JOURNAL OF VIROLOGY, Oct. 2010, p. 9995–10003
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 19
The Effect of Clade-Specific Sequence Polymorphisms on HIV-1
Protease Activity and Inhibitor Resistance Pathways?
Rajintha M. Bandaranayake,1Madhavi Kolli,1Nancy M. King,1Ellen A. Nalivaika,1Annie Heroux,2
Junko Kakizawa,3Wataru Sugiura,3,4and Celia A. Schiffer1*
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street,
Worcester, Massachusetts 016051; Biology Department, Brookhaven National Laboratory, Upton, New York 11973-50002;
Laboratory of Therapeutic Research and Clinical Science, AIDS Research Center, National Institute of Infectious Diseases,
4-7-1 Gakuenn, Musashimurayama, Tokyo 208-0011, Japan3; and Department of Infection and Immunology,
Clinical Research Center, Nagoya Medical Center, Nagoya, Japan4
Received 6 March 2010/Accepted 14 July 2010
The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01_AE
(AE) strain is seen principally in Southeast Asia. AE protease differs by ?10% in amino acid sequence from
clade B protease and carries several naturally occurring polymorphisms that are associated with drug resis-
tance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation
in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N
and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and
NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations
and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than
clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may
lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and
DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser
extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement
in the flap hinge region compared with those of clade B protease and suggests insights into the alternative
pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can
alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.
Human immunodeficiency virus type 1 (HIV-1) is classified
into three groups (M, N, and O), of which group M is further
classified into nine major clades (A, B, C, D, F, G, H, J, and K)
and 43 circulating recombinant forms (CRFs) based on viral
genomic diversity (32, 37). The majority of HIV-1 infections
across the globe result from non-B clade HIV-1 variants; clade
B accounts for only ?12% of infections (15). However, the
development of currently available anti-HIV therapies has
been based on the virology of clade B variants. In recent years,
several studies have shown that there are clear differences
between clades when it comes to viral transmission and the
progression to AIDS, an observation which raises questions
about the effectiveness of the currently available anti-HIV
therapies against the other clades and CRFs (16–18, 39).
HIV-1 protease has been an important drug target in the
global effort to curb the progression from HIV infection to
AIDS. However, the accumulation of drug-resistant mutations
in the protease gene has been a major drawback in using
HIV-1 protease inhibitors. The effects of mutations associated
with drug resistance in HIV-1 clade B protease have been
studied extensively over the years. For the most part, resistance
mutation patterns are very similar in HIV-1 clade B and non-B
clade proteases (19). However, several alternative resistance
pathways have been observed for non-B clade proteases com-
pared with those of clade B protease (1, 12, 13, 26). Limited
data are available on how sequence polymorphisms, some of
which are associated with drug resistance in clade B protease,
might influence the pathway to drug resistance in non-B clade
proteases. Furthermore, very little is understood about how
sequence polymorphisms in non-B clade proteases affect pro-
tease function and inhibitor binding.
HIV-1 CRF01_AE (AE) was the first CRF to be observed in
patient populations and is seen principally in Southeast Asia
(2, 10, 25). AE protease differs by ?10% in amino acid se-
quence from that of clade B protease (Fig. 1A). Interestingly,
AE protease develops a different resistance pathway from that
of clade B protease to confer resistance to the protease inhib-
itor nelfinavir (NFV) (1). In patients infected with AE, the
protease acquires predominantly the N88S mutation in re-
sponse to NFV therapy, whereas in patients with clade B in-
fection, the protease acquires the D30N/N88D mutations. The
fitness of AE viral strains is thought to be similar to that of
HIV-1 group M viral strains (11, 41). However, the effect of
AE-specific sequence variations as well as drug resistance sub-
stitutions on viral fitness has not been studied extensively.
In the present study, biochemical and biophysical methods
were used to determine the effect of sequence polymorphisms
in AE protease on enzyme activity and inhibitor binding.
Through determination of crystal structures and analysis of
changes in hydrogen bonding patterns, a structural rationaliza-
tion is described for the two different pathways observed for
clade B and AE proteases to attain resistance to NFV.
