Tài liệu Báo cáo khoa học: Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sites pdf

Effect of sequence polymorphism and drug resistance on two HIV-1
Gag processing sites
Anita Fehe
´
r
1
, Irene T. Weber
2
,Pe
´
ter Bagossi
1
,Pe
´
ter Boross
1
, Bhuvaneshwari Mahalingam
2
,
John M. Louis
3
, Terry D. Copeland
4
, Ivan Y. Torshin
5
, Robert W. Harrison
5
and Jo
´
zsef To¨ zse
´

r
1
1
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary;
2
Department of
Biology, Georgia State University, Atlanta, GA, USA;
3
Laboratory of Chemical Physics, National Institute of Diabetes,
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA;
4
NCI-Frederick, Frederick, MD, USA;
5
Department of Computer Science, Georgia State University, Atlanta, GA, USA
The HIV-1 proteinase (PR) has proved to be a good target
for antiretroviral therapy of AIDS, and various PR inhibi-
tors are now in clinical use. However, there is a rapid selec-
tion of viral variants bearing mutations in the proteinase that
are resistant to clinical inhibitors. Drug resistance also
involves mutations of the nucleocapsid/p1 and p1/p6 clea-
vage sites of Gag, both in vitro and in vivo. Cleavages at these
sites have been shown to be rate limiting steps for polypro-
tein processing and viral maturation. Furthermore, these
sites show significant sequence polymorphism, which also
may have an impact on virion infectivity. We have studied
the hydrolysis of oligopeptides representing these cleavage
sites with representative mutations found as natural varia-
tions or that arise as resistant mutations. Wild-type and five
drug resistant PRs with mutations within or outside the
substrate binding site were tested. While the natural varia-

tions showed either increased or decreased susceptibility of
peptides toward the proteinases, the resistant mutations
always had a beneficial effect on catalytic efficiency. Com-
parison of the specificity changes obtained for the various
substrates suggested that the maximization of the van der
Waals contacts between substrate and PR is the major
determinant of specificity: the same effect is crucial for
inhibitor potency. The natural nucleocapsid/p1 and p1/p6
sites do not appear to be optimized for rapid hydrolysis.
Hence, mutation of these rate limiting cleavage sites can
partly compensate for the reduced catalytic activity of drug
resistant mutant HIV-1 proteinases.
Keywords: HIV-1 proteinase; Gag processing sites; oligo-
peptide substrates; substrate specificity; molecular modeling.
All replication competent retroviruses code for an aspartic
proteinase (PR) whose function is critical for virion
replication (reviewed in [1]). The HIV-1 PR has proved to
be an excellent target for antiretroviral therapy of AIDS,
and various PR inhibitors are now in clinical use (reviewed
in [2]). However, as observed with reverse transcriptase
inhibitors, resistant viruses rapidly emerge in PR inhibitor
therapy. Moderate to high level of resistance (2- to 100-fold)
to PR inhibitors has been observed both in vitro and in vivo,
and has been attributed to the appearance of mutations in
the PR gene. Many of these mutations are located in the
substrate binding site of the PR, and these mutations
have considerable impact on PR activity and specificity.
Other resistant mutations alter residues outside of the
substrate binding site. The compromised catalytic capability
of the multiple drug resistant HIV-1 mutants is reflected by

impaired processing of Gag precursors in PR-mutated
virions [3,4] and by decreased in vitro catalytic efficiency of
the PR towards peptides representing natural cleavage sites
[5–8]. The development of high levels of resistance to PR
inhibitors, possibly requiring multiple mutations in the PR,
was therefore expected to be limited by the functional
constraints of the enzyme, which must cleave all precursor
cleavage sites during viral replication. Subsequently, a
second locus was found to be involved in drug resistance
to HIV PR inhibitors, both in vitro and in vivo,atthe
nucleocapsid (NC)/p1 and p1/p6 cleavage sites [9–14].
Evolution of PR cleavage sites other than NC/p1 and
p1/p6 in the internal (P2-P2¢) positions is limited, and
mutations are rarely observed even upon drug treatment
[12]. Cleavage at these sites appears to be a rate limiting step
in polyprotein processing [9,11]. Peptides representing these
sites have the lowest specificity constants (k
cat
/K
m
) among
all HIV-1 cleavage sites [15,16]. Furthermore, there is a
significant sequence polymorphism at these sites, which also
may have an impact on virion infectivity [17–19]. Natural
polymorphism and resistant mutations occurring at these
sites are shown in Fig. 1. Some of these amino acid
substitutions are frequently detected, others have been
found only in one clone including the P1 Asp, Ile and Lys
substituted NC/p1 sites, which are not expected to be
cleaved by the PR, based on previous extensive specificity

studies [20]. If cleavage at this site is important for virus
replication, as indicated by the mutations seen in resistance,
Correspondence to J. To
¨
zse
´
r, Department of Biochemistry and
Molecular Biology, Faculty of Medicine, University of Debrecen,
H-4012 Debrecen, PO Box 6, Hungary.
Fax: + 36 52 314989, Tel.: + 36 52 416432,
E-mail:
Abbreviations:MA,matrixprotein;CA,capsidprotein;NC,nucleo-
capsid protein; PR, proteinase.
Enzyme: retropepsin (EC 3.4.23.16).
Note: nomenclature of viral proteins is according to Leis et al. [50].
(Received 9 May 2002, revised 8 July 2002, accepted 11 July 2002)
Eur. J. Biochem. 269, 4114–4120 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03105.x
clones containing these Gag mutations should be replication
defective. Mutations outside of the P4-P3¢ region have also
been reported at these sites in PR inhibitor therapy [12,21].
However, those residues of a substrate are far from the
substrate binding subsites and are not expected to alter
the PR specificity directly, but they may alter the confor-
mation of the substrate region within the polyprotein or
may enhance viral fitness in a manner unrelated to the
PR-mediated processing rates.
Here we report kinetic studies using oligopeptides repre-
senting cleavage sites with representative, frequently occur-
ring mutations found as sequence polymorphisms and
in drug resistance, with wild-type and five drug-resistant

