positions 37, 38, and 40 were
found to cause T1249 resistance. In vivo resistance develop-
* Corresponding author. Mailing address: Laboratory of Experi-
mental Virology, Department of Medical Microbiology, Center for
Infection and Immunity Amsterdam (CINIMA), Academic Medical
Center of the University of Amsterdam, P.O. Box 22700, 1100 DE
Amsterdam, The Netherlands. Phone: 31-20-5664822. Fax: 31-20-
6916531. E-mail: firstname.lastname@example.org.
?Published ahead of print on 23 April 2008.
ment during treatment with T1249 was linked to mutations
G36D, V38E, Q40K, N43K, and A50V in HR1 and N126K and
S138A in HR2 (20, 38). Recently, third-generation fusion in-
hibitor peptides were described as having increased helical
structure and high HR1/HR2 bundle stability (T2635 and vari-
ants thereof) (17). These peptides are active against T20- and
T1249-resistant viruses, and no T2635 resistance has been re-
ported so far.
In this study, we selected T1249 drug-resistant HIV-1 vari-
ants in vitro. Our results demonstrate that the same amino acid
position, position 38, that is implicated in T20 and C34 resis-
tance is also involved in T1249 resistance, although different
substitutions are required. These findings indicate that T20
and T1249 exhibit very similar inhibition modes that trigger
similar but not identical escape routes. To evaluate the clinical
significance of the described mutations, the cross-resistance to
T20 and T2635 was determined. Interestingly, substitutions at
positions 38 caused increased susceptibility to T2635. In con-
trast, T1249 resistance mutations at positions 79 and 90 confer
cross-resistance to T2635.
MATERIALS AND METHODS
Peptide synthesis. Amino acids are indicated by the single-letter code. Pep-
tides were synthesized by solid-phase peptide synthesis by using a 4-(2,4-di-
methoxyphenyl-Fmoc (Rink-Amide) resin (BACHEM Biochemica, Heidelberg,
Germany) on a Syro synthesizer (MultiSynTech, Witten, Germany). All amino
acids were purchased from BACHEM Biochemica and used as N-?-(Fmoc)
protected with side chain functionalities protected as N-tert-butoxycarbonyl
(KW), O-tert-Butyl (DESTY), N-Trityl (HNQ), S-Trityl (C), S-2,2,4,6,7 (C), or
N-pentamethyl dihydrobenzofurane–5-sulfonyl (R) groups. A coupling protocol
using a 6.5-fold excess of HBTU-HOBt-amino acid-DIPEA (1:1:1:2) in NMP
with a 30-min activation time using double couplings was employed. Peptides
were cleaved from the resin by reaction with trifluoroacetic acid (TFA; 15 ml g?1
resin) containing 13.3% (by weight) phenol, 5% (by volume) thioanisole, 2.5%
(by volume) 1,2-ethanedithiol, and 5% (by volume) milliQ-purified H2O for 2 to
4 h at room temperature. The crude peptides were purified by reversed-phase
high-performance liquid chromatography (HPLC), either on a DeltaPack (25
mm or 40 mm [inner diameter] by 100 mm [length], 15-?m particle size, 100-Å
pore size; Waters, Milford, MA) or on an XTerra (50 mm by 4.6 mm [inner
diameter], 2.5-?m particle size; Waters, Milford, MA) RP-18 preparative C18
column with a linear AB gradient of 1 to 2% solvent B min?1where solvent A
was 0.05% TFA in water and solvent B was 0.05% TFA in acetonitrile. The
correct primary ion molecular weights of the peptides was confirmed by electron-
spray ionization mass spectrometry on a ZQ (Micromass, Almere, The Nether-
lands) or Quattro II (VG Organic, Cheshire, United Kingdom) mass spectrom-
In vitro selection of T1249-resistant HIV-1 variants. For the selection of
T1249-resistant viruses, SupT1 cells were transfected with 1 ?g DNA of either
the wild-type HIV-1LAImolecular clone or several T20-resistant variants: V38A,
V38G, V38W, and V38W/N126K mutants (5). Transfected cells were split 1 day
posttransfection into three to six separate culture flasks, and 0.5 ? 106fresh
SupT1 cells were added to initiate the evolution cultures. We started the selec-
tion with a concentration of 5 ng/ml T1249 for the T20-sensitive wild type and 20
ng/ml for T20-resistant variants, which is sufficient to reduce replication by
?90%. We initially split 100 ?l culture (cells and supernatant) when required
onto uninfected SupT1 cells. At each passage, the T1249 drug concentration was
increased on average 1.5 times. When HIV-induced cytopathic effects and in-
creased CA-p24 production were apparent, virus replication was maintained by
passage of cell-free culture supernatant onto uninfected SupT1 cells. We used
escalating volumes of cell-free culture supernatant to infect 5 ml fresh SupT1
cells (0.5 ? 106cells). Initially, we started by passaging 100 ?l cell-free super-
natant onto fresh cells. We used less supernatant in subsequent passages, from