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Pharmacology, University of Massachusetts Med-
ical School, 364 Plantation Street, Worcester, MA 01605. Phone: (508)
856-8008. Fax: (508) 856-6464. E-mail: Celia.Schiffer@umassmed.edu.
?Published ahead of print on 21 July 2010.
FIG. 1. (A) Amino acid sequence alignments of B-WT and AE-WT and NFV-resistant mutants. Residue positions that differ between clade
B and AE are indicated in red. NFV resistance mutations are indicated in blue. (B) Ribbon diagram superposition of DRVAE-WT(blue) and
DRVAE-N88S(gray). (C) Double-difference plot comparing DRVAE-WTand DRVAE-N88S. (D) Ribbon diagram superposition of clade DRVAE-WT
(magenta) and clade DRVB-WT(gray). (E) Double-difference plot comparing DRVAE-WTand DRVB-WT. The color contours in the double-
difference plots indicate distance differences of ?1.0 Å (black), 1.0 to 0.5 Å (green), 0.5 to 1.0 Å (blue), and ?1.0 Å (red).
MATERIALS AND METHODS
Protease gene construction. The clade B wild-type (B-WT) protease gene was
generated as previously described (34). The AE wild-type (AE-WT) protease
gene was synthesized in fragments (Integrated DNA Technologies, Coralville,
IA), with codons optimized for expression in Escherichia coli. The fragments
were ligated to form the complete gene, which was then inserted into the pET11a
expression vector (Novagen/EMD Chemicals, Gibbstown, NJ). The protease
sequence was confirmed by DNA sequencing. The NFV resistance mutations,
N88S in AE (AE-N88S) and D30N/N88D in clade B (B-D30N/N88D), were
generated by site-directed mutagenesis using a Stratagene QuikChange site-
directed mutagenesis kit (Agilent Technologies, La Jolla, CA). Mutagenesis was
confirmed by sequencing. The Q7K substitution was introduced to all protease
variants to prevent autoproteolysis (38).
Protein expression and purification. The clade B and AE variants were sub-
cloned into the heat-inducible pXC35 expression vector (American Type Culture
Collection [ATCC], Manassas, VA) and transformed into E. coli TAP-106 cells.
Protein overexpression, purification, and refolding were carried out as previously
described (20). Protein used for crystallographic studies was further purified with
a Pharmacia Superdex 75 fast-performance liquid chromatography column (GE
Healthcare, Chalfont St. Giles, United Kingdom) equilibrated with refolding
buffer (50 mM sodium acetate [pH 5.5], 10% glycerol, 5% ethylene glycol, and 5
Crystallization and structure determination. Protease solutions between 1.0
and 2.0 mg ml?1were equilibrated with a 5-fold molar excess of NFV, darunavir
(DRV), and amprenavir (APV) for 1 h on ice. Crystals were grown over a
reservoir solution consisting of 126 mM phosphate buffer (pH 6.2), 63 mM
sodium citrate, and 18% to 23% ammonium sulfate by the hanging-drop vapor
diffusion method. X-ray diffraction data for AE-WT were collected at a Bio-
CARS beamline 14-BM-C at the Advanced Photon Source (Argonne National
Laboratory, Argonne, IL) at a wavelength tuned to 0.9 Å with a Quantum 315
CCD X-ray detector (Area Detector Systems Corporation, Poway, CA). Diffrac-
tion data for AE-N88S were collected by using beamline X29A at the National
Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY) at a
wavelength tuned to 1.08 Å with a Quantum 315 charge-coupled-device (CCD)
X-ray detector (Area Detector Systems Corporation). Data for the B-D30N/
N88D variant was collected in-house with an R-Axis IV imaging plate system
(Rigaku Corporation, Tokyo, Japan) mounted on a rotating-anode X-ray source
(Rigaku Corporation). All data were collected under cryocooled conditions.
The data were indexed, integrated, and scaled using HKL/HKL-2000 software
(HKL Research, Charlottesville, VA) (29). Structure determination and refine-
ment were carried out using the CCP4 program suite (4) as previously described
(35). The tensor (T), libration (L), and screw (S) parameter files used in TLS
refinement were generated using the TLS motion determination server (30).