mutant PRs: M46L, V82A, I84V and L90M mutations that
appeared together with the Gag mutations in vivo,in
PR-inhibitor therapy [10,12]. V82S mutation was found in
ritonavir therapy [22]. I84V was found to be in vitro selected
against saquinavir and amprenavir [18], together with Gag
mutations against two BILA inhibitors [23], and in patients
treated with ritonavir [22] or indinavir [24]. Based on clinical
studies, the resistant Gag mutations may occur early in PR
inhibitor therapy, soon after the appearance of one (or a
few) critical PR mutations, therefore we have characterized
the effect of single protease mutations in response to Gag
cleavage site mutations. A similar approach could be used to
characterize the more complex inhibitor-resistant proteases
harboring all mutations appearing in vitro or in vivo in the
presence of PR inhibitors.
MATERIALS AND METHODS
Oligopeptide synthesis and characterization
Oligopeptides were synthesized by standard 9-fluorenyl-
methyloxycarbonyl chemistry on a model 430A automated
peptide synthesizer (Applied Biosystems, Inc.). All peptides
were synthesized with an amide end. Amino-acid compo-
sition of the peptides was determined with a Beckman 6300
amino-acid analyzer. Stock solutions and dilutions were
made in distilled water and the peptide concentrations were
determined by amino-acid analysis.
Construction and purification of HIV-1 PR mutants
HIV-1 PR (HIVHXB2CG) having stabilizing substitutions
(Q7K, L33I, L63I, C67A and C95A) was cloned into a pET
vector, expressed in Escherichia coli and purified to homo-
geneity as described [25]. The proteolytic activity of this

enzyme, designated as wild-type, was indistinguishable from
that of the native PR [25]. DNA derived from this clone was
used as a template for generating the mutant enzymes by site
directed mutagenesis. Mutations were confirmed by nucleic
acid sequencing and protein mass spectrometry. The mutant
enzymes were purified as described [26].
Enzyme assay with oligopeptide substrates
The PR assays were initiated by the mixing of 5 lL(0.05–
8 l
M
) purified wild-type or mutant HIV-1 PR with 10 lL
2x incubation buffer (0.5
M
potassium phosphate buffer,
pH 5.6, containing 10% glycerol, 2 m
M
EDTA, 10 m
M
dithiothreitol, 4
M
NaCl) and 5 lL0.5–7 m
M
substrate. The
reaction mixture was incubated at 37 °Cfor1hand
terminated by the addition of 180 lL 1% trifluoroacetic
acid. Substrates and the cleavage products were separated
using a reversed-phase HPLC method described previously
[15]. Kinetic parameters were determined by fitting the
data obtained at less than 20% substrate hydrolysis to the
Michaelis–Menten equation by using the

FIG
.
P
program
(Fig. P Software Corp.). For the NC/p1 peptide series the
specificity constants (k
cat
/K
m
) were determined by using
competitive assays [27] with substrate KTGVL-fl-VVQPK
[28], while for the p1/p6 peptide series, it was determined
under pseudo first order conditions using 0.02–0.15 m
M
concentration range, in which the activity was a linear
function of the substrate concentration. The standard error
for kinetic values was below 20%.
Active site titration
The active amount of enzyme used in the assays was
determined by active site titration using the potent HIV-1
PR inhibitor DMP-323 for the wild-type PR and for the
M46L, V82A, I84V, L90M mutants, while the V82S mutant
was titrated using amprenavir. Active site titrations were
performed by using the HPLC method [15], except 0.2 lL
aliquot of the inhibitor (0–10 l
M
) in dimethylsulfoxide was
added to the reaction mixture. The inhibition curves were
determined at three substrate concentrations, and the active
enzyme concentration was determined using the

DYNAFIT
program [29]. The standard error for the enzyme concen-
trations was below 20%.
Molecular modeling
All models were built from the high resolution crystal struc-
ture (PDB entry 1fgc) of HIV-1 PR-inhibitor complex
[26] by altering the appropriate residues of enzyme and
Fig. 1. Sequence around the NC/p1 and p1/p6 cleavage sites in HIV-1.
The sequence of HIV-1
IIIB
, a member of the B subtype, is shown. The
P4-P3¢ region of the cleavage site sequences are marked with an upper
line, these residues are expected to bind in the S4-S3¢ substrate binding
subsites of the enzyme. Residues appearing in other HIV-1 virus iso-
lates [18] are indicated under the sequence of HIV-1
IIIB
, while residues
appearing only in drug resistance are in bold [9–13]. Mutations
underlinedintheFigurealsoappearedindrugresistanceonapar-
ticular natural sequence background, however, the same residues have
also been found in other natural sequences [18], therefore these
mutations are considered to be due to sequence polymorphism, as they
can be observed without the selective pressure caused by the PR
inhibitors.
Ó FEBS 2002 Studies on mutant HIV-1 processing sites (Eur. J. Biochem. 269) 4115
inhibitor. All water molecules in the crystal structure were
used. A proton was placed between the carboxylate oxygens
of catalytic aspartates D25 and D25¢, as described previ-
ously [30]. Minimization with
AMMP

