From the Laboratory of Chemical Physics, NIDDK,
National Institutes of Health, Bethesda, Maryland 20892, and
Department of Biochemistry and Molecular Biology, University
Medical School of Debrecen, H-4012 Debrecen, Hungary, and
¶ Molecular Virology and Carcinogenesis Laboratory, ABL-Basic
Research Program, NCI-Frederick Cancer Research and Development
Center, Frederick, Maryland 21702-1201
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ABSTRACT |
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We have developed a system for expression and
purification of wild-type human T-cell leukemia virus type 1 (HTLV-1)
proteinase to attain sufficient quantities for structural, kinetic, and
biophysical investigations. However, similar to the human
immunodeficiency virus type 1 (HIV-1) proteinase, HTLV-1 proteinase
also undergoes autoproteolysis rapidly upon renaturation to produce two
products. The site of this autoproteolytic cleavage was mapped, and a
resistant HTLV-1 proteinase construct (L40I) as well as another
construct, wherein the two cysteine residues were exchanged to
alanines, were expressed and purified. Oligopeptide substrates
representing the naturally occurring cleavage sites in HTLV-1 were good
substrates of the HTLV-1 proteinase. The kinetic parameters
kcat and Km were nearly
identical for all the three enzymes. Although three of four peptides
representing HTLV-1 proteinase cleavage sites were fairly good
substrates of HIV-1 proteinase, only two of nine peptides representing
HIV-1 proteinase cleavage sites were hydrolyzed by the HTLV-1
proteinase, suggesting substantial differences in the specificity of
the two enzymes. The large difference in the specificity of the two
enzymes was also demonstrated by inhibition studies. Of the several
inhibitors of HIV-1 or other retroviral proteinases that were tested on
HTLV-1 proteinase, only two inhibit the enzyme with a
Ki lower than 100 nM.
The human T-cell leukemia virus type 1 (HTLV-1)1 is a retrovirus
that has been etiologically associated with human adult T-cell leukemia
(1, 2), HTLV-1-associated myelopathy, tropical spastic paraparesis (3,
4), and a number of other chronic diseases (5). Although it has not
been shown to be directly linked to the development of leukemia,
several recent studies indicate that viral replication is critical for
the development of HTLV-1-associated myelopathy. Initial studies have
reported the treatment of this syndrome using 5'azidothymidine
(6).
All replication-competent retroviruses including HIV and HTLV-1 code
for an aspartic proteinase (PR). Unlike HIV-1, HTLV-1 PR is coded
through a mechanism of expression in separate gag-pro and
gag-pro-pol open reading frames (7). The level of expression of the Gag and Gag-Pro polyproteins in HTLV-1 is comparable with that
of Gag and Gag-Pol polyproteins in HIV-1. The function of the mature
125-amino acid-long HTLV-1 PR is crucial for virion replication (for a
review, see Ref. 8). The HIV-1 PR proved to be a promising target of
antiretroviral therapy of AIDS, and various PR inhibitors are now in
clinical use (for a review, see Ref. 9).
As in the case of treatment of patients with reverse transcriptase
inhibitors, selection of viral variants that are resistant to
inhibitors of PR also develops rapidly (9). Some of the amino acid
changes in the HIV-1 PR that are responsible for drug resistance are
found in equivalent positions of other retroviral proteinases,
including HTLV-1 (see Fig. 1). A
comparative study of various retroviral proteinases is expected to
reveal the common features of their specificity. These studies will aid
in the rational design of inhibitors effective against different
retroviral proteinases, which may reduce the frequency of occurrence of
drug-resistant mutants. In this respect, many inhibitors designed
against HIV-1 PR have much reduced potency against the closely related
HIV-2 PR (10) as well as against the PR of lentiviral equine infectious anemia virus (11). Furthermore, a recent study demonstrated that all
four HIV-1 PR inhibitors currently used in therapy failed to block
HTLV-1 Gag processing in vitro (12).
INTRODUCTION
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Abstract
Introduction
References
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Fig. 1.
Sequence comparison of HIV-1 and HTLV-1
proteinases. The three conserved regions of proteinases of
HIV-1HXB2 and HTLV-1 (from clone CS95) are shown in
bold. Residues of HTLV-1 PR that are found in equivalent
positions of drug-resistant variants of HIV-1 proteinases are
underlined. Additionally, three frequently occurring
drug-resistant mutations in HIV-1, G48V, A71V, and V82F, are
represented by residues L, L, and W in
equivalent positions in HTLV-1 PR.