Model building and real-space refinement were carried out with Coot molecular
graphics software (8). Structure comparisons were made by superposing the
structures using the C? atoms of the terminal regions (residues 1 to 9 and 86 to
99) from the two monomers. In the case of the AE complexes, which have
multiple orientations for the inhibitor, only the orientation common with the
clade B structures was used for analysis. Structures were visualized using PyMol
molecular graphics software (6).
Double-difference plots were generated for AE and clade B protease struc-
tures to graphically visualize structural differences between the clades, as previ-
ously described (35). Briefly, distances between all C? atoms within the dimer
were calculated for each complex. A distance difference matrix was then com-
puted for each atom for a given pair of complexes. The distance difference matrix
was then plotted as a contour plot using the gnuplot plotting software (44).
Nomenclature. The following nomenclature format will be used to refer to
each crystal structure: inhibitorprotease variant. Thus, DRV in complex with AE-
WT, clade B-WT, AE-N88S, and AE-D30N/N88D protein are designated
DRVAE-WT, DRVB-WT, DRVAE-N88S, and DRVB-D30N/N88D, respectively. Prime
notation is used to distinguish the two monomers in the protease dimer. For
example, residue 30 from the first monomer would be referred to as Asp30, and
the same residue from the second monomer would be referred to as Asp30?.
ITC. Binding affinities and thermodynamic parameters of inhibitor binding to
clade B and AE variants were determined by isothermal titration calorimetry
(ITC) with a VP isothermal titration calorimeter (MicroCal, LLC, Northampton,
MA). The buffer used for all protease and inhibitor solutions consisted of 10 mM
sodium acetate (pH 5.0), 2% dimethyl sulfoxide, and 2 mM tris[2-carboxyethyl]
phosphine. Binding affinities for all protease variants were obtained by compet-
itive displacement titration using acetyl-pepstatin as the weaker binder. A solu-
tion of 30 to 45 ?M protease was titrated with 10-?l injections of 200 ?M
acetyl-pepstatin to saturation. The pepstatin was then displaced by titrating 36
8-?l injections of 200 ?M APV or NFV or 41 7-?l injections of 40 ?M DRV.
Heats of dilution were subtracted from the corresponding heats of reaction to
obtain the heat resulting solely from the binding of the ligand to the enzyme.
Data were processed and analyzed with the ITC data analysis module (Microcal)
for Origin 7 data analysis and graphing software (OriginLab, Northampton,
MA). Final results represent the average of at least two measurements.
Measurement of protease activity. Protease activity was assayed by following
each variant’s ability to hydrolyze the fluorogenic substrate HiLyte Fluor
488-Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala-Met-Ser-Lys (QXL-520) (AnaSpec,
Inc., Fremont, CA) that corresponds to the HIV-1 CA-p2 substrate. The CA-p2
cleavage site was used since it is conserved between HIV-1 clades (7). The assay
was carried out in a 96-well plate, and the enzymatic reaction was initiated by
adding 20 ?l of a solution of 100 to 250 nM protease to 80 ?l of substrate
solution. The buffer used in all reactions consisted of 10 mM sodium acetate (pH
5.0), 2% dimethyl sulfoxide, and 2 mM tris[2-carboxyethyl]phosphine. Final
concentrations in each experiment were 0 to 40 ?M substrate and 20 to 50 nM
protease. Accurate concentrations of properly folded active protease were de-
termined by carrying out ITC experiments for each variant with acetyl-pepstatin
as described in the previous section. Fluorogenic response to protease cleavage
was monitored at 23°C using a Victor3microplate reader (PerkinElmer, Wal-
tham, MA) by exciting the donor molecule at 485 nM and recording emitted light
at 535 nM. Data points were acquired every 30 s. The data points in relative
fluorescence units (RFU) were converted into concentrations using standard
calibration curves generated for HiLyte Fluor 488 at each substrate concentra-
tion. In addition to the conversion of RFUs to concentrations, the generation of
calibration curves at each substrate concentration allowed us to correct for the
inner filter effect (5). Rates of each enzymatic reaction were determined from the
linear portion of the data and were fitted against substrate concentrations to
determine Kmand catalytic turnover rate (kcat) values using VisualEnyzmics
enzyme-kinetics software (SoftZymics, Princeton, NJ). Final results for each
variant represent the average from at least two experiments.