included short runs of
molecular dynamics as described previously [31]. Analysis of
hydrophobic interactions was performed with
INTG
[32].
RESULTS AND DISCUSSION
Kinetic parameters for wild-type and mutant HIV-1
proteinase-catalyzed hydrolysis of matrix/capsid Gag
cleavage site substrate
Previous specificity studies based on the substrate repre-
senting the matrix/capsid (MA/CA) cleavage site of HIV-1
suggested that this site is evolutionarily optimized for
efficient hydrolysis, as most of the substitutions in the
sequence resulted in less efficient cleavage, and none of the
substitutions resulted in substantially improved hydrolysis
[33–36]. We have tested the mutant HIV-1 proteinases on
this substrate (Table 1). The K
m
values were increased for
all mutants to varying degrees. This is in good agreement
with previous studies showing that binding site mutants
tend to increase K
m
values [5]. However, the k
cat
values for
the mutants were also improved, in some cases (e.g. I84V)
dramatically, yielding higher specificity constants (k
cat
/K

m
)
for these enzymes (Table 1).
Crystal structures of the inhibitor bound HIV-1 PR
with the studied mutations have been reported previously
[8,26,37–39]. Met 46 forms part of the flap and Leu at this
position could reduce its mobility, as suggested for the Ile
mutant from molecular dynamics simulations [40]. PR
with M46L mutation alters both K
m
and k
cat
for the
HIV-1 MA/CA substrate, but yields a specificity constant
similar to that of the wild-type enzyme (Table 1). V82
mutations to Ala or Ser increase the size of the internal
substrate binding subsites, particularly S1 and S1¢,result-
ing in larger K
i
values for inhibitors and increased K
m
values for the substrates. These mutants were also less
efficient on other Gag cleavage site substrates [5,8,41]. The
I84V mutation also influences the internal ligand binding
sites [38], consistent with the observed improvement in the
hydrolysis of the MA/CA substrate, as reported previ-
ously for a CA/p2 substrate [42]. Others reported
decreased catalytic efficiency for this mutant [5,43].
However, PR with I84V mutation did not substantially
alter Gag processing and did not affect virion replication

[23]. Leu 90 has no direct contact with the ligands,
however, it is close to the dimer interface of the PR, and
Met at this position substantially decreased protease
stability under low ionic strength conditions [8,44].
However, this effect is not seen in the kinetics of the
MA/CA hydrolysis. In the high ionic strength of the
assays the dimerization is strengthened as compared to
low ionic strength conditions [45].
Kinetic parameters for wild-type HIV-1 proteinase-
catalyzed hydrolysis of NC/p1 and p1/p6 cleavage site
substrates
The oligopeptides were chosen to represent the naturally
occurring NC/p1 and p1/p6 cleavage sites in HIV-1
HXB2
,a
representative isolate of the B subtype [18]. The kinetic
parameters were determined for hydrolysis of the oligopep-
tides by the wild-type HIV-1 PR (Table 2). The two peptides
had low specificity constants (k
cat
/K
m
), as compared to
peptides representing other Gag and Gag-Pol cleavage sites
tested under identical conditions (Table 1, [15]), in good
agreement with the hypothesis that cleavage at these sites
might be the rate limiting step of Gag processing [9]. Others
also reported that peptides representing these cleavage sites
are not efficient substrates of PR [7,16]. Although the
specificity constants for the two substrates were similar, the

K
m
and k
cat
values differed remarkably: the NC/p1 peptide
exhibited low K
m
and k
cat
values, while the p1/p6 cleavage
site showed both higher K
m
and k
cat
values (Table 2).
Increasing the concentration of the NC/p1 substrate above
the K
m
resulted in a decreased velocity, suggesting the
possibility of increased nonproductive binding at the
substrate binding site. Nevertheless, the specificity constants
determined with a competition assay for the NC/p1
substrate, as well as under pseudo first order conditions for
the p1/p6 substrate were in good agreement with the values
calculated from the Michaelis–Menten curve (Table 2).
Processing of peptides representing NC/p1 cleavage
site sequences by wild-type and mutant HIV-1
proteinases
Oligopeptides including both natural sequence polymor-
phisms and resistant mutations of the NC/p1 cleavage site

Table 1. Kinetic parameters for the hydrolysis of the oligopeptide rep-
resenting the matrix/capsid cleavage site of HIV-1 (VSQNY-fl-PIVQ).
Enzyme
K
m
(m
M
)
k
cat
(s
)1
)
k
cat
/K
m
(m
M
)1
Æs
)1
)
Wild-type 0.15 6.9 46.0
M46L 0.56 18.4 32.9
V82S 1.34 13.4 10.0
V82A 0.42 7.0 16.7
I84V 1.02 93.5 92.0
L90M 0.64 33.0 51.6
Table 2. Processing of peptides representing wild-type HIV-1 Gag NC/p1 and p1/p6 cleavage sites by wild-type HIV-1 proteinase.

Site
Substrate
sequence
K
m
(m
M
)
k
cat
(s
)1
)
k
cat
/K
m
a
(m
M
)1
Æs
)1
)
k
cat
/K
m
b
(m

M
)1
Æs
)1
)
1. NC/p1
ERQAN-fl-FLGKI 0.17 0.15 0.9 1.0
7. p1/p6
RPGNF-fl-LQSRP 1.20 0.98 0.8 0.8
a
Values calculated from the individual K
m
and k
cat
values.
b
Value determined for peptide 1 using a competitive assay with substrate
KTGVL-fl-VVQPK [28] and under pseudo first order reaction condition for peptide 7 using a substrate concentration range (0.02–0.15 m
M
)
in which the activity was a linear function of the substrate concentration.
4116 A. Fehe
´
r et al. (Eur. J. Biochem. 269) Ó FEBS 2002
sequences were assayed as substrates of HIV-1 PR using
competitive assays (Table 3). Natural variations of this
cleavage site (peptides 2 and 3) exhibited either decreased or
increased specificity constants with the wild-type PR
(Table 3). The mutated residues in this sequence are in the
outer regions of the cleavage site sequence recognized by the

