From the Departments of Virology and ¶ Chemical
and Physical Sciences, Experimental Station, DuPont Pharmaceuticals,
Wilmington, Delaware 19880 and the
Protein Chemistry Laboratory,
SAIC Frederick, NCI-Frederick Cancer Research and Development
Center, National Institutes of Health, Frederick, Maryland 21702
Received for publication, September 25, 2000, and in revised form, February 19, 2001
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ABSTRACT |
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A full-length and C-terminally truncated version
of human endogenous retrovirus (HERV)-K10 protease were
expressed in Escherichia coli and purified to homogeneity.
Both versions of the protease efficiently processed HERV-K10 Gag
polyprotein substrate. HERV-K10 Gag was also cleaved by human
immunodeficiency virus, type 1 (HIV-1) protease, although at different
sites. To identify compounds that could inhibit protein processing
dependent on the HERV-K10 protease, a series of cyclic ureas that had
previously been shown to inhibit HIV-1 protease was tested. Several
symmetric bisamides acted as very potent inhibitors of both the
truncated and full-length form of HERV-K10 protease, in subnanomolar or
nanomolar range, respectively. One of the cyclic ureas, SD146, can
inhibit the processing of in vitro translated HERV-K10 Gag
polyprotein substrate by HERV-K10 protease. In addition, in virus-like
particles isolated from the teratocarcinoma cell line NCCIT, there is
significant accumulation of Gag and Gag-Pol precursors upon treatment
with SD146, suggesting the compound efficiently blocks HERV-K Gag
processing in cells. This is the first report of an inhibitor able to
block cell-associated processing of Gag polypeptides of an endogenous retrovirus.
The human genome contains a large number of endogenous retroviral
sequences that are virtually all highly defective because of multiple
termination codons, deletions or the lack of a 5' long terminal repeat
(1, 2). It is assumed that at some time during the course of human
evolution, exogenous progenitors of human endogenous retroviruses
(HERVs)1 integrated into the
cells of the germ line and thereby obtained the ability to be inherited
by offspring of the host as a mendelian trait (3).
HERVs are grouped into at least a dozen single and multiple copy number
families and are classified according to the tRNA that they use as
primer for reverse transcription (1, 4). The retroviral element that
carries a primer binding site complementary to the 3' end of a lysine
tRNA is called HERV-K. HERV type K represents the biologically most
active form of a variety of retroviral elements present in the human
genome (1, 5). Although the HERV-K group comes closest of all known
HERVs to containing infectious virus, no corresponding
replication-competent virus has so far been described (1, 3). Although
humans harbor several dozen proviral copies of HERV type K per haploid
genome (4, 6, 7), some of which code for the characteristic retroviral
proteins Gag, Pol, and Env (8, 9), recent studies raised a suggestion that no complete proviral copy of HERV-K exists (10, 11); the issue
remains to be clarified. In terms of infectious virion production,
HERV-K could be defective at multiple levels, including the observed
arrest during budding, inefficient RT enzyme activity, and incomplete
Env expression and processing (1).
HERV-K elements exhibit restricted cell type expression, observed
mainly in germ cell tumors (including testicular teratocarcinoma cell
lines) and their testicular precursor lesions (8, 12, 13). Typically
the coding regions of HERV-K elements are far less disrupted by
mutations than other HERV families, and protein synthesis has been
observed for all the main retroviral genes. The HERV-K Gag precursors
are cleaved into major core, matrix, and nucleocapsid components
(14-16), presumably by HERV-K protease, because functional activity
has been demonstrated for this enzyme (15, 17).
Detailed electron microscopic surveys have revealed the existence of
retrovirus-like particles in breast carcinoma and teratocarcinoma cell
lines (18-20). The phenotype of human teratocarcinoma-derived retrovirus particles has been correlated with complex mRNA
expression of HERV-K sequences in those cells, reminiscent of the
mRNA expression pattern observed after exogenous retrovirus
infection with, for example, lenti- or spumavirus strains (8, 9).
Several hypotheses have so far been proposed about possible implication
of HERV expression in certain pathogeneses, including autoimmune
diseases such as insulin-dependent diabetes mellitus (21),
tumor development, and even cardiovascular disease (22). In addition,
numerous possible roles have been proposed for HERVs in reproductive
physiopathology (reviewed in Ref. 23). In the study published by Sauter
et al. (16), authors reported that HIV-1-infected patients
and especially patients with seminomas exhibit elevated titers of
anti-HERV-K10 Gag antibodies. Towler et al. (25) reported
that HERV-K10 protease is highly resistant to a number of clinically
used HIV-1 protease inhibitors, including ritonavir, indinavir, and
saquinavir. They reported the protease to be a homodimer with a pH
optimum at 4.5 and with a higher enzymatic activity and stability at
elevated ionic strengths. The authors raised an interesting speculation
that HERV-K protease might somehow complement HIV-1 protease under
conditions where the latter activity is impaired because of either the
presence of drug resistance mutations or the presence of potent HIV-1
protease inhibitor.
The aim of this study was to identify potent inhibitors of HERV-K10
protease and to demonstrate their action in virus-producing cells. The
results shown in this report indicate that some members of the cyclic
urea class can act as very potent inhibitors of this protease in a
nanomolar range and are capable of blocking processing of HERV Gag
in vitro as well as in the teratocarcinoma cell line NCCIT.