Although several studies report expression of HTLV-1 PR in Escherichia coli (13-17) as well as its chemical synthesis (18), its biochemical and kinetic properties and specificity have not been well described. This can be attributed to the difficulty in protein purification and to the autoproteolysis that is known to occur with native retroviral proteinases (19, 20). Peptides representing some of the natural cleavage sites of HIV-1 are hydrolyzed by HTLV-1 PR (16, 18), although no kinetic parameters were reported for these peptides.
The crystal structures of HIV-1 and several other retroviral
proteinases have been determined (21-23). However, to date no structural information is available for the HTLV-1 protease.
Furthermore, insight into the mechanism of autocatalytic maturation of
the mature PR from the precursor is limited to HIV-1 (24, 25). Here we
report the high level expression in E. coli and rapid purification of wild type and autoproteolysis-resistant HTLV-1 proteinases. The specificities of these two enzymes were compared utilizing synthetic peptides that correspond to known Gag and Pol
cleavage sites of HIV-1 and HTLV-1. Several potent inhibitors of HIV-1
PR and other retroviral proteinases have been assessed for their
inhibition of the HTLV-1 PR. We also report the effect of substituting
the two cysteine residues in HTLV-1 PR and define a chromogenic
substrate for assaying this enzyme.
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MATERIALS AND METHODS |
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Cloning, Expression, and Site-directed Mutagenesis of the HTLV-1 Proteinase-- The PR coding region of the SacI fragment of an infectious HTLV-1 clone, pCS-HTLV-I (26), derived by polymerase chain reaction amplification was cloned at the NdeI and BamHI sites of pET-11a vector (Novagen, WI). Mutagenesis of the Leu40 to Ile was performed by using the overlap extension method (27). The C90A and C109A mutations were introduced into the L40I-mutated HTLV-I PR using the Quick-Change mutagenesis protocol (Stratagene, CA). Triple mutant L40I,C90A,C109A PR was prepared by using the L40I,C109A double mutant as the template.
Cells bearing the construct for the expression of the wild-type HTLV-1
PR and its mutants were grown in Luria-Bertani medium supplemented with
1% glucose at 37 °C, 30% pO2, pH 7.0, in a 3-l Braun model MD
fermentor. When the cell suspension reached an absorbance of 0.5-0.6
at 600 nm, protein expression was induced by the addition of 2 mM isopropyl -D-thiogalactopyranoside for 3-4 h. Cells (4 g) were suspended in 20 volumes of buffer A (50 mM Tris-HCl, pH 8.2, 1 mM DTT, 1 mM
EDTA) containing 100 µg/ml lysozyme and lysed by sonication. The
insoluble recombinant protein (inclusion bodies) was washed twice by
resuspension in buffer A containing 1 M urea and 1% Triton
X-100 and pelleted by centrifugation at 20,000 × g for
30 min. The final pellet was solubilized in 50 mM Tris-HCl,
pH 8.0, 7.5 M guanidine HCl, 5 mM DTT, and 5 mM EDTA and applied to a Superdex 75 column (HiLoad 26 × 60, Amersham Pharmacia Biotech, NJ) equilibrated in 50 mM Tris-HCl, pH 7.5, 4 M guanidine HCl, 5 mM EDTA, and 5 mM DTT at a flow rate of 3 ml/min. Peak fractions were further purified by reverse-phase HPLC on
POROS RII resin (PerSeptive Biosystems, MA). HPLC fractions were
combined, and aliquots were dried, assayed for protein content as
described previously (28), and subjected to mass spectroscopic and
N-terminal sequence analyses.
Protein Folding-- The HPLC-purified protein fractions were dialyzed in large excess of 25 mM formic acid, pH 2.8, and subsequently into 50 mM sodium acetate buffer, pH 5.0, 1 mM, DTT, 1 mM EDTA. For kinetic studies, a portion of the protein in 25 mM formic acid was quenched with at least 100-fold excess of 20 mM PIPES buffer, pH 7.0, containing 150 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM DTT, and 0.5% Nonidet P-40.