PR, and the respective S4 and S3 binding sites could accept
a variety of residues [33,34,46]. When compared to peptides
having the same natural sequence background (peptides 4
and1,5and2,6and3),theAlatoValmutationatP2,seen
in resistance, increased the specificity constant by two to
tenfold. As the Ala to Val mutation had a varying effect on
the specificity constant depending on the peptide sequence,
this result further supports the view that substrate binding
subsites of the HIV-1 PR do not act independently of each
other [35,46].
Based on molecular modeling, the P2 Val fits much better
than Ala in the S2 binding site due to more favorable van
der Waals contacts with Val32, Ile47 and Ile84 (not shown).
P2 Val also shifts the sequence toward a type 2 consensus
sequence, where beta-branched residues were found to be
the best at this position [46]. P3 Arg is often disordered in
HIV-1 PR-inhibitor crystal structures, however, when it is
ordered it interacts with Arg8 and Glu21 through water
molecules and makes hydrophobic interactions with Phe53,
Pro81 and Val82 [26]. P3 Arg could interact favorably with
Asp29 and P1 Asn, which may contribute to its beneficial
effect in specificity. The 20-fold increased specificity con-
stant obtained for the doubly substituted peptide is within
the range of good Gag cleavage site substrates [15].
The same substrate set was also tested with the mutant
PRs. M46L, V82S and V82A mutants gave lower specificity
constants for each substrate as compared to the wild-type
enzyme. These enzymes were also less efficient on the MA/
CA cleavage site (Table 1). Similar to wild-type PR, the
resistant mutation of P2 Ala to Val always increased the

specificity constant when compared to peptides with
the same natural sequence. While the combined P3 Arg
and P2 Val mutations (peptide 6) provided the best
combination for the M46L mutant, the P2 Ala to Val
mutation alone provided the best specificity constant
(peptide 4) for the V82 mutants (Table 3). In an in vivo
study using indinavir, the V82A mutation arose with the P2
Ala to Val mutation, without the appearance of P3 Arg
mutation [10]. In these mutants the positive effect of P3 Arg
is offset due to the loss of favorable hydrophobic interaction
with the smaller Ala or Ser side chain. These results agree
with the observations that the naturally existing sequence
variation in the virus at this cleavage site may be important
for developing resistance [19].
The I84V mutant gave somewhat better specificity con-
stant with the wild-type substrate and with some of its
variants. This enzyme was also more efficient than the wild-
type PR on the MA/CA substrate. The L90M mutant was
also more efficient than the wild-type enzyme on the
substrates. As seen with the wild-type enzyme and M46L,
the P2 Val mutation in the respective natural sequence
background always increased the specificity constant for
these mutants.
Processing of peptides representing p1/p6 cleavage
site sequences by wild-type and mutant HIV-1
proteinases
Although previous studies indicated that the p1/p6 cleavage
site is rather conserved and the p2/NC cleavage site is the
most variable [17], analysis of various HIV isolates in the
database suggested that substantial sequence polymorphism

occurs at this site, especially at positions close to the site
of cleavage (see Fig. 1). To our knowledge, the effect of
these variations on the susceptibility towards PR cleavage
has not been reported yet. Strikingly, there are several
variants at P1 and P1¢ positions, which are important
determinants of specificity [34,46]. However, the natural
variations we examined did not substantially change the
specificity constants for the wild-type PR, except for the P1
Leu substitution (Table 4), which provided a very inefficient
processing. This result raises the question of whether viral
proteins having this mutation could be processed at this site
and whether viruses harboring this mutation could be
replication competent. The only sequence reported to have
this residue (C.BR92BR025) is not from a full-length clone
[18]. In contrast to the natural variations, the P1¢ Phe
substitution, which is seen only in resistant virus, was a
substantially better substrate for the wild-type enzyme.
Differences in free energy of enzyme–substrate inter-
action can be related to kinetic data by the equation
DG ¼ –RTln(k
cat
/K
m
) from the transition state theory. The
logarithmic value of the specificity constant showed a strong
correlation with the volume of the P1¢ residue (correlation
coefficient R ¼ 0.90) and the number of hydrophobic
contacts the side chain formed with residues of the S1¢
subsite (R ¼ 0.98). The fit of various P1¢ residues into the
S1¢ binding site of HIV-1 PR is shown in Fig. 2. The results

Table 3. Processing of peptides representing wild-type and mutant HIV-1 Gag NC/p1 cleavage sites by HIV-1 proteinase. Ratios of specificity
constants for substituted over wild-type substrate are shown in parentheses.
Substrate
sequence
a
Type of
mutation
b
k
cat
/K
m
(m
M
)1
Æs
)1
)
Wild-type M46L V82S V82A I84V L90M
1.
ERQAN-fl-FLGKI (–) 1.0 (1.0) 0.3 (1.0) 0.4 (1.0) 0.7 (1.0) 1.6 (1.0) 2.5 (1.0)
2. EGQAN-fl-FLGKI (P) 0.4 (0.4) 0.2 (0.8) 0.2 (0.5) 0.03 (0.04) 0.4 (0.3) 1.0 (0.4)
3.
ERRAN-fl-FLGKI (P) 1.7 (1.7) 0.6 (2.0) 0.3 (0.8) 0.5 (0.7) 3.2 (2.0) 4.2 (1.7)
4. ERQ VN-fl-FLGKI (R) 2.6 (2.6) 2.4 (8.0) 1.7 (4.3) 2.1 (3.0) 5.9 (3.7) 9.6 (3.8)
5.
EGQVN-fl-FLGKI (R) 0.8 (0.8) 0.7 (2.3) 0.4 (1.0) 0.7 (1.0) 2.8 (1.8) 4.1 (1.6)
6.
ERRVN-fl-FLGKI (R) 20.1 (20.1) 11.7 (39.0) 0.8 (2.0) 1.1 (0.9) 7.9 (4.9) 45.4 (18.2)
a