Cloning of Truncated Version of HERV-K10 Protease--
Genomic
DNA was extracted from the buffy coat fraction of fresh human blood
(24). DNA coding for core region of HERV-K10 protease (25) was then
amplified by polymerase chain reaction with Taq DNA
polymerase (PerkinElmer Life Sciences). Oligonucleotides 5'-CTAGGAAGCTTCATATGGACTATAAAGGCGAAATTCAA-3' (PRT-A) and
5'-GCTGTGGATCCTTACTACATGGTGATTTCCGCACC-3' (PRT-B) were used as
sense and antisense primer, respectively. PCR product was cloned into
mammalian expression vector pcDNA3.1(+) (Invitrogen) via
HindIII and BamHI restriction sites. DNA
sequencing of several clones revealed presence of substantial
polyporphism. The clone with the DNA sequence identical to that
published by Ono et al. (6) was chosen for further
experiments. This clone was subjected to another round of PCR
amplification, this time with oligonucleotides
5'-AGACTGGATCCGACTATAAAGGCGAAATTCAA-3' and 5'-ACAGATCTCGAGCATGGTGATTTCCGCACC-3'. The amplification product was cloned into Escherichia coli expression plasmid
pET21a(+) (Novagen) via BamHI and XhoI
restriction sites.
Cloning of the Full-length Version of HERV-K10 Protease--
The
cloning of the full-length version of HERV-K10 protease into pET21a(+)
and its site-directed mutagenesis was described previously (25).
Cloning of HERV-K10 gag--
Plasmid pcG3gag (a gift
from Dr. Ralf R. Tönjes, Paul-Ehrlich-Institut, Langen, Germany)
(26) was used as a template for PCR amplification of HERV-K10
gag region. Oligonucleotides
5'-AACACGGATCCATGGGGCAAACTAAAAGTAAA-3' and
5'-AGATGAATTCCTACTGCTGCACTGCCGCTTG-3' were used as sense and antisense
primer, respectively. PCR product was cloned into pcDNA3.1(+) (Invitrogen) via BamHI and EcoRI restriction sites.
Expression and Purification of 13-kDa Form of HERV-K10
Protease--
Luria-Bertani broth (1 L) supplemented with ampicillin
(200 µg/ml) was inoculated with 5 ml of overnight culture of E. coli BL21(DE3) expression strain (Novagen) harboring
pET21a(+)/HERV-K10 protease construct. When an
A600 value of 0.6 was reached, the expression of
HERV-K10 protease was induced by addition of
isopropyl-1-thio- Expression and Purification of Full-length Forms of HERV-K10
Protease--
E. coli BL21(DE3) strain was transformed with
expression plasmids containing either wild type form or active site
mutant (D26N) of the 18-kDa version of HERV-K10 protease.
Overnight culture was diluted 1:50 into 1 liter of LB broth. At an
A600 value of 0.6, 1 mM
isopropyl-1-thio- Expression and Purification of HIV-1 Protease--
HIV-1
protease was expressed in E. coli and then renatured from
inclusion bodies as described previously (28).
N-terminal Amino Acid Sequence Analysis--
The N-terminal
sequence was determined using the Hewlett Packard G1005A protein
sequencing system with on-line PTH analysis. All methods, reagents, and
consumables used were those recommended by the manufacturer.
Mass Spectrometry--
Matrix-assisted laser desorption
ionization mass spectrometry data were obtained on a PerSeptive
Biosystems Voyager DE-Pro mass spectrometer. The spectra were acquired
in the linear mode with delayed extraction. External calibration was
performed using calibrant 3 supplied by the manufacturer. The sample
was diluted 1:10 in sinapinic acid matrix solution. The matrix was
prepared by dissolving 10 mg/ml sinapinic acid in aqueous 30%
acetonitrile containing 0.3% trifluoroacetic acid.
Generation of Anti-HERV-K10 Protease Antiserum--
1 mg of
truncated version of HERV-K10 protease was loaded on SDS-PAGE,
and the band was excised from the gel. The gel slice was covered with
phosphate-buffered saline and emulsified with a syringe through a
23-gauge needle. The emulsion was then used directly to immunize
rabbits with 100 µg/dose.
Enzyme Assay--
To measure the inhibitory potency of
compounds, the discontinuous HPLC method described in Erickson-Viitanen
et al. (34) was used. The synthetic fluorescent cationic
peptide substrate 2-aminobenzoyl-Ala-Thr-His-Gln-Val-Tyr-Phe(NO2)-Val-Arg-Lys-Ala (28) was incubated with truncated or full-length HERV-K10 at 25 °C in an assay buffer containing 50 mM MES, pH 5.0, 1 M NaCl, 20% glycerol, 1 mM EDTA. The synthesis
of the substrate has been described elsewhere (28). The enzymatic
reaction was terminated with 0.2 M ammonium hydroxide.
Enzymatic hydrolysis of the substrate yielded the fluorescent anionic
product, (2-aminobenzoyl)-ATHQVY. The extent of hydrolysis was
determined using anion-exchange HPLC. An Amersham Pharmacia Biotech
HR5/5 MonoQ column eluted at 1.0 ml/min with 0-70% buffer B for 10 min was used to separate the fluorescent cleavage product from the
fluorescent substrate. The mobile phase buffer A contained 20 mM Tris/HCl, 0.02% sodium azide, and 10% acetonitrile at
pH 9.0, whereas buffer B consisted of buffer A plus 0.5 M
ammonium formate at pH 9.0. The column was washed with 100% buffer B
for 5 min and then stepped down to 0% buffer B to recycle the gradient
for the next injection. The cleavage product was measured at an
emission wavelength of 430 nm and excitation wavelength of 330 nm.
Linearity of enzymatic activity with time was first established, and
based on the results, reactions involving the truncated or full-length
HERV-K10 protease were quenched after 20 in or 40 min, respectively
(Fig. 1).
The Km values were determined with fixed enzyme
concentration (0.5 nM) and substrate concentrations of
0.5-50 µM; the data were fitted directly to
Michaelis-Menten equation with GraFit software version 4.0.10 (Erithacus Software Ltd.). In the next step, potent inhibitors were
identified as described below. The active site concentrations of the
proteases were determined by titrating the enzymes with different
concentrations of SD146. This data then enabled us to convert
vmax values into those for kcat.