100 ?l in the second passage to a minimum of 10 ?l. Cells and supernatant
samples were taken at regular time points and stored at ?70°C. Cell culturing,
transfections, and CA-p24 determination were performed as previously reported
Proviral DNA isolation, PCR amplification, and sequencing. HIV-1-infected
cells (1 ml culture) were pelleted by centrifugation at 4,000 rpm for 4 min and the
supernatant was analyzed for CA-p24 content and stored at ?70°C. The cell
pellet was lysed in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.5% Tween 20
and incubated with 500 ?g proteinase K/ml at 56°C for 60 min and heat inacti-
vated at 95°C for 10 min. The complete env genes from proviral DNA sequences
were PCR amplified from solubilized cellular DNA by using the Expand high-
fidelity PCR system according to the manufacturer’s instructions (Roche, Mann-
heim, Germany). Briefly, after incubation for 5 min at 94°C, the reaction mixture
was subjected to 35 PCR cycles in a type 9700 DNA thermal cycler (Perkin
Elmer, Waltham, MA), with each cycle including a denaturation step for 30 s at
94°C, an annealing step for 30 s at 60°C, and an extension step for 3 min at 68°C.
This was followed by a final extension step of 7 min at 68°C. The PCR was
FIG. 1. Schematic of gp160 and the gp120 and gp41 subunits and a close-up of the gp41 ectodomain. Indicated are the positions and amino
acid residues of peptide-based fusion inhibitors. Bolded amino acid residues indicate deviations from the prototype HXB2 sequence. The GIV
sequence in HR1 which includes amino acid 38 of gp41 is bold and underlined. Amino acid positions implicated in T1249 resistance are marked
with filled circles. FP, fusion peptide.
VOL. 82, 2008 T1249-RESISTANT HIV-16679
performed with 50 ng sense and antisense primers (WS1 [5?-ATAAGCTTAGC
AGAAGACA-3?] and 3?envMD4 [5?-GCAAAATCCTTTCCAAGCCC-3?]) in a
50-?l PCR. DNA products were analyzed on a 1% agarose gel that was
prestained with ethidium bromide. PCR products were sequenced directly using
the DNA BigDye Terminator sequencing kit (ABI, Foster City, CA) and an ABI
377 automated sequencer.
Construction of HIV-1LAImolecular clones. The full-length molecular clone of
HIV-1LAI(pLAI) was used to produce wild-type and mutant viruses (41). We
already described the wild-type variant with the GIV-SNY sequence (V38) as
observed in a patient isolate (different from the GIV-NNY sequence that is
present in the HIV-1LAImolecular clone) and the T20-resistant V38A, V38G,
and V38W variants (5). The GIV-SNY (N125S) variant shows a slight decrease
in fitness compared to the GIV-NNY variant (data not shown). The plasmid
pRS1, designed to subclone mutant env genes, was described previously (45).
Mutations were introduced into pRS1 by using the QuikChange mutagenesis kit
(Stratagene, La Jolla, CA), and the entire env gene was verified by DNA se-
quencing. Mutant env genes (corresponding to V38E, V38R, V38K, N43K,
Q79E, and K90E mutations) in pRS1 were cloned back into pLAI as SalI-BamHI
fragments. Because of the common appearance of charged amino acids at posi-
tion 38, we also made a V38K mutant, even though we did not observe this
mutant in the evolution experiments.
Transfections and CA-p24 determination. The SupT1 T-cell line was main-
tained in RPMI 1640 supplemented with 10% fetal calf serum and penicillin and
streptomycin (both at 100 U/ml) and incubated at 37°C with 5% CO2. SupT1
cells were transfected with HIV-1 molecular clones by electroporation. Briefly,
5 ? 106cells were washed in RPMI 1640 with 20% fetal calf serum, mixed with
1 ?g of DNA in 0.4-cm cuvettes, and electroporated at 250 V and 975 ?F,
followed by resuspension of cells in RPMI 1640 with 10% fetal calf serum. The
transfected cells were split at day 1 posttransfection and cultured with 100
ng/ml T20, 100 ng/ml C34, and 25, 100, or 200 ng/ml T1249. CA-p24 produc-
tion was determined from culture supernatant taken at various days post-
IC50and infectivity determination. The TZM-bl reporter cell line (15, 51)
stably expresses high levels of CD4 and HIV-1 coreceptors CCR5 and CXCR4
and contains the luciferase and ?-galactosidase genes under the control of the
HIV-1 long-terminal-repeat promoter. The TZM-bl cell line was obtained
through the NIH AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH (TZM-bl from John C. Kappes, Xiaoyun Wu, and Tranzyme
Inc. [Durham, NC]). One day prior to infection, TZM-bl cells were plated on a
96-well plate in Dulbecco’s modified Eagle’s medium containing 10% fetal bo-
vine serum, 1? minimum essential medium nonessential amino acids, and pen-
icillin-streptomycin (both at 100 units/ml) and incubated at 37°C with 5% CO2.