Substituted residues are in bold.
b
The type of mutation: natural polymorphism (P), mutation appearing only in drug resistance (R, see
Fig. 1). If two mutations occurred and one of them was occurring only in drug resistance, the cleavage site was considered as R type.
Ó FEBS 2002 Studies on mutant HIV-1 processing sites (Eur. J. Biochem. 269) 4117
suggest that the maximization of the van der Waals
interactions of P1¢ with S1¢ residues may be the most
important feature determining the efficiency of cleavage.
Similar effects were observed for P2 substitution in the NC/
p1 site. The specificity changes obtained with the mutants
for the substituted peptides tend to be similar to those
obtained for the wild-type enzyme, but some exceptions
were also observed. For example, M46L preferred P1Y and
P1¢F substantially more than the wild-type enzyme, while
the same substrate mutations were much less preferred by
the V82S mutant. For all mutant PRs the P1¢F substitution
provided the best substrate within the series. Similar results
were reported for P1¢F substitution in the CA/p2 cleavage
site [42].
CONCLUSIONS
HIV-1 grown in cultured cells in the presence of PR
inhibitors produces multiple PR mutants of lower suscep-
tibility to inhibitors. Furthermore, mutants selected with
one inhibitor are often cross-resistant to other inhibitors
(reviewed in [47]). The number of mutations increases with
the time of therapy. Schock et al. [6] proposed that
mutations located in the binding cleft of the enzyme can
Table 4. Processing of peptides representing wild-type and mutant HIV-1 p1/p6 Gag cleavage sites by HIV-1 proteinase. Ratios of specificity
constants for substituted over wild-type substrate are shown in parentheses.
Substrate

sequence
a
Type of
mutation
b
k
cat
/K
m
(m
M
)1
Æs
)1
)
Wild-type M46L V82S V82A I84V L90M
7.
RPGNF-fl-LQSRP (–) 0.8 (1.0) 0.6 (1.0) 1.2 (1.0) 1.2 (1.0) 1.2 (1.0) 2.1 (1.0)
8.
RPGNY-fl-LQSRP (P) 1.3 (1.6) 2.6 (4.3) 0.9 (0.8) 2.2 (1.8) 1.2 (1.0) 0.6 (0.3)
9.
RPGNL-fl-LQSRP (P) 0.01 (0.01) 0.05 (0.08) 0.03 (0.03) 0.09 (0.08) < 0.07 (< 0.01) 0.07 (0.03)
10.
RPGNF-fl-PQSRP (P) 0.3 (0.4) 0.3 (0.5) 0.06 (0.05) 0.2 (0.2) 0.8 (0.7) 0.5 (0.2)
11.
RPGNF-fl-VQSRP (P) 0.8 (1.0) 0.5 (0.8) 0.7 (0.6) 1.2 (1.0) 1.2 (1.0) 2.1 (1.0)
12.
RPRNF-fl-LQSRP (P) 0.5 (0.6) 0.4 (0.7) 0.6 (0.5) 0.7 (0.6) 0.8 (0.7) 1.3 (0.6)
13.
RQGNF-fl-LQSRP (P) 0.3 (0.4) 0.05 (0.1) 0.3 (0.3) 0.4 (0.3) 0.4 (0.3) 0.4 (0.2)

14. RPGNF-fl-FQSRP (R) 7.6 (9.5) 8.7 (14.5) 2.2 (1.8) 11.0 (9.2) 8.3 (6.9) 22.3 (10.6)
a
Substituted residues are in bold.
b
Type of mutation: natural polymorphism (P), mutation appearing in drug resistance (R).
Fig. 2. Fitting of various P1¢ residues in the p1/p6¢ substrate sequence into the S1¢ binding site of HIV-1 PR. Space filling models of the P1¢ residue of
peptides 7, 10, 11 and 14 (Table 4) are shown occupying the S1¢ binding site of wild-type HIV-1 PR.
4118 A. Fehe
´
r et al. (Eur. J. Biochem. 269) Ó FEBS 2002
lead to the development of drug resistance by increasing K
i
of the inhibitors at the expense of impaired proteinase
function, while non-active-site mutations may act by
enhancing the catalytic efficiency. However, reduced cata-
lytic efficiency has been reported for nonactive site muta-
tions L90M and N88D [8], and both increases and decreases
in catalytic efficiency have been observed for active site
mutants of HIV-1 PR [8,42]. This variation is confirmed by
our results: the active site mutant I84V enzyme had higher
specificity constant than the wild-type PR, while the
nonactive-site mutations M46L and L90M did not sub-
stantially improve the catalytic efficiency of the PR, and
even resulted in reduced activity on some substrates.
Resistant mutants of HIV-1 PR must possess sufficient
proteolytic activity (k
cat
/K
m
) to support viral replication by