In order to determine the biochemical fitness of a particular variant in the
presence of a given inhibitor, vitality values were calculated using the following
equation, based on the vitality function described previously, where Kdis the
dissociation constant and kcat/Kmis the catalytic efficiency (14, 43).
?Kd? ?kcat/Km??clade B-WT
The calculated vitality value for B-WT for a particular inhibitor would be 1.0, and
vitality values greater than 1.0 would indicate that a given variant had a selective
advantage over the same inhibitor, while values lower than 1.0 would indicate
that the variant did not have a selective advantage.
Crystal structures. The AE-WT and NFV-resistant clade B
and AE variants were cocrystallized with NFV, DRV, and
APV to reveal the structural basis for the altered NFV resis-
tance pathways. In addition, the effects of background poly-
morphisms in AE-WT on inhibitor binding compared with that
of clade B-WT were discerned. Crystals of AE protease in
complex with NFV and APV did not diffract to a high
resolution; therefore, structural comparisons were carried
out for AE and clade B protease in complex with DRV. The
structure of DRVB-WTwas solved previously in the laboratory
and was used for structural comparisons (Protein Data Bank
[PDB] code 1T3R). Both DRVAE-WTand DRVAE-N88Scrys-
tallized with DRV bound in two orientations in the active
site. Crystallographic data and refinement statistics for
DRVAE-WT, DRVB-WT, DRVAE-N88S, and DRVB-D30N/N88D
are given in Table 1.
Structural comparisons were carried out for AE and clade B
DRV complexes by pairwise structural superposition and
double-difference plots (Fig. 1B to E). The DRVAE-WTand
DRVAE-N88Scomplexes were structurally similar (Fig. 1B and C).
Although the DRVAE-WTand DRVB-WTcould be superim-
posed on each other very well (root mean square deviation
VOL. 84, 2010HIV-1 PROTEASE, POLYMORPHISMS, AND RESISTANCE PATHWAYS9997
[RMSD] of 0.21 Å), there were clear and significant differences
between the variants in the main chain at the flap hinge region
(residues 33 to 39) and the protease core region (residues 16 to
22) (Fig. 1D and Fig. 2A to D). These differences were further
evident by the presence of significant peaks in the double-
difference plot (Fig. 1E). The Ile36 side chain in DRVAE-WT
packs well against the core region through favorable van der
Waals interactions and is shorter than the Met36 in DRVB-WT
(Fig. 2C). In addition, the shorter Asp35 in DRVAE-WTfurther
enhances the packing by being flipped inward against the core,
while in DRVB-WT, the longer Glu35 is flipped outward into
the solvent and forms a salt bridge with Arg57 (Fig. 2D). The
packing of the flap hinge and core regions in DRVAE-WTis
further stabilized by a hydrogen bond between the carbonyl
oxygen of Asp35 and Lys20 NZ atom and is not present in
The Asp30? side chain of DRVB-WTdoes not directly form a
hydrogen bond with DRV but indirectly interacts with the N1
atom of DRV through a water molecule-mediated hydrogen
bond network (Fig. 3A). In contrast, the Asp30? side chain of
DRVAE-WTforms a direct hydrogen bond with the N1 atom of
DRV (Fig. 3B). Residue 30 of both NFV-resistant variants also
interacts with the N1 atom of DRV through water molecule-
mediated hydrogen bonding (Fig. 3C and D). However, in
addition to this interaction, Asn30 of DRVB-D30N/N88Dand
Asp30 of DRVAE-N88Sare oriented away from the active site,
enabling them to form hydrogen bonds with Asp88 and
Ser88, respectively. In both cases, the NFV resistance mu-
tations stabilize residue 30 away from the active site via
Binding thermodynamics. To determine the effects of back-
ground sequence polymorphisms and NFV resistance muta-
tions on inhibitor binding, the binding thermodynamic param-
eters of NFV, DRV, and APV binding to WT and resistant AE
and clade B variants were determined by isothermal titration
calorimetry (Table 2). The AE-WT protease had a 6.9-fold-
weaker affinity for NFV and a 2.7-fold-weaker affinity for DRV
TABLE 1. Crystallographic statistics
Result for indicated variant
Cell dimensions (Å)
Total no. of
No. of unique
55,05610,326 12,493 17,277
Bond length (Å)
aZ, number of molecules in the unit cell; RMSD, root mean square deviation;
Rsymm? ?hkl?Ihkl? ?Ihkl??/?hklIhkl; I/?I, signal-to-noise ratio; Rwork? ??Fobs?