Each virus variant was produced in C33A cells by calcium phosphate precipita-
tion as previously described (14). A fixed amount of virus (1 ng CA-p24) was
preincubated for 30 min at room temperature with 0, 0.15, 0.46, 1.37, 4.12, 12.35,
37.04, 111.1, 333.3, 1,000 or 3,000 ng/ml of T20 or T1249 or 0, 0.051, 0.15, 0.46,
1.37, 4.12, 12.35, 37.04, 111.1, 333.3, or 1,000 ng/ml of T2635. This mixture was
added to the cells in the presence of 400 nM saquinavir (Roche, Mannheim,
Germany) and 40 ?g/ml DEAE in a total volume of 200 ?l. Two days postin-
fection, the medium was removed and cells were washed once with phosphate-
buffered saline (PBS) and lysed in reporter lysis buffer (Promega, Madison, WI).
Luciferase activity was measured using a luciferase assay kit (Promega, Madison,
WI) and a Glomax luminometer according to the manufacturer’s instructions
(Turner BioSystems, Sunnyvale, CA). All infections were performed in dupli-
cate, and luciferase measurements were also performed in duplicate. Uninfected
cells were used to correct for background luciferase activity. The infectivity of
each mutant without inhibitor was set at 100%. Nonlinear regression curves were
determined and 50% inhibitory concentrations (IC50s) were calculated using
Prism software version 4.0c. The relative infectivities of molecular clones com-
pared to HIV-1LAIwere calculated for all T1249 escape mutants. Luciferase
activity without inhibitor of quadruple infections was measured in duplicate and
corrected for background luciferase activity. Infectivity of HIV-1LAIwild type
was normalized to 100% and relative infectivity for the other mutants was
Protein expression, purification, and proteolysis. The recombinant N36(L6)C34
model peptide and its variants were expressed in the Escherichia coli strain
BL21(DE3)/pLysS by using a modified pET3a vector (Novagen, San Diego, CA).
The sequence of N36(L6)C34 is SGIVQQQSNL LRAIEAQQHL LQLTVW
GIKQ LQARVLSGGR GGWMDWEREI SNYTKQIYTL IEESQNQQEK NE
QELL (with the six-residue linker underlined). Substitutions were introduced
into the pN36/34 plasmid (5) by the method used by Kunkel (30) and were
verified by DNA sequencing. The cells were grown at 37°C in LB medium to an
optical density of 0.7 at 600 nm and harvested by centrifugation 4 h postinduction
with 0.5 mM isopropylthio-?-D-galactoside. Cells were lysed by glacial acetic acid
and centrifuged to separate the soluble fraction from inclusion bodies. The
soluble fraction containing peptide was subsequently dialyzed into 5% acetic acid
overnight at 4°C. Peptides were purified from the soluble fraction to homoge-
neity by reverse-phase HPLC (Waters, Milford, MA) on a C18preparative
column (Vydac, Hesperia, CA) by using a water-acetonitrile gradient in the
presence of 0.1% TFA and lyophilized. Peptide identities were confirmed by
electrospray mass spectrometry (Voyager Elite; PerSeptive Biosystems, Fram-
ingham, MA). Protein concentrations were determined by the method of Edel-
hoch (19). Proteinase K digestion was performed with protease/protein ratios of
1:100 (wt/wt) at room temperature in PBS (pH 7.0). Proteolysis was quenched by
addition of phenylmethylsulfonyl fluoride to a final concentration of 2 mM.
Proteolytic fragments were analyzed by reverse-phase HPLC as described above
and identified by N-terminal sequencing and mass spectrometry. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis was carried out on 16.5% polyacryl-
amide gels using a Tris-Tricine buffer system (46).