correctly cleaving Gag and Gag-Pol precursors. The order of
Gag cleavage is important for infectivity [48]. The most
efficiently cleaved sites are not mutated in resistance, and the
catalytically compromised mutant HIV-1 PRs can still cleave
these substrates sufficiently for viral replication. In fact, it
may be difficult to improve the cleavage by mutation of these
sites, as they appear to be optimized for rapid hydrolysis (for
example the MA/CA or CA/p2 cleavage sites [34,36]). On the
other hand, the NC/p1 and p1/p6 sites do not appear to be
optimized for rapid hydrolysis by wild-type PR, as substan-
tially improved specificity constants were obtained with
resistant cleavage site mutations. Therefore, it is possible to
increase the cleavage rate by mutation at these sites when the
PR activity is diminished due to the accumulation of PR
mutations. The P2 Val and P1¢ Phe residues appearing in
drug resistance at these sites were the most frequent residues
in efficiently cleaved substrates selected by screening a phage
display library [49]. The observed improvement in hydrolysis
of the substrates harboring these mutations was more
pronounced for the wild-type enzyme and nonactive-site
mutants (L90M and M46L) than for the active-site mutants.
Consequently, mutation of the rate limiting cleavage sites,
NC/p1 and p1/p6, can partly compensate for the reduced
catalytic activity of resistant PR mutants.
ACKNOWLEDGEMENTS
We thank Dr Bruce Korant (DuPont Pharmaceuticals) for providing
DMP-323 and amprenavir for the active site titrations, Szilvia Peto
¨
for
technical assistance and Suzanne Specht for peptide synthesis and

amino-acid analysis. Research sponsored in part by the Hungarian
Science and Research Fund (OTKA T 30092; F34479), by the United
States Public Health Service Grant GM62920, by the National Cancer
Institute, DHHS under contract with ABL, by the Intramural AIDS
Targeted Antiviral Program of the Office of the Director of NIH and
by AIDS FIRCA Grant TW01001. The contents of this publication
do not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by the U.S.
Government.
REFERENCES
1. Oroszlan, S. & Luftig, R.B. (1990) Retroviral proteinases. Curr.
Top. Microbiol. Immunol. 157, 153–185.
2. Swanstrom, R. & Eron, J. (2000) Human immunodeficiency virus
type-1 protease inhibitors: therapeutic successes and failures,
suppression and resistance. Pharmacol. Ther. 86, 145–170.
3. Mammano, F., Petit, C. & Clavel, F. (1998) Resistance-associated
loss of viral fitness in human immunodeficiency virus type 1:
phenotypic analysis of protease and gag coevolution in protease
inhibitor-treated patients. J. Virol. 72, 7632–7637.
4. Zennou,V.,Mammano,F.,Paulous,S.,Mathez,D.&Clavel,F.
(1998) Loss of viral fitness associated with multiple Gag and Gag-
Pol processing defects in human immunodeficiency virus type 1
variants selected for resistance to protease inhibitors in vivo.
J. Virol. 72, 3300–3306.
5. Gulnik, S.V., Suvorov, L.I., Liu, B., YuB., Anderson, B., Mitsuya,
H. & Erickson, J.W. (1995) Kinetic characterization and cross-
resistance patterns of HIV-1 protease mutants selected under drug
pressure. Biochemistry 34, 9282–9287.
6. Schock, H.B., Garsky, V.M. & Kuo, L.C. (1996) Mutational

anatomy of an HIV-1 protease variant conferring cross-resistance
to protease inhibitors in clinical trials. Compensatory modulations
of binding and activity. J. Biol. Chem. 271, 31957–31963.
7.Ermolieff,J.,Lin,X.&Tang,J.(1997)Kineticpropertiesof
saquinavir-resistant mutants of human immunodeficiency virus
type 1 protease and their implications in drug resistance in vivo.
Biochemistry 36, 12364–12370.
8. Mahalingam, B., Louis, J.M., Reed, C.C., Adomat, J.M., Krouse,
J., Wang, Y.F., Harrison, R.W. & Weber, I.T. (1999) Structural
and kinetic analysis of drug resistant mutants of HIV-1 protease.
Eur. J. Biochem. 263, 238–245.
9. Doyon, L., Croteau, G., Thibeault, D., Poulin, F., Pilote, L. &
Lamarre, D. (1996) Second locus involved in human immuno-
deficiency virus type 1 resistance to protease inhibitors. J. Virol.
70, 3763–3769.
10. Zhang, Y.M., Imamichi, H., Imamichi, T., Lane, H.C., Falloon, J.,
Vasudevachari, M.B. & Salzman, N.P. (1997) Drug resistance
during indinavir therapy is caused by mutations in the pro-
tease gene and its Gag substrate cleavage sites. J. Virol. 71, 6662–
6670.
11. Robinson, L.H., Myers, R.E., Snowden, B.W., Tisdale, M. &
Blair, E.D. (2000) HIV type 1 protease cleavage site mutations and
viral fitness: implications for drug susceptibility phenotyping
assays. AIDS Res. Hum. Retrovir. 16, 1149–1156.
12. Coˆ te
´
,H.C.,Brumme,Z.L.&Harrigan,P.R.(2001)Human
immunodeficiency virus type 1 protease cleavage site mutations
associated with protease inhibitor cross-resistance selected by
indinavir, ritonavir, and/or saquinavir. J. Virol. 75, 589–594.