Inhibition Kinetics--
Samples of the HIV-1 protease
inhibitors indinavir (MK-639), saquinavir (Ro 31-8959), and ritonavir
(ABT-538) were synthesized at DuPont Pharmaceuticals. Pepstatin A was
purchased from Sigma-Aldrich. The cyclic ureas were prepared as
described elsewhere (29-32). All inhibitors were dissolved in dimethyl
sulfoxide and stored at
On the basis of previous studies with HIV-1 protease (34), the mode of
inhibition was assumed to be competitive. To verify this assumption,
the dose response data were obtained for SD146 as a representative
compound at four substrate concentrations; IC50 values
increased linearly with increasing substrate concentration, indicating
the competitive nature of inhibition (35).
Effect of SD146 on the Cleavage of in Vitro Translated HERV Gag
Polyprotein by HERV-K10 Protease--
Plasmid pcDNA3.1(+)/HERV-K10
gag was used as template in TnT® Quick Coupled
Transcription/Translation (Promega) reactions to produce
[35S]methionine-labeled HERV Gag polyprotein that then
served as a substrate for HERV-K10 protease. The in vitro
translation product was incubated together with 0.54 µM
HERV-K10 protease (truncated form) and various concentrations of SD146
(0-1 µM) in 20 mM PIPES, pH 6.5, 0.1 M NaCl, 1 mM DTT, 10% glycerol, for 1 h
at 37 °C. The substrate and cleavage products were separated on
NuPage SDS-polyacrylamide gel (Novex) and autoradiographed.
Subsequently, the dried gel was scanned for radioactivity with a
Bio-Rad Molecular Imager FX, and the HERV Gag polyprotein bands were
quantitated using QuantityOne software (Bio-Rad).
Mammalian Cell Cultures and Collection of Particulate
Material--
Human teratocarcinoma cell lines NCCIT, PA-1, and
NTERA-2, as well as the embryonic kidney line 293 (all purchased from
American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
calf serum, 2 mM glutamine, and antibiotics (100 units/ml penicillin, 100 µg/ml streptomycin). Cell cultures were subcultured routinely twice per week. NCCIT cell line was treated with several concentrations of SD146 (up to 2 µM) or left untreated,
and aliquots of culture supernatants were taken at time 0 and after 1 day. HERV-K particles were recovered by ultracentrifugation. After a
10-min centrifugation in a Sorvall RT6000B table top centrifuge at 1500 rpm to remove unbroken cells and large cell debris, the samples were
centrifuged for 3 h in a Sorvall RC80 ultracentrifuge at
78,000 × g at 8 °C. Medium was discarded. The virus
pellets were resuspended in a minimal volume of 1% SDS, 1%
mecaptoethanol, and 7% glycerol, heated at 56 °C for 1 min, and
loaded onto 10% polyacryamide gels.
Immunoblotting--
Protein samples were separated with SDS-PAGE
and transferred to Immobilon-P polyvinylidene diflouride membranes
(Millipore) using semidry method. The membranes were probed with either
anti-HERV-K10 protease antiserum at a dilution of 1:250 or polyclonal
anti-HERV-K Gag antiserum (15) at a dilution of 1:10,000. Blots were
stained indirectly by using horseradish peroxidase-conjugated donkey
anti-rabbit antibodies and subsequent chemiluminescence detection
(PerkinElmer Life Sciences).
Viral RNA Isolation and RT-PCR--
RNA was isolated from
concentrated virus particles with QIAamp Viral RNA Mini Kit (Qiagen).
Eluted RNA was treated with RNase-free DNase I to digest any
contaminating cell genomic DNA and repurified with the same kit. RNA
was eluted in 60 µl of diethylpyrocarbonate-treated water.
Reverse transcription was performed in a volume of 20 µl containing 5 µl of viral RNA, 0.5 mM dNTP mix, 10 units of RNAsin, 100 ng of primer PRT-B, and 4 units of Omniscript reverse transcriptase (Qiagen). The reaction was carried out for 1 h at 37 °C. 5 µl of RT reaction was used in PCR amplification, together with 0.1 µM primers PRT-A and PRT-B, 1.5 mM
MgCl2, 0.2 mM dNTP mix, and 1 unit of
Taq DNA polymerase (PerkinElmer Life Sciences). The reaction
mix was initially denatured at 94 °C for 5 min and then subjected to
30 cycles of denaturation at 94 °C, annealing at 50 °C, and
extension at 72 °C. An aliquot of PCR reaction was used directly in
DNA sequencing, with either PRT-A or PRT-B as a primer.
Molecular Modeling of HERV-K10 Protease--
The
three-dimensional homology model of the truncated version of HERV-K10
protease was constructed using coordinates of HIV-1 protease complexed
with SD146 as a template (Protein Data bank file 1QBT.pdb; Ref. 36)
with the program Molecular Operating Environment (Chemical Computing
Group Inc.). The sequence was aligned initially maximizing the homology
but later adjusted to accommodate the insertion at position 39 (HIV
numbering) at the elbow of the flap and the insertion at position 80 (HIV numbering) at the active site, mimicking the three-dimensional
structure of feline immunodeficiency virus protease. The
homology algorithm of the Molecular Operating Environment software
created 10 models, each of which was generated by making a series of
Boltzmann-weighted choices of side chain rotamers and loop
conformations from a set of protein fragments of high resolution
protein structures. An average model was potential energy-minimized
using AMBER forcefield.
Expression and Purification of HERV-K10 Proteases.--
Two
versions of HERV-K10 protease were expressed in E. coli. The
amino acid sequences of polypeptide chains that were expressed are
shown in Fig. 3. The C-terminal boundary
of the truncated version was chosen on the basis of sequence homology
with the mature HIV-1 protease (25). An additional 58 amino acid
residues included at the N-terminal end of the protein were expected to be cleaved off in an autocatalytic manner. This N-terminal flanking portion was expressed to allow us to readily monitor autoprocessing activity (28). The nucleotide sequence of the clone that was chosen for
E. coli expression was in complete agreement with the cDNA sequence of HERV-K10 protease ORF published in Ono et
al. (6). The truncated, "core" protease was expressed as a
185-amino acid precursor at a high level in form of insoluble
cytoplasmic inclusion bodies. During the renaturation step with
dialysis all of the precursor (20 kDa) was autocatalytically processed
to give rise to mature, enzymatically active 13-kDa form. The site of N-terminal autoprocessing was determined with N-terminal amino acid
sequencing. It was shown to be GKAAY-WASQ with the dash designating the scissile bond and was in agreement with previous findings (25).