Biophysical analysis. Circular dichroism (CD) experiments were performed
on an 62A/DS (Aviv Associates, Lakewood, NJ) spectropolarimeter equipped
with a thermoelectric temperature control at 10 ?M peptide concentration in
PBS (50 mM sodium phosphate, pH 7.0, 150 mM NaCl). CD spectra were
collected from 260 to 200 nm at 4°C, using an average time of 5 s, a cell path
length of 0.1 cm, and a bandwidth of 1 nm. An ellipticity value at 222 nm ([?]222)
value of ?33,000 degrees cm2dmol?1was taken to correspond to 100% helix
(11). Thermal stability was determined by monitoring [?]222as a function of
temperature. Thermal melts were performed in two-degree intervals with a
2-min equilibration at the desired temperature and an integration time of 30 s.
All melts were reversible. Superimposable folding and unfolding curves were
observed, and ?90% of the signal was regained upon cooling. Temperatures of
midpoint unfolding transitions (Tm) were estimated by evaluating the maximum
of the first derivative of [?]222in relation to the temperature data (8). Equilib-
rium ultracentrifugation measurements were carried out on an XL-A analytical
ultracentrifuge equipped with an An-60 Ti rotor (Beckman Coulter, Fullerton,
CA) at 20°C as described previously (48). Protein solutions were dialyzed over-
night against PBS (pH 7.0), loaded at initial concentrations of 10, 30, and 100
?M, and analyzed at rotor speeds of 20,000 and 23,000 rpm. Data were acquired
at two wavelengths per rotor speed setting and processed simultaneously with a
nonlinear least-squares fitting routine (26). Solvent density and protein partial
specific volume were calculated according to solvent and protein composition,
respectively (31). The apparent molecular masses of all N36(L6)C34 variants
were within 10% of that calculated for an ideal trimer, with no systematic
deviation of the residuals.
In vitro selection of resistance to T1249. T1249-resistant
HIV-1 isolates were selected by repeated passage of HIV-1LAI
TABLE 1. HIV-1 evolution cultures
Input virus amino acid
gp41 amino acid
aUnderlining indicates the mutated nucleotide.
6680EGGINK ET AL.J. VIROL.
on the SupT1 T-cell line in the presence of increasing concen-
trations of T1249. We multiple independent cultures with dif-
ferent HIV-1 variants (V38 wild-type virus and T20-resistant
variants V38A, V38G, V38W, and V38W/N126K). Cultures
were started by transfection of the HIV-1LAImolecular clones
into SupT1 cells. T1249 was added at concentrations of 5 ng/ml
for the T20-sensitive wild-type virus (V38) and 20 ng/ml for all
T20-resistant variants, a drug level that is sufficient to reduce
virus replication by ?90%. Drug pressure was increased on
average 1.5-fold at each passage, and virus and cell samples
were stored at several time points. Viral replication was mon-
itored via CA-p24 measurements and the appearance of virus-
induced syncytia. Sequence analysis was performed on day 46
for cultures started with the wild-type virus and on day 33 for
FIG. 2. Replication of wild-type (WT) and escape viruses. (a) Viral replication curves of the indicated HIV-1 variants over an 8-day period.
SupT1 cells were transfected at day 0 with the molecular clones of variant viruses. (b) Replication of wild-type virus or molecular clones of variant
viruses in the presence of fusion inhibitor. The open-circle curves represent replication in the absence of fusion inhibitor; the other curves were
obtained with the indicated inhibitors. T1249 was tested at three concentrations. Shown are the results of a representative experiment; similar
results were obtained in three separate transfection experiments.
VOL. 82, 2008 T1249-RESISTANT HIV-16681
cultures started with the T20-resistant viruses (Table 1). The
wild-type and T20-resistant viruses replicated efficiently with
40 and 55 ng/ml T1249, respectively.
Genotypic analysis of T1249-resistant HIV-1. We PCR am-
plified the gp41 ectodomain from proviral DNA and analyzed
the sequence for acquired mutations. Sequence analysis of the
viral population revealed the acquisition of mutations within
the HR1 region, but no changes in HR2 were detected. Ten of
the 19 cultures acquired mutations at position 38, which is also
implicated in T20 and C34 resistance (one V38E mutant, two
A38E mutants, three G38R mutants, and four W38R mutants).
Interestingly, V38 and V38A evolve to E (observed three
times), whereas V38G and V38W (alone and combined with
N126K) evolve to R (observed seven times). This pattern is
governed by the underlying codon changes (Table 1; see Dis-
cussion for details). We also observed other gp41 changes:
Q79E (culture V5), K90E (culture V1), and N43K (in combi-
nation with V38A; culture A1). A double mutation, A38E/
A96T, was also observed in culture A2. Cultures V2, V4, V6,
W3, WK3, and WK4 did not reveal any mutational changes
within the gp41 ectodomain, suggesting that T1249 resistance
in these variants is caused by changes outside the gp41 ectodo-
main, for example in gp120. We constructed the HIV-1LAI
V38E, V38R, Q79E, K90E, and N43K mutants. The N43K
mutant was generated in the wild-type context, thus, without
the V38A substitution. Because of the common appearance of
charged amino acids at position 38, we also constructed a
V38K mutant, although we did not observe this mutant during
the evolution experiments.