13. Maguire,M.F.,MacManus,S.,Griffin,P.,Guinea,C.,Harris,
W.,Richards,N.,Wolfram,J.,Tisdale,M.,Snowden,W.&
Kleim, J.P. (2001) Interaction of HIV-1 protease and gag gene
mutations in response to amprenavir-selective pressure exerted in
amprenavir-treated subjects – contribution of Gag p6 changes
L449F and P453L. Antivir. Ther. 6 (Suppl. 1), 48.
14. Catamancio, S.S., Pasquale, M.P., Citterio, P., Kurtagic, S., Galli,
M. & Rusconi, S. (2001) In vitro evolution of the human
immunodeficiency virus type 1 Gag-protease region and main-
tenance of reverse transcriptase resistance following prolonged
drug exposure. J. Clin. Microbiol. 39, 1124–1129.
15. To
¨
zse
´
r, J., Bla
´
ha, I., Copeland, T.D., Wondrak, E.M. & Oroszlan,
S. (1991) Comparison of the HIV-1 and HIV-2 proteinases using
oligopeptide substrates representing cleavage sites in Gag and
Gag-Pol polyproteins. FEBS Lett. 281, 77–80.
16. Wondrak, E.M., Louis, J.M., de Rocquigny, H., Chermann, J.C.
& Roques, B.P. (1993) The gag precursor contains a specific
HIV-1 protease cleavage site between the NC (P7) and P1 proteins.
FEBS Lett. 333, 21–24.
17. Barrie, K.A., Perez, E.E., Lamers, S.L., Farmerie, W.G., Dunn,
B.M., Sleasman, J.W. & Goodenow, M.M. (1996) Natural vari-
ation in HIV-1 protease, Gag p7 and p6, and protease
cleavage sites within Gag/Pol polyproteins: amino acid substitu-
tions in the absence of protease inhibitors in mothers and children

Ó FEBS 2002 Studies on mutant HIV-1 processing sites (Eur. J. Biochem. 269) 4119
infected by human immunodeficiency virus type 1. Virology 219,
407–416.
18.Korber,B.,Kuiken,C.,Foley,B.,Hahn,B.,McCuthan,F.,
Mellors, J. & Sodroski, J., eds (1998) Human Retroviruses and
AIDS. Los Alamos Laboratory, Los Alamos, NM, USA
19. Bally, F., Martinez, R., Peters, S., Sudre, P. & Telenti, A. (2000)
Polymorphism of HIV type 1 gag p7/p1 and p1/p6 cleavage sites:
clinical significance and implications for resistance to protease
inhibitors. AIDS Res. Hum. Retrovir. 16, 1209–1213.
20. Dunn, B.M., Gustchina, A., Wlodawer, A. & Kay, J. (1994)
Subsite preferences of retroviral proteinases. Methods Enzymol.
241, 254–278.
21. Delphin, N., Schneider, V., Nicolas, J.C. & Rozenbaum, W.
(1999) Variations in the p7/p1 gag cleavage region and virological
response to HIV protease inhibitor. Antivir. Ther. 4 (Suppl. 1), 58.
22. Molla, A., Korneyeva, M., Gao, Q., Vasavanonda, S., Schipper,
P.J., Mo, H.M., Markowitz, M., Chernyavskiy, T., Niu, P.,
Lyons, N., Hsu, A., Granneman, G.R., Ho, D.D., Boucher,
C.A.B., Leonard, J.M., Norbeck, D.W. & Kempf, D.J. (1996)
Ordered accumulation of mutations in HIV protease confers
resistance to ritonavir. Nat. Med. 2, 760–766.
23. Croteau, G., Doyon, L., Thibeault, D., McKercher, G., Pilote, L.
& Lamarre, D. (1997) Impaired fitness of human immuno-
deficiency virus type 1 variants with high-level resistance to pro-
tease inhibitors. J. Virol. 71, 1089–1096.
24. Condra, J.H., Holder, D.J., Schleif, W.A., Blahy, O.M.,
Danovich, R.M., Gabryelski, L.J., Graham, D.J., Laird, D.,
Quintero, J.C., Rhodes, A., Robbins, H.L., Roth, E., Shiva-
prakash, M., Yang, T., Chodakewitz, J.A., Deutsch, P.J., Leavitt,

R.Y., Massari, F.E., Mellors, J.W., Squires, K.E., Steigbigel, R.T.,
Teppler, H. & Emini, E.A. (1996) Genetic correlates of in vivo
viral resistance to indinavir, a human immunodeficiency virus type
1 protease inhibitor. J. Virol. 70, 8270–8276.
25. Louis,J.M.,Clore,G.M.&Gronenborn,A.M.(1999)Autopro-
cessing of HIV-1 protease is tightly coupled to protein folding.
Nat. Stuct. Biol. 6, 868–875.
26. Mahalingam, B., Louis, J.M., Hung, J., Harrison, R.W. & Weber,
I.T. (2001) Structural implications of drug-resistant mutants
of HIV-1 protease: high-resolution crystal structures of the mutant
protease/substrate analogue complexes. Proteins 43, 455–464.
27. Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd edn. pp.
111–112. W.H. Freeman, New York.
28. To
¨
zse
´
r, J., Zahuczky, G., Bagossi, P., Louis, J.M., Copeland,
T.D., Oroszlan, S., Harrison, R.W. & Weber, I.T. (2000) Com-
parison of the substrate specificity of the human T-cell leukemia
virus and human immunodeficiency virus proteinases. Eur.
J. Biochem. 267, 6287–6295.
29. Kuzmic, P. (1996) Program DYNAFIT for the analysis of enzyme
kinetic data: Application to HIV proteinase. Anal. Biochem. 237,
260–273.
30. Weber, I.T. & Harrison, R.W. (1996) Molecular mechanics cal-
culations on HIV-1 protease with peptide substrates correlate with
experimental data. Protein Eng. 9, 679–690.
31. Weber, I.T. & Harrison, R.W. (1999) Molecular mechanics ana-
lysis of drug resistant mutants of HIV protease. Protein Eng. 12,