When analyzed with mass spectroscopy, the protein showed a molecular
mass that was in agreement with the expected size and that also
suggested that no C-terminal autoprocessing occurred (data not shown).
In addition to the monomer, a peak representing the mass of a dimerized
protease was present. Affinity chromatography with pepstatin A as an
immobilized ligand was used efficiently to purify the 13-kDa form of
HERV-K10 protease (37). The method described by Wondrak et
al. (37) for HIV-1 protease purification was adjusted to ensure
the best yield of HERV-K10 protease. Several NaCl and
(NH4)2SO4 concentrations in
pepstatin A binding buffer were tested. The majority of HERV-K10
protease was bound to pepstatin A in the presence of 0.5 M
NaCl and complete absence of
(NH4)2SO4. Bound protease was
eluted with no salt buffer and appeared to be homogenous as assessed
with SDS-PAGE, isoelectric focusing, and native PAGE (data not
shown).
The expression plasmid for full-length HERV-K10 protease was
constructed so that only five additional amino acids (in addition to T7
tag) were present at N terminus because the presence of longer flanking
region was observed not to be necessary for proper autoprocessing. At
the C terminus the protease extends all the way to the termination
codon that is present in full-length HERV-K10 provirus (6), which
accounts for additional 50 amino acid residues not present in the
truncated, core protease version. Nucleotide and deduced amino acid
sequence of the clone that coded for full-length protease differ from
that published by Ono et al. (6) as described previously
(25). The full-length version differs from the truncated form also in
the residue at position 65 (mature HERV protease numbering; Fig. 3);
this residue is not positioned close to the active site or in the flaps
and is believed not to be important for substrate or inhibitor binding.
Metal chelation chromatography and subsequent ion exchange
chromatography were used to purify both mature wild type full-length
HERV-K10 protease and its active site mutant (D26N). Soluble fraction
of E. coli cells was applied to nickel resin, and His
tag-containing protease was bound. After elution with high imidazole
buffer, the protease was further purified to homogeneity with cationic
ion exchange chromatography on MonoS column. The mature wild type
enzyme had a molecular mass of 18.2 kDa (including His tag), whereas
active site mutant showed a molecular mass of 20 kDa because of the
presence of T7 tag and remaining N-terminal pentapeptide that was not
cleaved off because of lack of enzymatic activity of the protein.
Enzymatic Activity of HERV-K10 Proteases--
Enzymatic activity
of the enzymes was quantitatively assessed by determining kinetic
constants for the hydrolysis of
2-aminobenzoyl-Ala-Thr-His-Gln-Val-Tyr-Phe(NO2)-Val-Arg-Lys-Ala. First, the Km values were determined. After
identifying compounds with potent inhibitory activity, the active sites
of the protein preparations were titrated, and the
kcat values were then calculated from
vmax. As can be seen in Table
I, the Km value for
the truncated version of HERV-K10 protease was about 20 times lower
than that of the full-length counterpart. Similar ratio was observed
previously for the hydrolysis of a different peptide substrate and
under slightly different reaction conditions; in that report the
Km for the truncated version was about 10 times
lower than that of the full-length enzyme (25). The turnover capacity
(kcat) of the full-length protease was about 10 times higher than that of the truncated form, resulting in a catalytic
efficiency that was twice higher for the 13-kDa protein than what one
could observe with the 18-kDa form. The ratio of kcat values of both protease forms differs from
that in a previous report (25); the difference is probably to be
attributed to different substrates and to slightly different reaction
conditions that were used in the assays.
Enzymatic activities of both versions of HERV-K10 protease were then
tested against polyprotein substrate. Radioactively labeled HERV-K10
Gag polypeptide was shown to be successfully cleaved by both versions
of the protease, as well as by recombinant HIV-1 protease (Fig.
4A). The specificities of
full-length and truncated forms seemed to be identical as suggested by
similar cleavage patterns. HIV-1 protease, however, cleaved HERV-K10
Gag polypeptide at different sites, suggesting different substrate
specificity under the reaction conditions that we used in our assay.
Active site mutant of full-length HERV-K10 protease was not
enzymatically active, as expected. These results seem to be consistent
with differential cleavage of HIV-1 Gag and Pol precursors by HERV-K10 protease in the context of chimeric virions, where the HERV enzyme cleaved HIV-1 polyproteins at both apparently authentic as well as
nonauthentic sites (17, 38).
Identification of Potent HERV-K10 Inhibitors--
To evaluate the
capacity of potent HIV-1 protease inhibitors to inhibit HERV-K10
protease, Ki (app) values for a series
of P2,P2'-substituted cyclic ureas were determined. In addition,
pepstatin and three Food and Drug Administration-approved HIV-1
protease inhibitors, ritonavir, saquinavir, and indinavir, were
tested. The apparent inhibition constants for both
versions of HERV-K10 protease are shown in Table
II, together with previously reported
values for inhibition of wild type HIV-1 protease (39). Although
potent inhibitors of wild type HIV-1 protease activity, the three Food
and Drug Administration-approved compounds turned out to be weak
inhibitors of both versions of HERV-K10 protease. The linear peptidyl
mimetic inhibitors had Ki (app) values
ranging from 0.6 to 5.7 µM.