Resistance and fitness of T1249-resistant HIV-1. We tested
the impact of the observed gp41 mutations on in vitro virus rep-
lication and resistance to T1249, T20, and C34. Viral DNA con-
structs were transfected into the SupT1 T-cell line and cultured in
in the absence of drug indicated that some variants have a re-
duced replication capacity (e.g., V38K, V38R, Q79E, and K90E
variants) (Fig. 2a). However, the V38E and N43K variants repli-
cated at levels similar to those of the wild type.
As expected, replication of the wild-type virus (V38) was
strongly inhibited by all inhibitors. In contrast, all HR1 variants
with substitutions at amino acid position 38 (V38E, V38R, and
V38K) showed resistance to T1249 and cross-resistance to T20
and C34. The V38E variant could replicate efficiently without
drug and at T1249 concentrations up to 25 ng/ml but was
inhibited at 100 ng/ml T1249. This mutant was fully resistant to
T20 and C34 at concentrations of 100 ng/ml. The V38R and
V38K variants were more resistant to T1249 than V38E and
fully resistant to 100 ng/ml of T20 and C34. The HR1 mutant
Q79E exhibited reduced replication capacity and showed mod-
est resistance to T1249 with slight cross-resistance to T20 and
C34. The K90E mutant with a substitution outside the HR1
region in the loop of gp41 displayed very low replication ca-
pacity but was able to replicate at low levels in the presence of
25 ng/ml T1249. In fact, levels of replication of the Q79E and
K90E variants were improved in the presence of drug, suggest-
ing a drug-stimulatory effect as described previously (3, 5).
N43K mutant virus has been observed in T20- and T1249-
treated patients (33, 38) and indeed had a low level of T20
resistance but no apparent T1249 or C34 resistance, although
we tested this mutant without V38A, the context in which it
was selected in the evolution experiment.
Infectivity and resistance in single-cycle infection experi-
ments. To quantitate the infectivity of the T1249 escape vari-
ants, we performed single-cycle infection assays (Fig. 3). We
also included our previously described T20-resistant V38A,
V38W, and V38G mutants (5) for comparison. Luciferase ac-
tivity in TZM-bl reporter cells was measured 2 days postinfec-
tion. Infectivity of HIV-1LAIwild-type virus was normalized to
100%, and the relative infectivities of the HIV-1LAImutants
were calculated. All mutants showed diminished infectivity,
with V38R, V38K, K90E, and Q79E mutants showing the most
profound infectivity defects. These results are generally in con-
cert with the replication capacities (Fig. 2).
We tested the susceptibility of wild-type virus to three spec-
tra of peptide inhibitors (Fig. 4a and Table 2). T20 inhibited
wild-type virus with an IC50of ?45 ng/ml. The IC50of T1249
was fourfold lower (?12 ng/ml), and the IC50of T2635 was
again fourfold lower (?3.3 ng/ml). We next established the
levels of T1249 resistance and cross-resistance to the first- and
third-generation fusion inhibitors T20 and T2635 of the in vitro
selected virus variants (Fig. 4b, c, and d and Table 2). All
mutants with an amino acid substitution at position 38 of gp41
showed high levels of resistance to T20, but only the T1249
escape mutants were resistant to T1249, with the V38E mutant
showing the highest level of resistance (24-fold) (Table 2). The
V38R mutant, which was selected in several evolution cultures,
and the newly constructed V38K mutant showed moderate
resistance to T1249 (6.7-fold and 2.9-fold, respectively). Again,
the N43K mutant did not confer T1249 resistance (in the
absence of V38A). The T20- and T1249-resistant V38 variants
showed no cross-resistance to T2635, and some (V38A, V38W,
V38G, and V38E mutants) appeared to be even more sensitive
to inhibition by T2635. Interestingly, the Q79E and K90E vari-
ants, which were somewhat resistant to T20 and T1249 (1.6-
and 4.0-fold to T20 and 3.4- and 3.0-fold to T1249, respec-
tively), were modestly resistant to T2635 (4.1-fold and 6.8-fold,
Biophysical properties of gp41 six-helix bundle variants.
During HIV-1 entry, the HR1 and HR2 segments in gp41
associate to form a highly thermostable six-helix bundle (Fig.