469–474.
32. Torshin, I. (1999) Activating oligomerization as intermediate level
of signal transduction: analysis of protein-protein contacts and
active sites in several glycolytic enzymes. Front Biosci. 4, D557–
D570.
33. To
¨
zse
´
r, J., Gustchina, A., Weber, I.T., Bla
´
ha, I., Wondrak, E.M.
& Oroszlan, S. (1991) Studies on the role of the S
4
substrate
binding site of HIV proteinases. FEBS Lett. 279, 356–360.
34. To
¨
zse
´
r, J., Weber, I.T., Gustchina, A., Bla
´
ha, I., Copeland, T.D.,
Louis, J.M. & Oroszlan, S. (1992) Kinetic and modeling studies of
S
3
-S
3
¢ subsites of HIV proteinases. Biochemistry 31, 4793–4800.
35. To

¨
zse
´
r, J., Bagossi, P., Weber, I.T., Louis, J.M., Copeland, T.D. &
Oroszlan, S. (1997) Studies on the symmetry and sequence context
dependence of the HIV-1 proteinase specificity. J. Biol. Chem. 272,
16807–16814.
36. Boross, P., Bagossi, P., Copeland, T.D., Oroszlan, S., Louis, J.M.
&To
¨
zse
´
r, J. (1999) Effect of substrate residues on the P2¢ prefer-
ence of retroviral proteinases. Eur. J. Biochem. 264, 921–929.
37. Baldwin, E.T., Bhat, T.N., Gulnik, S., Liu, B., Topol, I.A., Kiso,
Y., Mimoto, T., Mitsuya, H. & Erickson, J.W. (1995) Structure of
HIV-1 protease with KNI-272, a tight-binding transition-state
analog containing allophenylnorstatine. Structure 3, 581–590.
38. Ala, P.J., Huston, E.E., Klabe, R.M., McCabe, D.D., Duke, J.L.,
Rizzo, C.J., Korant, B.D., DeLoskey, R.J., Lam, P.Y., Hodge,
C.N. & Chang, C.H. (1997) Molecular basis of HIV-1 protease
drug resistance: structural analysis of mutant proteases complexed
with cyclic urea inhibitors. Biochemistry 36, 1573–1580.
39. Mahalingam, B., Boross, P., Wang, Y.F., Louis, J.M., Fischer,
C.C., To
¨
zse
´
r, J., Harrison, R.W. & Weber, I.T. (2002) Combining
mutations in HIV-1 protease to understand mechanisms of resis-

tance. Proteins 48, 107–116.
40. Collins, J.R., Burt, S.K. & Erickson, R.W. (1995) Flap opening in
HIV-1 protease simulated by ÔactivatedÕ molecular dynamics. Nat.
Struct. Biol. 2, 334–338.
41. Lin, Y., Lin, X., Hong, L., Foundling, S., Heinrikson, R.L.,
Thaisrivongs, S., Leelamanit, W., Raterman, D., Shah, M.,
Dunn, B.M. & Tang, J. (1995) Effect of point mutations on the
kinetics and the inhibition of human immunodeficiency virus type
1 protease: relationship to drug resistance. Biochemistry 34, 1143–
1152.
42. Ridky, T.W., Kikonyogo, A., Leis, J., Gulnik, S., Copeland, T.,
Erickson, J., Wlodawer, A., Kurinov, I., Harrison, R.W. &
Weber, I.T. (1998) Drug resistant HIV-1 proteases identify enzyme
residues important for substrate selection and catalytic rate.
Biochemistry 37, 13835–13845.
43. Wilson, S.I., Phylip, L.H., Mills, J.S., Gulnik, S.V., Erickson,
J.W., Dunn, B.M. & Kay, J. (1997) Escape mutants of HIV-1
proteinase: enzymic efficiency and susceptibility to inhibition.
Biochem. Biophys. Acta 1339, 113–125.
44. Xie, D., Gulnik, S. & Gustchina, E., YuB., Shao, W., Qoronfleh,
W., Nathan, A. & Erickson, J.W. (1999) Drug resistance muta-
tions can affect dimer stability of HIV-1 protease at neutral pH.
Protein Sci. 8, 1702–1707.
45. Szeltner, Z. & Polga
´
r, L. (1996) Conformational stability and
catalytic activity of HIV-1 protease are both enhanced at high salt
concentration. J. Biol. Chem. 271, 5458–5463.
46. Griffiths, J.T., Phylip. L.H., Konvalinka, J., Strop, P., Gustchina,
A., Wlodawer, A., Davenport, R.J., Briggs, R., Dunn, B.M. &

Kay, J. (1992) Different requirements for productive interaction
between the active site of HIV-1 proteinase and substrates con-
taining -hydrophobic*hydrophobic- or -aromatic*Pro- cleavage
sites. Biochemistry 31, 5193–5200.
47. Miller, V. (2001) International perspectives on antiretroviral
resistance. Resistance to protease inhibitors. J. Acquir. Immune
Defic. Syndr. 26 (Suppl. 1), S34–S50.
48. Pettit, S.C., Sheng, N., Tritch, R., Erickson-Viitanen, S. &
Swanstrom, R. (1998) The regulation of sequential processing of
HIV-1 Gag by the viral protease. Adv. Exp. Med. Biol. 436, 15–25.
49. Beck, Z.Q., Hervio, L., Dawson, P.E., Elder, J.H. & Madison,
E.L. (2000) Identification of efficiently cleaved substrates for
HIV-1 protease using a phage display library and use in inhibitor
development. Virology 274, 391–401.
50. Leis, J., Baltimore, D., Bishop, J.M., Coffin, J., Fleissner, E.,
Goff, S.P., Oroszlan, S., Robinson, H., Skalka, A.M., Temin,
H.M. & Vogt, V. (1988) Standardized and simplified nomen-
clature for proteins common to all retroviruses. J. Virol. 62, 1808–
1809.
4120 A. Fehe
´
r et al. (Eur. J. Biochem. 269) Ó FEBS 2002