A series of 13 compounds of the cyclic urea class was tested, all of
them being P2,P2'-substituted. The symmetric substituted cyclic ureas
in general fared better in inhibition assay than the five asymmetric
compounds tested. From the latter, compound Q8467 exhibited the weakest
activity, with the apparent inhibition constants being 16 and 61 nM for truncated and full-length HERV-K10 protease,
respectively. The remaining asymmetric ureas (SD152, SD145, XW805, and
XV651) did not differ significantly from each other, their
Ki (app) values being in the range of
about 3-8 nM for 13-kDa protein and about 30-40
nM for 18-kDa form. Among cyclic C2 symmetric
ureas the compound with the smallest, cyclopropyl side groups, XK234,
fared the worst, the Ki (app) values
being about 0.7 and 1.9 µM. This compound had also turned out to be less efficient in inhibiting HIV-1 protease than the bulkier
members of this group. XM412, also known as DMP450, containing m-aminomethylbenzyl groups, exhibited more inhibitory
potency toward HERV-K10 proteases, although with apparent inhibition
constants of about 90 and 400 nM, it was still much less
potent than the remaining six cyclic ureas. XV643, XV644, SD146, XV648,
and XV652 were capable of inhibiting 13-kDa protease in subnanomolar
range, with the Ki (app) values ranging
from 0.10 nM for XV648 to 0.52 nM for XV652.
The group of these five compounds inhibited the 18-kDa enzyme in
nanomolar range; apparent inhibition constants were 2.3-4.3
nM. In general, Ki (app) values for the full-length form of HERV-K10 protease were about 3-20
times higher than those for the truncated counterpart; however, the
compounds that acted as weak inhibitors with one version of the
protease were also weak with the other and vice versa. The differences
in Ki (app) values between both
versions of the protease were consistent with the lower
Km value obtained for the 13-kDa form and were
observed also in a previous report where compounds KNI-227 and KNI-272
were measured (25). The differences are very likely to be attributed to
50-amino acid C-terminal extension present in the full-length enzyme;
however, in the absence of x-ray data it is not possible to provide a
more detailed explanation.
Inhibition of HERV-K10 Gag Processing--
SD146 was previously
reported (40) to have potent activity in cells to block HIV-1 Gag
processing by a variety of HIV-1 protease mutants. Because of this and
its excellent potency against HERV-K10 proteases (Table II), it was
chosen for detailed studies of HERV-K10 Gag processing. To estimate the
range of concentration at which which SD146 inhibits processing of
HERV-K Gag polyprotein, we first tested the system with recombinant
HERV-K10 protease and in vitro translated HERV-K10 Gag
polyprotein substrate. SD146 inhibited the processing of HERV Gag, with
the dose response data shown in Fig. 4B. On the basis of the
quantitation of substrate disappearance, the IC50 was
estimated to be 0.35 µM.
Among the cell lines we examined, the only one in which we could detect
synthesis/release of HERV Gag polypeptides was NCCIT (Fig.
5), although PA-1 appeared to express
small quantities of complete and partially processed intracellular HERV
Gag (not shown). NCCIT cells released HERV-K Gag polypeptides that were
detected mainly at 30 kDa, although also observed were varying amounts of larger polypeptides of 39 and 76 kDa (Fig. 5A) (1, 16). When 1 or 2 µM SD146 was added to the cells, and cells
were incubated for 1 day or more, the pattern changed drastically, with
the released particles containing little or no 30-kDa polypeptide and
correspondingly greater amounts of the 76-kDa full-length Gag precursor
(Fig. 5A). Also seen in inhibitor-treated NCCIT cells and
virus particles were forms of Gag-related antigens larger than 76 kDa,
suggesting possible accumulation of Gag-Pol precursors. This was
concurrent with disappearance of the processed 30-kDa core polypeptide.
The difference in cell lysates was present but much less obvious than that in virion samples, mainly because only a small quantity of processing of HERV Gag is intracellular. In cells treated with 1 µM SD146 we could observe the disappearance of p30 (Fig.
5B). Dose response data were obtained for the inhibition of
HERV Gag and HERV protease processing (Fig. 5, C and
D, respectively). The size of the mature HERV-K
protease seemed to be slightly higher than 18 kDa (Fig. 5D).
On the basis of the quantitation of product appearance, we estimated
the IC50 to be 0.37 µM in the case of Gag
processing and 0.42 µM in the case of protease
maturation. Taken together, the results in Fig. 5 show that the HIV-1
protease inhibitor SD146 is able to effectively block HERV-K10 Gag
processing, both in a teratocarcinoma cell line and in the released
particles, as predicted from our enzyme inhibition cell-free results
(Table II and Fig. 4B).
RT-PCR and DNA Sequencing of NCCIT-derived Virions--
To verify
that the particles derived from NCCIT cell line are indeed HERV-K
encoded, viral RNA was isolated from the cell culture medium and its
protease region RT-PCR amplified. A single product of expected size
(~500 base pairs) was obtained. Direct DNA sequencing of the PCR
product resulted in a single sequence and revealed that this region
differs from the HERV-K10 clone published by Ono et al. (6)
in 2 nucleotides. Neither of the substitutions (T3545C and C3572T;
numbering as in Ref. 6) lead to an amino acid change. When BLAST search
was performed against all nucleotide sequences deposited in
GenBankTM to that date, the amplified region of RNA of
NCCIT-derived HERV particles completely matched only the HERV protease
region of a recently deposited Homo sapiens chromosome 5 clone CTB-69E10 (GenBankTM accession number AC016577).
Retroviral proteins are synthesized in the form of Gag or Gag/Pol
precursors that are then processed by the action of a virus-encoded aspartic protease. The existence of a functional HERV-K protease was
inferred from the presence of processed Gag proteins in teratocarcinoma cells (5). Direct evidence for a functional protease activity came from
expression of different clones in E. coli (15, 17, 25,
38).
Recently, a hypothesis has been proposed that HERV-K10 encoded aspartic
protease might complement HIV-1 protease during infection and thereby
interfere with clinical antiviral therapy because it is highly
resistant to currently approved HIV-1 protease inhibitors (25). To
identify low molecular weight compounds that could inhibit proteolytic
activity of this enzyme, we first expressed two versions of this
enzyme in an E. coli expression system. The N termini
of both enzymes were the result of autocatalytic processing by the
protease. The C terminus of smaller, core form was chosen on the basis
of sequence homology with mature HIV-1 protease. The C-terminal
boundary of full-length version corresponds to that found in
prt-ORF of proviral DNA (6); this version has 50 additional
amino acids on its C terminus. Whether it is the full-length enzyme
that is biologically relevant or additional C-terminal processing
occurs to give rise to smaller molecular species remains to be seen.