FIG. 3. Single-cycle infection assay of wild-type (WT) and escape
viruses. Relative infectivities compared to that of HIV-1LAIwere cal-
culated for all T1249 escape mutants. Luciferase activity without in-
hibitor of duplicate infections was measured in duplicate and corrected
for background luciferase activity. Infectivity of HIV-1LAIwas normal-
ized to 100%, and the relative infectivities of the other mutants were
6682 EGGINK ET AL.J. VIROL.
5b and c) (9, 34, 49, 52). The formation of this six-helix bundle
is thought to be mechanistically and thermodynamically cou-
pled to HIV-1 membrane fusion (18). Prior to bundle forma-
tion, a prehairpin intermediate containing the HR1 coiled-coil
trimer which is the target for the HR2-based inhibitors T20,
C34, T1249, and T2635 is present (18). T20 resistance muta-
tions in HR1 destabilize the six-helix bundle, suggesting that a
decreased T20-HR1 association is underlying the resistance
phenotype (5). To determine the effects of the T1249 resis-
tance mutations on the folding, stability, and conformation of
the gp41 core, we introduced each of the V38E, V38R, V38K,
and N43K substitutions into the soluble N36(L6)C34 six-helix
bundle model peptide that is formed by covalent attachment of
the N36 (HR1) and C34 (HR2) peptides by a short flexible
linker (Fig. 5a).
CD spectroscopy analysis was used to measure the extent of
FIG. 4. Inhibition of wild-type and T20 and T1249 escape viruses by T20, T1249, and T2635. Single-cycle infection experiments were performed
as described in Materials and Methods and the legend to Fig. 3 in the presence of a peptide concentration range. (a) Increased potencies of second-
and third-generation fusion inhibitors are shown. (b) Inhibition of wild-type and T20- or T1249-resistant viruses by T20, T1249, and T2635.
VOL. 82, 2008 T1249-RESISTANT HIV-16683
?-helical structure. Monitoring of the typical ellipticity at 222
nm indicated that the wild-type and T1249-resistant variants
contain ?90% helical structure (Table 3). Sedimentation equi-
librium experiments suggest that all peptides exist in a dis-
cretely trimeric state over a 10-fold protein concentration
range (10 to 100 ?M) (shown for V38E in Fig. 5d; Table 3). We
conclude that the introduction of T1249 resistance mutations
into the HR1 region of gp41 does not perturb the overall
folding and structure of the six-helix bundle, in agreement with
the essential role of bundle formation during fusion.
The thermal unfolding of each variant at 10 ?M protein
concentration was also monitored by CD. The sigmoidal tran-
sitions observed at 222 nm (Fig. 5e) indicate a cooperative
disruption of the helical structure with increasing temperature.
The midpoints (Tmvalues) of the transition of V38K, V38R,
V38E, and N43K are 74, 74, 72, and 78°C, respectively, com-
pared to a Tmof 80°C for the wild-type molecule, indicating
that the substitutions cause a slight destabilization of the six-
helix bundle (Table 3). Intriguingly, the pretransitional slopes
and the levels of steepness of the main transition differ greatly
for the wild-type bundle and its variants, indicating that the
introduction of the charged side chains at positions 38 and 43
alter the mechanism of thermal unfolding. We have not ob-
served this phenomenon with the neutral T20 resistance mu-
tations at position 38 (5).
Proteolytic degradation of gp41 six-helix bundle variants.
To probe structural consequences of the introduction of a
charged amino acid in HR1, we compared the sensitivities of
the N36(L6)C34 variants to proteolytic degradation by protein-
ase K (Fig. 6). Consistent with a lower thermal stability, the
V38K, V38R, and V38E variants exhibited increased sensitivity
to proteolysis compared to the wild-type peptide, as indicated
by the rapid disappearance of the undigested peptide band and
proteolytic products. In contrast, the N43K mutant shows a
slight decrease in protease sensitivity (Fig. 6). Moreover, the
proteolytic fragmentation patterns of the V38K, V38R, and
V38E peptides differs greatly from those of the wild-type and
N43K peptides. Digestion of these two peptides yields N36/
HR1 (residues 34 to 70) (observed mass, 4,083 Da; expected
mass, 4,082 Da) and C34/HR2 (residues 117 to 150) including
the N-terminal linker Ser-Gly-Gly-Arg-Gly-Gly (observed
mass, 4,788 Da; expected mass, 4,789 Da). Limited proteolysis
of V38K, V38R, and V38E variants generates N7(L6)C22,
spanning residues 64 to 70 (N7) and 117 to 138 (C22) con-
nected by the linker (observed mass, 4,115 Da; expected mass,
4,116 Da). These results indicate that the N-terminal end of
N36/HR1, which contains the charged residue at position 38, is
not properly folded and more susceptible to proteolysis. We
conclude that the T1249 resistance mutations V38K, V38R,
and V38E do not affect the overall formation of the gp41 core
structure but destabilize the six-helix bundle conformation.