Fcal?/?Fobs; Rfree? ?test(?Fobs? ? Fcal?)2/?test?Fobs?2.
bSee King et al.(21) and Surleraux et al. (40).
FIG. 2. (A) Ribbon diagram superposition of DRVAE-WT(blue) and DRVB-WT(gray). The red box indicates the region of the protease
molecule highlighted in panels B to D. (B) Structural rearrangement of the flap hinge and core regions between DRVAE-WT(blue) and DRVB-WT
(gray). (C) Flap hinge and core regions of DRVAE-WT. (D) Flap hinge and core regions of DRVB-WTprotease. Hydrogen bond interactions are
indicated by red dashed lines.
9998BANDARANAYAKE ET AL. J. VIROL.
than the affinities of B-WT protease for NFV and DRV, re-
spectively (Table 2). This result indicates that the AE-WT
protease has an inherently weaker affinity for NFV and DRV.
No significant differences in the enthalpy of NFV binding
were observed among any of the variants. Although the bind-
ing of DRV to all protease variants was enthalpically favorable,
the enthalpic contributions were reduced with the AE variants
(?10.1 kcal mol?1for AE-WT and ?5.1 kcal mol?1for AE-
FIG. 3. Protease inhibitor hydrogen bonding interactions. DRV is shown in orange, and hydrogen bond interactions are indicated by red
dashed lines. Since the charged states of the Asp25 carboxyl groups and the position of the O18 hydroxyl hydrogen of DRV are not known,
all possible hydrogen bond interactions between the Asp25 carboxyl groups and O18 of the DRV molecules are shown. (A) DRVB-WT(gray).
(B) DRVAE-WT(blue). (C) DRVB-D30N/N88D(salmon). (D) DRVAE-N88S(green).
TABLE 2. Binding thermodynamic parameters for NFV, DRV, and APV binding to AE and clade B variantsa
?H (kcal mol?1)
?T?S (kcal mol?1)
?G (kcal mol?1)
(2.6 ? 0.5) ? 109
(1.2 ? 0.4) ? 108
(3.7 ? 1.0) ? 108
(5.8 ? 1.2) ? 107
0.39 ? 0.07
8.1 ? 2.8
2.7 ? 0.7
17.2 ? 3.5
4.4 ? 0.1
6.7 ? 0.3
5.0 ? 0.3
6.2 ? 0.7
?12.6 ? 0.1
?10.8 ? 0.2
?11.5 ? 0.2
?10.4 ? 0.1
(2.2 ? 1.1) ? 1011
(3.7 ? 0.7) ? 1010
(9.1 ? 0.3) ? 1010
(1.1 ? 0.8) ? 1010
0.004 ? 0.002
0.026 ? 0.005
0.0109 ? 0.0003
0.087 ? 0.062
?12.1 ? 0.9
?12.5 ? 0.4
?10.1 ? 0.5
?5.1 ? 3.6
?15.2 ? 0.3
?14.2 ? 0.1
?14.7 ? 0.02
?13.5 ? 0.4
(2.6 ? 1.3) ? 109
(1.2 ? 0.2) ? 1010
(3.1 ? 0.2) ? 109
(1.3 ? 0.9) ? 1010
0.39 ? 0.20
0.08 ? 0.01
0.32 ? 0.02
0.08 ? 0.06
?7.3 ? 0.9
?10.2 ? 1.5
?5.5 ? 0.3
?5.0 ? 3.6
?12.6 ? 0.3
?13.5 ? 0.09
?12.70 ? 0.03
?13.6 ? 0.4
aKa, association constant; Kd, dissociation constant; H, enthalpy; T, temperature; S, entropy; G, Gibbs free energy.