Initial studies suggest that some limited cleavage of 13 amino acid
residues at the C terminus occurs after prolonged incubation (25,
38).
The DNA sequence of HERV-K10 protease ORF strongly suggests that this
protease belongs to the group of aspartic proteases, because the ORF
contains sequence motif LVDTGAXX(T/S)(V/I). Furthermore, a
sequence GLVGIG, a so-called "flap," is found downstream of the
active center. In addition, the sequence GRDLL conserved in aspartic
proteases, is found at nucleotide position 3723-3737 (Ref. 15;
numbering as in Ref. 6). Schommer et al. (17) showed that
presence of high concentration of HIV-1 protease inhibitor Ro 31-8959
(saquinavir) can inhibit autoprocessing of HERV-K10 protease in
E. coli expression broth, suggesting a similarity between
active sites of the two viral proteases. We therefore decided to test a
series of our cyclic ureas, second generation HIV protease inhibitors
(reviewed in Ref. 41), for their ability to inhibit HERV-K10 protease.
Although as a whole the cyclic urea class has relatively poor
pharmacokinetic properties, mostly because of low water and oil
solubility (41), these compounds are extremely potent against HIV-1
protease in vitro, and some of them have very good
resistance profiles. At least one of the cyclic ureas, DMP450 (42), is
presently in human clinical trials versus HIV. Several
symmetric bisamides exhibited high potency against both versions of
HERV-K10 protease. In the absence of any available structural data, we
built a homology three-dimensional model of the 13-kDa form of this
enzyme to be able to understand the mode of action of the compounds.
The cyclic urea substituents at P1, P1', P2, and P2' are optimized for
good potency against HIV-1 protease. In this enzyme, P1 and P1'
residues form van der Waals' contacts with Pro81,
Val82, and Ile84, whereas P2 and P2' groups
form contacts with Ile47, Ile50, and
Ile84. Cyclic urea inhibitors with smaller P2 and P2' were
shown to be less potent against HERV-K10 protease than HIV-1 protease
(XK234, XM412). However, cyclic urea amides containing P3 and P3'
groups are as potent against HERV-K10 protease as HIV-1 protease
(e.g. XV652, XV643, XV644, SD146, and XV648). Most of the
hydrogen bond contacts between the cyclic urea amide inhibitor and
HIV-1 protease complexes are predicted to be maintained in the cyclic
urea amide and HERV-K10 protease complexes (Fig.
6). The potency of cyclic ureas increase
with the increasing potential of forming hydrogen bonds. For example,
SD146 (HERV-K10 Ki (app) = 0.15 nM), which is capable of forming 12 hydrogen bonds, is
~4500 times more potent than XK234 (HERV-K10
Ki (app) = 670 nM). Besides
the interaction with hydrogen bonds, the hydrophobic interaction is
predicted to be important for the good potency of the cyclic urea
amides. For instance, the substitution of Ile47 in HIV-1
protease for Leu52 in HERV-K10 protease is predicted to
result in loss of van der Waals' interactions with P2 or P2' groups,
but at the same time this change results in increased van der Waals'
interactions between Leu52 HERV and P3, P3' groups of
cyclic urea amides. A similar effect caused by the hydrophobic
interactions was observed previously in case of double mutant V82F/I84V
of HIV-1 protease (40, 42).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (Sigma) to a final
concentration of 0.4 mM. After 3 h at 37 °C the
bacterial cells were pelleted by centrifugation at 6000 × g for 10 min. The cells were resuspended in 50 ml of 5× TE
buffer (0.1 M Tris/HCl, 5 mM EDTA, pH 7.5) and
subjected to sonication (6 × 30 s, 40 W, microtip). The
soluble fraction was discarded. Inclusion bodies were washed twice with
20 ml of 5× TE buffer and then dissolved in 100 ml of 8 M
urea, 0.1 M Tris/HCl, pH 7.5, 1 mM DTT.
Refolding of HERV-K10 protease was achieved by dialyzing the solution
against 4 liters of 20 mM PIPES, pH 6.5, 1 M
NaCl, 1 mM DTT, at 4 °C for 3 h and then against 4 liters of fresh buffer overnight. During the renaturation procedure the
precursor form of HERV-K10 protease (20 kDa) completely autoprocessed
to give rise to the mature, catalytically active 13-kDa form. The
solution was centrifuged for 10 min to eliminate the precipitated
proteins and then further clarified by filtration through a 0.45-µm
membrane. The solution was then mixed 1:1 with buffer A (50 mM PIPES, pH 6.5, 1 M NaCl, 1 mM
EDTA, 1 mM NaK tartrate, 10% glycerol). Pepstatin A-agarose suspension (Sigma) was then added, and the flask was left
overnight at 4 °C. An Amersham Pharmacia Biotech column was packed
with the slurry and then connected to a fast protein liquid chromatography system (ÄKTA, Amersham Pharmacia Biotech). The column was washed with 5 column volumes of buffer A at 1 ml/min. The
bound proteins were eluted with buffer B (0.1 M Tris/HCl, pH 8.0, 1 mM NaK tartrate, 10% glycerol, 5% ethylene
glycol). The protease containing fractions were pooled and concentrated with Amicon stir cell over YM3 membrane to about 2 ml. Protease concentration was determined with UV spectrophotometry (27). Calculated
molar absorption coefficient of 29850 M
1
cm
1 was used. The protein solution was aliquoted and
stored at
80 °C.