In this study, we selected HIV-1 variants resistant to
T1249 in vitro. Interestingly, gp41 amino acid position 38,
implicated in T20 resistance, is also involved in T1249 resis-
tance. Although the interface of the peptide inhibitor with
HR2 is quite large, the resistance mutations primarily appear
at or near position 38. Because of the large contact surface, the
virus can perhaps easily compensate for point mutations, and
therefore mutations at the docking site of the peptide inhibitor
will have a more dramatic impact on peptide binding. The
LLSGIV stretch has been shown to be a critical docking site for
T20 (50), and this may explain the critical role of position 38 in
resistance development. Whereas T20 resistance is mediated
by hydrophobic and noncharged amino acid substitutions at
position 38 in HR1 (V38A, V38G, and V38W), T1249 resis-
tance appears to require charged amino acids (V38E, V38R,
and V38K). These results indicate that T20 and T1249 exhibit
very similar inhibition modes that call for similar but not iden-
tical mechanisms of resistance. T1249 is a more potent inhib-
itor than T20, likely due to its higher HR1 binding affinity. A
different gp41 amino acid substitution (involving charged res-
idues) is apparently needed to prevent T1249 binding as op-
posed to T20 binding. A randomized mutagenesis study that
focused on gp41 residues 37 and 38 previously showed the
importance of residue 38 in T1249 resistance (3, 5, 5a). Our
viral escape study, without an a priori bias for any specific
residue, confirms the importance of residue 38 but also dem-
onstrates that other changes can confer T1249 resistance. Be-
sides mutations at position 38, substitutions at the C terminus
of the HR1 domain (Q79E) and in the loop (K90E) were
found to cause resistance to T1249 as well.
In single-cycle infection experiments, we measured up to
24-fold T1249 resistance for the V38E mutant and lower levels
TABLE 2. Resistance in single cycle infection assaysa
T20 T1249 T2635
Viral resistance (n-fold)IC50(SD)
Viral resistance (n-fold)IC50(SD)
Viral resistance (n-fold)
aData were derived from the experiment whose results are shown in Fig. 4. WT, wild type.
bValues, presented in ng/ml, were calculated as described in Materials and Methods.
6684 EGGINK ET AL. J. VIROL.
FIG. 5. Biophysical characterization of T1249-resistant six-helix bundle variants. (a) Schematic representation of gp41 and the N36(L6)C34
peptide used for biophysical experiments. FP, fusion peptide. (b) Lateral (left) and axial (right) views of a six-helix bundle formed by the N36 (HR1;
green) and C34 (HR2; red) peptides (PDB accession no. 1AIK). The N termini of the N36 helices point toward the top of the page, and those
of the C34 helices point toward the bottom. (c) Helical wheel representations of N36 and C34. Heptad repeat positions are labeled “a” through
“g.” The “a” and “d” residues in the C peptide (red) interact with the exposed “e” and “g” side chains on the central N peptide coiled coil (green).
Position 38 is located at an “e” position. (d) Equilibrium sedimentation data (20,000 rpm) for the V38E mutant (30 ?M) collected in PBS (pH
7.0) at 20°C. The deviation in the data from the linear fit for a trimeric model is plotted (upper panel). (e) Thermal melting curves of wild-type
N36(L6)C34 (filled circles), V38K (open squares), V38R (open triangles), V38E (open circles), and N43K (open diamonds) variants monitored
by the CD signal at 222 nm in PBS (pH 7.0) at a peptide concentration of 10 ?M.
of resistance (3.0- to 6.7-fold) for the V38R, V38K, Q79E, and
K90E variants. The previously described T20-resistant V38A,
V38G, and V38W variants provided only a low level of T1249
resistance (2.1- to 2.7-fold) (Table 2). Interestingly, none of the
V38 variants provide cross-resistance to the third-generation
fusion inhibitor T2635. In fact, some position 38 variants were
found to be more susceptible to T2635 than the wild type was.
In contrast, the Q79E and K90E mutants exhibited modest
levels of resistance to all three spectra of peptide inhibitors.
These observations suggest that resistance to T2635 differs
mechanistically from T20 and T1249 resistance. While the po-
sition 38 substitutions directly affect the HR1-peptide interac-
tion, this is probably not the case for the Q79E and K90E
substitutions because they are located outside the actual pep-
tide binding site. Possibly they accelerate the HR1-HR2 asso-
ciation and thereby restrict the time frame in which the pep-
tides can act.
