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
´

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

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

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

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

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

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

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

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

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

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

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

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.
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