-D-galactopyranoside was added, and the
culture was then incubated in a 37 °C shaker for 1 h. Cells were spun down and washed with 50 mM Tris/HCl, pH 8.0, 5 mM EDTA. Cells were then resuspended in 25 ml of lysis/wash
buffer (40 mM phosphate buffer, pH 7.0, 0.3 M
NaCl, 20 mM imidazole). Lysozyme was added to 0.2 µg/ml,
and the suspension was incubated on ice for 30 min. Cells were then
sonicated (6 × 30 s) and then centrifuged at 10,000 × g for 30 min. Supernatant was filtered through 0.45-µm syringe filter, and the pellet was discarded. One ml of Qiagen nickel-nitrilotriacetic acid Superflow resin was put in a 10-ml Bio-Rad
disposable column and then equilibrated with 10 ml of lysis/wash
buffer. The soluble fraction was applied to the column and allowed to
enter by gravity flow. 20 ml of lysis/wash buffer were used to wash the
resin. The protease was eluted with 6 ml of elution buffer (40 mM phosphate buffer, pH 7.0, 0.3 M NaCl, 300 mM imidazole) and then further purified by ion exchange
chromatography. Nickel-nitrilotriacetic acid purified material was
dialyzed against 1 liter of buffer C (20 mM sodium acetate,
2 mM EDTA, 2 mM DTT, pH 5.0) and then applied
to an Amersham Pharmacia Biotech MonoS HR 5/5 column. The resin was
washed with buffer C, and the protease was eluted with a linear NaCl
gradient from 0-1 M NaCl. The fractions containing
protease were pooled. Protein concentration was determined with UV
spectrophotometry. Molar absorption coefficients of 33,690 and 34970 M
1 cm
1 were used for wild type
and active site mutant forms, respectively.
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Fig. 1.
Linearity of HERV-K10 protease activity with
time. Full-length (18 kDa) protease and its truncated (13 kDa)
version were incubated at 25 °C at a concentration of 0.5 nM in an assay buffer containing 50 mM MES, pH
5.0, 1 M NaCl, 20% glycerol, 1 mM EDTA, with 2 µM fluorogenic peptide substrate for various times. The
amount of product found during each incubation was measured by HPLC as
described under "Experimental Procedures" and is shown as peak
height. Based on these observations the assay time of 20 or 40 min was
chosen for the 13- and 18-kDa HERV proteases, respectively.
20 °C. Their chemical structures are shown
in Fig. 2. The activity of the proteases
was measured in the absence and presence of seven different
concentrations of inhibitor at a fixed concentration of both enzyme and
substrate. The proteases were preincubated 5 min at 25 °C with
inhibitors. Substrate was then added to the final concentration of 2 µM, and the assay was carried out as described above.
Fractional activities ranging from 0.2 to 0.8 relative to uninhibited
control were fitted directly to the following Morrison equation (33).
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Fig. 2.
Chemical structures of inhibitors
tested in this study. Food and Drug Administration-approved
compounds Ro 31-8959 (saquinavir), ABT-538 (ritonavir), and MK-639
(indinavir) are all linear peptidomimetics. The second group of
compounds are P2, P2'-substituted cyclic ureas.
In this equation, [I] is inhibitor concentration,
[Et] is the concentration of active enzyme,
vi is the activity at a particular inhibitor
concentration, vo is activity of uninhibited
enzyme, vi/vo is
fractional activity, and Ki (app) is the estimated apparent inhibition constant.
(Eq. 1)
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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Fig. 3.
Amino acid sequence alignment of mature HIV-1
protease and all three versions of HERV-K10 protease expressed.
The HERV proteins shown here represent the E. coli
expression products. N-terminal T7 tag and C-terminal His tag are
underlined. The DTG signature motif of aspartic proteases is
shown in gray. Sites of N-terminal autoprocessing of
HERV-K10 protease are denoted with an arrow. ASM,
active site mutant; WT, wild type. Because of two
insertions, the numberings of amino acid residues of mature HIV-1 and
HERV-K10 proteases differ. The numbering system that is used throughout
the text starts with the first amino acid of mature enzyme,
P in case of HIV-1 and W in case of HERV-K10
(these residues are designated in bold type).
Kinetic parameters for the cleavage of substrate
(2-aminobenzoyl)-ATHQVYF(NO2)VRKA
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Fig. 4.
HERV-K10 Gag polyprotein cleavage.
Radioactively labeled HERV-K10 Gag was prepared by in vitro
transcription/translation reaction as described under "Experimental
Procedures." A, HERV Gag was incubated in 20 mM PIPES, pH 6.5, 0.1 M NaCl, 1 mM
DTT, 10% glycerol for 1 h with 0.4 µg of either HIV-1 protease
(lane 2), 13- or 18-kDa version of HERV-K10 protease
(lanes 3 and 4, respectively), or active site
mutant of full-length HERV-K10 protease (lane 5). Lane
1 represents noncleaved HERV-K10 Gag. B, effect of
SD146 on the cleavage of HERV-K10 Gag polyprotein by HERV-K10 protease.
The in vitro translated HERV Gag was incubated with 13-kDa
form of HERV-K10 protease in presence of various concentrations of
SD146 as described under "Experimental Procedures." Lanes
1-7, HERV Gag incubated with the protease and different
concentrations of SD146 (from left to right: 0, 0.1, 0.2, 0.3, 0.5, 0.7, and 1.0 µM); lane 8,
HERV Gag incubated without the protease.
Inhibition of HERV-K10 protease and HIV-1 protease activity
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Fig. 5.
Western blot analysis of NCCIT cells and
HERV-K virions released from this cell line. Teratocarcinoma cell
line NCCIT was treated with SD146 as described under "Experimental
Procedures." HERV virions were recovered from the culture medium and
analyzed with antisera directed against HERV Gag (A-C) or
HERV-K10 protease (D). A, samples of HERV
particles. Lane 1, virions from untreated cells. Lanes
2 and 3, virions from cells treated with 1 and 2 µM SD146, respectively. B, NCCIT cell lysates.