VOL. 84, 2010HIV-1 PROTEASE, POLYMORPHISMS, AND RESISTANCE PATHWAYS9999
N88S) compared with those for the clade B variants (?12.1
kcal mol?1for B-WT and ?12.5 kcal mol?1for B-D30N/
N88D). As expected, the NFV-resistant variants showed a sig-
nificant reduction in binding affinity for NFV compared to that
of the wild-type variants. With the AE-N88S variant, the af-
finity for NFV was reduced 44.1-fold (Kd? 17.2 nM) and was
far more significant than the D30N/N88D mutations in clade B
protease, which reduced the affinity for NFV 20.7-fold (Kd?
8.1 nM). Similarly, the AE-N88S variant had a 21.8-fold-
weaker affinity (Kd? 0.087 nM) for DRV compared to a
6.5-fold-weaker affinity (Kd? 0.026 nM) with the B-D30N/
N88D variant. Thus, the single N88S substitution in the AE
protease has a profound effect on the binding of NFV and
In contrast to NFV and DRV, clade-specific sequence dif-
ferences and NFV resistance mutations had only a minimal
effect on the affinities for APV of both AE and clade B pro-
tease. Despite this, there were some differences in energy pa-
rameters. The binding of APV to the clade B variants appeared
to be more enthalpically favorable than that to the AE variants.
This was compensated for by an increase in the entropic com-
ponent to the binding energy for the AE proteases.
Protease activity and vitality. The enzyme-kinetic parame-
ters determined for each clade B and AE variant with the
CA-p2 fluorogenic substrate analog are summarized in Table
3. The Kmvalue for B-D30N/N88D protease (35.9 ?M) was
2.1-fold greater than that for B-WT protease (16.7 ?M). How-
ever, the Kmvalues for the AE protease variants (17.5 ?M for
AE-WT and 19.0 ?M for AE-N88S) were similar to that of
B-WT protease. The turnover rate for B-D30N/N88D protease
(kcat? 0.13 s?1) was significantly lower than that of B-WT
protease (kcat? 1.79 s?1). Turnover rates for AE-WT (kcat?
0.7 s?1) and AE-N88S (kcat? 0.2 s?1) were 2.5 and 8.5-fold
lower, respectively, than that of clade B-WT. The kcat/Kmval-
ues, or catalytic efficiency values, for B-D30N/N88D and AE
variants were lower than that of B-WT protease. Therefore,
the reduction in catalytic efficiency of the B-D30N/N88D pro-
tease compared with that of B-WT protease resulted from the
combined effects of the Kmand kcatvalues. However, for the
AE variants, the lower turnover rates alone were responsible
for the reduced catalytic efficiencies. Overall, these results in-
dicate that the polymorphic sequence differences in AE pro-
tease can alter the activity profile of the enzyme compared to
results with the clade B protease.
Vitality values were calculated to determine if the protease
variants had a selective advantage over NFV, DRV, and APV.
AE-WT and AE-N88S protease had calculated vitality values
of 2.52 and 4.01 for NFV, respectively, compared with 0.76 for
B-WT (Table 4). However, vitality values for DRV were not
significantly different from that of B-WT protease. Vitality
values for APV were significantly lower for all variants than for
B-WT protease. These results indicate that AE-WT may have
a selective advantage over NFV compared to B-WT but that
the AE variants may not have a significant selective advantage
against DRV or APV relative to B-WT.
Although the majority of HIV-1 patients are infected with
non-B forms of the virus, molecular studies have been carried
out predominantly with clade B variants. The AE protease has
several polymorphisms that are associated with inhibitor resis-
tance in clade B. AE also shows altered patterns of drug re-
sistance to NFV. We have performed detailed studies to de-
termine the effects of sequence polymorphisms on enzyme
structure, activity, and inhibitor binding. These analyses led to
a structural rationalization for the altered pathways for drug
AE-WT protease has an inherently weaker affinity for NFV
and DRV than that of B-WT, as is evident from the thermo-
dynamic data (Table 2). The weaker affinity observed for NFV
is consistent with previously published data for another AE
protease variant (3), as well as for clade A protease (42), which
is closely related. The inherent weaker affinity for NFV likely
allows the AE protease to gain resistance to NFV through a
single nonactive-site substitution, N88S. The clade B protease,
in contrast, which has a relatively stronger affinity for NFV,
requires a combination of an active-site mutation (D30N) and
a nonactive-site mutation (N88D) to gain NFV resistance. The
ability of the AE-N88S protease to maintain affinity for sub-
strates is evident from our enzyme kinetics data (Table 4), in
which the Kmvalue for AE-N88S was comparable to that of
AE-WT and B-WT protease. The Kmvalue for clade B-D30N/
N88D, on the other hand, was significantly worse than that of
the B-WT, likely reflecting the effect of the altered active site.