Similar to the HR1-T20/T1249 interaction, the HR1-HR2
interaction can be affected by the drug resistance mutations.
Indeed, as for the V38A T20-resistant mutant (5), a decrease
in melting temperature of the six-helix bundle was seen for the
V38E, V38R, and V38K variants. Consistent with these results,
limited proteolytic experiments reveal not only a decrease in
overall proteolysis resistance relative to that of the wild type
but also a major change in the proteolytic pattern. This sug-
gests that charged side chains at position 38 of gp41 perturb
the six-helix bundle structure more dramatically than non-
charged residues. Indeed, we measured a significantly destabi-
lized six-helix bundle, reduced infectivity, and delayed replica-
tion for the resistant variants.
We analyzed the gp41 sequences after only one month of
culturing under T1249 pressure. Upon prolonged culturing, we
expect that further evolution will take place. It is likely that
additional and/or compensatory mutations in gp41 or gp120
may provide further resistance to T1249 and/or improve viral
fitness. This possibility is currently under investigation.
We initiated our in vitro T1249 escape studies with wild-type
and T20-resistant virus variants. The input type of amino acid
38 appears to determine the outcome of evolution. Specifically,
the V38R variant was generated exclusively from the T20-
resistant V38G (three mutants) and V38W (four mutants)
variants, whereas 38E was derived exclusively from the V38
wild type (one mutant) and the T20-resitant V38A variant (two
mutants). Inspection of the underlying codon changes provides
a likely explanation (Table 1). Evolution of a 38E-encoding
codon is relatively easy starting from V38-encoding and 38A-
encoding codons (GTG3GAG and GCG3GAG, respec-
tively), which require only a single transversion (T-to-A and
C-to-A, respectively) (6, 27, 28). However, both codons require
(GTG3CGG or AGG for V38; GCG3CGG or AGG for
38A). Interestingly, the situation is reversed for the G38- and
W38-encoding codons, which prefer to evolve toward 38R. The
G38E change requires only a single transition (GGG3GAG),
but there are two simple routes toward R (GGG3CGG or
AGG). The GGG3AGG change was in fact seen exclusively
(three mutants), and it is linked to the most frequent G-to-A
mutation that is needed (6, 7, 27, 28). Starting with a 38W-
encoding codon also provides a route to a 38E-encoding codon
(TGG3GAG, a double mutation) that is more difficult than
that to 38R-encoding codon (TGG3CGG or AGG), two sin-
gle mutation routes, of which the transition type (T to C; three
mutants) is preferred over the transversion type (T to A; one
mutant). Thus, the mutational bias of HIV-1 determines the
precise evolution path toward drug resistance (28).
The findings reported here are of potential clinical relevance
as T20 therapy may trigger the selection of resistant viruses
that influence resistance development under subsequent T1249
therapy. Although the further clinical development of T1249
has been halted (23, 37), the same selection phenomenon may
occur with new entry inhibitors that use a similar mechanism of
action. However, our observation that T2635 is not affected by
T20 and T1249 resistance mutations at position 38 may dis-
prove this argument. The results of this study also underscore
the possibility that HIV-1 will lose fitness in the process of
FIG. 6. Proteolytic degradation of T1249-resistant six-helix bundle
variants. The proteinase K digestion products of wild-type (WT)
N36(L6)C34 and V38K, V38R, V38E, and N43K mutants are shown
(see Materials and Methods). Lanes 1 to 4, digestion after 0, 10, 20,
and 30 min, respectively. Samples containing 2.5 ?g of protein were
analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
on 16.5% Tricine gels with Coomassie blue staining. Proteolytic frag-
ments corresponding to the HR1 and HR2 regions are indicated.
TABLE 3. Summary of physicochemical analysis
aMobs/Mmonomeris the apparent molecular mass determined from sedimenta-
tion equilibrium data divided by the expected mass of a monomer.
6686 EGGINK ET AL.J. VIROL.
becoming resistant to potent fusion inhibitors, which may im-
pact disease progression. Newer fusion inhibitors, including
T2635, may reduce resistance development by a combination
of improved potency and loss of Env function upon the acqui-
sition of resistance. As such, the further development of this
class of antivirals is warranted.
We thank Trimeris and Roche for providing us with the T20 and
T1249 peptides. We are grateful to Ilja Bontjer and Stef Heynen for
This research was supported in part by grant number 2005021 from the
AIDS Fund (Amsterdam, The Netherlands) to B.B. and the National
Institutes of Health grant AI42382 to M.L. R.W.S. is a recipient of an
Anton Meelmeijer fellowship, a VENI fellowship from The Netherlands
amfAR Mathilde Krim research fellowship.
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