Lane 1, untreated cells. Lane 2, cells treated
with 1 µM SD146. C, HERV Gag processing as a
function of SD146 concentration. Lanes 1-5, virions from
cells treated with 0, 0.2, 0.5, 0.75, and 1 µM SD146.
D, processing of HERV protease as a function of SD146
concentration. Samples were probed with a rabbit antiserum raised
against HERV-K10 protease at a dilution 1:250. Lanes 1-5,
virions from cells treated with 0, 0.2, 0.5, 0.75, and 1 µM SD146.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 6.
Schematic representation of hydrogen bonds
between HIV-1/HERV-K10 protease and the SD146. Hydrogen bonds
between HIV-1 protease and SD146 were determined by x-ray
crystallography (36), and those for HERV-K10 protease were modeled. In
the model of HERV-K10 protease complexed with SD146, all hydrogen bonds
are predicted to be preserved except that between the side chain of
Asp30 and ring nitrogen atom of the inhibitor
(thicker line), because Asp30 is replaced with
Val31 in HERV-K10 protease. HERV residues are in
parentheses and in bold type. All distances are
in Å.
The question of activity of the cyclic ureas in cells was addressed. In this study we demonstrated that HERV-K Gag processing in a cell environment can be blocked by synthetic protease inhibitors, as could be seen by substantially reduced proportion of HERV Gag precursor being cleaved to smaller polypeptides in NCCIT cell line treated with SD146 (Fig. 5A). To our knowledge, this is the first report of inhibition of HERV-K Gag maturation in cell milieu. Given the inability of cyclic ureas to inhibit cellular proteases (34), our results strongly support a model in which the aspartic protease of HERV-K10 processes homologous Gag polypeptides in human teratocarcinoma cells. Much of the HERV-K10 Gag within NCCIT cells is unprocessed. This is different from the case with HIV-1-infected cells, where a significant percent of HIV-1 Gag is cleaved. In contrast, processing of extracellular HERV Gag appears to be efficient, implying the HERV-K10 protease is inactive or unavailable except in maturing virions. HIV-1 protease is toxic to a variety of mammalian cells (43), but clearly human cells, including some teratocarcinoma cell lines, are not damaged by endogenous retroviral proteases. The question of whether the cells have evolved to resist the action of the endogenous viral proteases or the enzymes are sequestered/inactive until packaging/exit should be addressed.
The extracellular particle yield, as estimated by Western blotting of total viral proteins, was roughly the same in presence of the protease inhibitor, indicating that HERV-K Gag polypeptide processing is not a limiting step for particle release. Similar results obtained with viral RNA isolation from the particulate material of NCCIT cell culture medium and its subsequent quantification with RT-PCR amplification support this observation (data not shown). These data are consistent with the observation that HIV-1 protease inhibitors block the processing of Gag and Gag-Pol precursor polyproteins in HIV-1-infected cells but do not markedly alter either the number of particles released from the infected cells (44, 45) or the amount of packaged viral RNA (46, 47). In addition to using antigen-specific immunoblotting, we verified the identity of NCCIT released virions by checking the nucleotide sequence of packaged RNA. The sequence of the 500-nucleotide protease region that was RT-PCR amplified unequivocally shows that the virions belong to HERV-K family. However, additional regions would have to be sequenced for an exact clone number to be assigned, especially with regard to the fact that the recent estimates based on BLAST searches and phylogenetic analyses show that there could be as many as 170 HERV-K elements present in human genome (4). The protease amino acid sequence deduced from the obtained nucleotide sequence was identical to that of HERV-K10 clone (6).
Although cyclic ureas act as potent inhibitors of HIV-1 and HERV-K
protease, they do not inhibit mammalian, nonretroviral cellular
aspartic proteases (34). However, a question arises whether cell
processes could be affected because of HERV-K protease inhibition. The
fact that HERVs remain a constitutive part of the genome and the notion
that ORFs for all major viral proteins exist and have retained coding
capacity despite extensive deleterious effects normally associated with
endogenization of retroviruses suggest that they may confer certain
positive traits to the host (48). HERV encoded proteins, including
HERV-K protease, might well be involved in normal cell physiology and
pathophysiology. Our results in which the activity of HERV protease and
inhibition of viral protein processing could be efficiently
accomplished in teratocarcinoma cells may help to clarify the role of
HERVs in cell physiology.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Beverly C. Cordova for providing us with the human lymphocyte fraction and Drs. Ralf Tönjes and Reinhard Kurth (Paul-Ehrlich-Institut, Langen, Germany) for kindly supplying pcG3gag clone. We are especially grateful to Ronald M. Klabe and Dr. James L. Meek for excellent technical advice with HPLC enzyme assay. We thank Leah A. Breth and Jennifer E. Kochie for raising anti-HERV-K10 protease antiserum, Jeanne I. Corman for N-terminal amino acid sequencing and mass spectroscopy analysis, and Wilfred Saxe for help with modeling of HERV-K10 protease. Thanks also to Drs. Lee T. Bacheler and Robert A. Copeland for helpful discussions and to Dr. Susan K. Erickson-Viitanen for continuing support and useful suggestions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the DuPont Pharmaceuticals Company Postdoctoral Program. Permanent address: Dept. of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
** Present address: Bayer Corp., Dept. of Protein Biosciences, 400 Morgan Lane, West Haven, Connecticut 06516.
Present address: Lilly Research Labs. Indianapolis, IN 46285.
§§ To whom correspondence should be addressed: DuPont Pharmaceuticals, Dept. of Virology, Experimental Station E336/22, P.O. Box 80336, Wilmington, DE 19880-0336. Tel.: 302-695-9493; Fax: 302-695-9420; E-mail: bruce.d.korant@dupontpharma.com.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M008763200
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ABBREVIATIONS |
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The abbreviations used are: HERV, human endogenous retrovirus; HIV, human immunodeficiency virus; MES, morpholineethanesulfinic acid; ORF, open reading frame; PIPES, piperazine-N,N'-bis[2-ethanesulfonic acid]; RT, reverse transcriptase; PCR, polymerase chain reaction; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
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