As an active-site residue, Asp30 plays a key role in substrate
recognition by interacting with substrates through side chain-
mediated hydrogen bonds with the MA-CA, CA-p2, p1-p6, and
p2-NC cleavage sites (36). Therefore, as is evident from our
enzyme kinetics data, the D30N/N88D mutations in clade B
will likely affect substrate binding and processing. Several stud-
ies have observed substrate coevolution in instances in which
the protease mutates active-site residues in order to confer
inhibitor resistance (22, 23). However, since the AE-N88S pro-
tease variant has no active-site mutations, the enzyme retains
the ability to effectively recognize substrates while conferring
NFV resistance. Therefore, the presence of the N88S sub-
stitution in AE protease is unlikely to induce coevolution of
TABLE 3. Enzyme kinetics parameters for clade B and AE-WT
and NFV-resistant variants
Result for indicated variant
16.7 ? 6.0
1.79 ? 0.28
0.11 ? 0.04 0.004 ? 0.002 0.04 ? 0.01 0.010 ? 0.001
35.9 ? 0.1
0.13 ? 0.09
17.5 ? 4.0
0.70 ? 0.08
19.0 ? 0.8
0.20 ? 0.02
TABLE 4. Vitality values for clade B and AE WT and
Result for indicated variant
10000 BANDARANAYAKE ET AL. J. VIROL.
the viral substrates in order to maintain effective enzymatic
Despite having Kmvalues that were comparable to that of
B-WT protease, both AE-WT and AE-N88S had significantly
lower catalytic turnover rates (kcat) than that of the B-WT
protease (Table 3). As a result, the catalytic efficiency of the
AE variants is lower than that of the B-WT protease. The
lower turnover rates of the AE variants could be a direct result
of the reduced flexibility of the flap hinge (residues 33 to 39)
and core regions (residues 16 to 22) of the protein. Molecular
dynamics studies have revealed that hydrophobic sliding of the
core region facilitates substrate binding through the opening of
the active site (9). The unique hydrogen bonds observed be-
tween the flap hinge and the core in the AE variants alter
movement of the core, thus impacting the ability of the active
site to open up for substrate binding and product release.
Based on our enzyme kinetics data, this altered flexibility of the
flap hinges in the AE variants has little effect on substrate
binding but rather affects the catalytic step of the reaction by
slowing down product release.
The higher vitality value observed for AE-WT with NFV
provides supporting evidence for the reduction in the efficacy
of NFV against the AE protease compared with that of clade
B (Table 4). This result is consistent with previous vitality
calculations for the clade A protease (42). In addition, these
results further highlight the idea that background polymorphic
sequence variations in the AE protease can affect the potency
of NFV. The suboptimal efficacy of NFV against the AE-WT
protease likely permits a nonactive-site variant, AE-N88S, to
emerge over variants with active-site mutations to effectively
confer resistance to NFV.
The impact on other inhibitors, however, is complex. APV
and DRV are chemically very closely related compounds, and
similar susceptibility and resistance patterns have been ob-
served for these two inhibitors (31). However, this pattern is
not evident for this series of resistant variants. Both the N88S
mutation in the AE and the D30N/N88D mutations in the
clade B proteases result in hypersusceptibility to APV. Similar
results have been observed also for a B-N88S protease variant
(24, 45). In contrast, the same substitutions in the protease give
rise to even greater resistance to DRV. However, since DRV
presents a greater genetic barrier to resistance than APV (33),
the in vivo implications of weaker affinity for DRV in the AE
variants are likely negligible. Indeed, our calculated vitality
values indicate that DRV maintains its potency against the AE
variants despite having a weaker affinity for AE-WT and AE-
N88S relative to clade B protease.
A close look at the NFVB-WTprotease complex reveals an
important interaction between the Asp30 residue side chain
and the inhibitor bound in the active site. (PDB code 3EKX)
(Fig. 4A and B). One of the side chain oxygen atoms of Asp30
forms a direct hydrogen bond with the O38 atom of NFV. Our
crystal structures of the NFV-resistant variants show that N88S
in AE and N88D in clade B have the ability to interact with
residue 30 and orient it away from the active site (Fig. 3B and
D) and thereby disrupt the interaction between residue 30 and
the inhibitor. These structural observations are similar to in-
terpretations made in previous molecular dynamics studies
involving NFV-protease complexes (27, 28). Thus, NFV resis-
tance is likely caused in large part due to the loss of this
interaction in the NFV-resistant variants.
Overall, mutations that emerge in response to inhibitor ther-
FIG. 4. Hydrogen bond network involving residue 88. (A) Asn88 bridges the terminal helix with Asp30 from the active site and Thr74 from one
of the outer beta strands. The red box indicates the region of the protease molecule highlighted in panels B to D. (B) Asn88 in NFVB-WT(PDB
code 3EKX). (C) Asp88 in DRVB-D30N/N88D. (D) Ser88 in DRVN88S-WT. Hydrogen bond interactions are indicated by red dashed lines.
VOL. 84, 2010HIV-1 PROTEASE, POLYMORPHISMS, AND RESISTANCE PATHWAYS10001
apy need to have a minimal impact on protease structure and
activity to maintain the enzyme’s function. The D30N substi-
tution, which is associated with NFV resistance, is one of the
few drug-resistant mutations that involve a change in charge.
The additional substitution of N88D likely helps preserve the
net charge on the protein. In AE, resistance to NFV occurs
indirectly with the N88S mutation. Likewise, the sole NFV-
resistant alteration, N88S, in the AE protease does not change
the overall electrostatics. Thus, in both clade B and AE, NFV
resistance is attained with no change to the net charge of the
enzyme. In the wild-type variants, Asn88 is one of the few
internal hydrogen bonding side chains in the core of the pro-
tease monomer. The side chain of Asn88 has a key role in the
protease structure bridging the terminal helix, with residues 30
and 31 coming from the active site to the backbone of Thr74 in
the center of one of the outer beta strands (Fig. 4A and B).
With the substitutions of Asp in clade B and Ser in AE for Asn
at position 88 in the NFV-resistant protease variants, the hy-
drogen bonding network is preserved through the coordination
of some key water molecules in the core of the protease mono-
mer (Fig. 4B to D). Thus, mutations confer resistance to NFV
through a series of interdependent changes that preserve the
structural and electrostatic properties of HIV-1 protease.
In conclusion, protease activity and the response to protease
inhibitors can be affected by clade-specific sequence differ-
ences. Our findings likely extend beyond HIV-1 protease to
other drug targets within HIV and underscore the need to
consider clade-specific polymorphisms when developing new
drugs and formulating treatment plans. Furthermore, drug re-
sistance pathways observed in the context of clade B viruses
cannot be assumed to hold true for other HIV-1 clades.
This work was supported by grants from the National Institutes of
Health (P01-GM66524) and Tibotec, Inc., to C.A.S. Additionally, this
study was supported by a Grant-in-Aid for AIDS research from the
Ministry of Health, Labor, and Welfare of Japan (H19-AIDS-007)
We thank William Royer, Moses Prabu-Jayabalan, and Madhavi
Nalam for helpful discussions and Christina Ng and Brendan Hilbert
for assistance with data collection.
We gratefully acknowledge the Mail-In Data Collection Program of
the National Synchrotron Light Source, Brookhaven National Labo-
ratory, for collecting X-ray data at the X29A beamline, for which
financial support comes principally from the Offices of Biological and
Environmental Research and of Basic Energy Sciences of the U.S.
Department of Energy and from the National Center for Research
Resources of the National Institutes of Health. Use of the Advanced
Photon Source for X-ray data collection was supported by the U.S.
Department of Energy, Basic Energy Sciences, Office of Science, under
contract DE-AC02-06CH11357. Use of the BioCARS Sector 14 was
supported by the National Institutes of Health, National Center for
Research Resources, under grant RR007707.
The protease inhibitors used in this study were obtained through the
NIH AIDS Research and Reference Reagent Program, Division of
AIDS, National Institute of Allergy and Infectious Diseases, NIH.
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