Oligopeptide Synthesis and Characterization-- Oligopeptides were synthesized by standard tert-butoxycarbonyl or 9-fluorenylmethyloxycarbonyl chemistry on a model 430A automated peptide synthesizer (Applied Biosystems, Inc.) or a semiautomatic Vega peptide synthesizer (Vega-Fox Biochemicals). All peptides were synthesized with C-terminal amides. Amino acid composition of the peptides was determined with a Beckman 6300 amino acid analyzer. Stock solutions and dilutions were made in distilled water (or in 10 mM DTT for peptides containing Cys or Met residues), and the peptide concentrations were determined by amino acid analysis.
Proteinase Inhibitors-- Substrate-based inhibitors of HIV-1 PR assayed as inhibitors of HTLV-1 PR were Ro-31-8959 (29), DMP 323 (30), UK-88,897 (31), KH 164 (32), and Compound 3 (33). Pepstatin A was obtained from Sigma, and reduced peptide bond inhibitor (N-1460) was purchased from Bachem BioScience Inc., King of Prussia, PA. Dr. Ivo Bláha (Ferring Leciva, Prague) prepared the statine- and phenylstatine-containing peptide inhibitors.
Enzyme Assay with Oligopeptide Substrates--
Purified HIV-1 PR
was prepared as described previously
(34).2 Active-site titration
for the HIV-1 PR was performed with Compound 3 (33). The enzyme
concentrations for HTLV-1 PR preparations were determined by amino acid
analysis of the stock solutions, and kcat values
were calculated by assuming 100% activity for the PR dimers. The PR
assays were initiated by mixing 5 µl of HTLV-1 or HIV-1 PR (8-140
nM), 10 µl of 2× incubation buffer (0.5 M
potassium phosphate buffer, pH 5.6, containing 10% glycerol, 2 mM EDTA, 10 mM DTT, 4 M NaCl), and
5 µl of 0.01 to 0.40 mM substrate. The range of substrate
concentration was selected depending on the approximate
Km values. The reaction mixture was incubated at
37 °C for 1 h and terminated by the addition of guanidine HCl to give a final concentration of 6 M. The solution was
acidified by the addition of trifluoroacetic acid, and an aliquot was
injected onto a Nova-Pak C18 reversed-phase chromatography
column (3.9 × 150 mm, Waters Associates, Inc.) using an automatic
injector. Substrates and the cleavage products were separated using an
increasing water-acetonitrile gradient (0-100%) in the presence of
0.05% trifluoroacetic acid. Cleavage products were identified by amino acid analysis. 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.). To assay
the inhibitors, the volume of substrate KTKVLVVQPK was reduced to
4.8 µl (0.25 mM final concentration), and 0.2 µl of inhibitor (in water or in Me2SO) was added. Inhibitors were
initially tested at 10 µM or higher concentration.
Me2SO was also added at the same concentration to the
appropriate control experiments. When inhibition was observed in
initial testing, Ki values were determined according
to Dixon (35). A competitive mode of inhibition was observed, and the
standard error for Ki values was estimated to be
below 20%.
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RESULTS AND DISCUSSION |
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Expression and Purification of HTLV-1 Proteinase--
The
375-base pair coding region derived from an infectious clone of HTLV-1
by polymerase chain reaction was cloned into pET-11a vector and
expressed in E. coli. Fig. 2
(lanes 1 and 2) shows the analysis of the total
cell lysate before and after isopropyl -D-thiogalactopyranoside induction. Two products were
observed, the larger molecular weight product corresponding to the
full-length mature PR and a smaller product with an apparent size of
roughly 7 kDa. The latter product was expected to be the result of
self-degradation (autoproteolysis) of the mature PR rather than by
premature termination during expression.
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As in the case of HIV-1 PR (28, 36), expression of HTLV-1 PR results in its accumulation in the form of inclusion bodies. Lanes 3, 4, and 5 show the supernatant upon lysis of cells and the supernatant and the pellet derived after 1 M urea treatment of the initial insoluble pellet, respectively. A comparison of lanes 4 and 5 shows that 1 M urea partially solublizes the full-length PR but not the truncated protein. The insoluble fraction after 1 M urea treatment of the inclusion bodies (see lane 5) was solubilized in guanidine hydrochloride and subjected to size-exclusion chromatography under denaturing conditions. The peak fractions of both the full-length PR and the truncated protein were further subjected to reverse-phase HPLC chromatography. The two proteins that were >98% pure were analyzed for their mass and N-terminal sequence. The full-length mature PR clearly corresponded to the expected N-terminal sequence and mass (m/z = 13475). The truncated protein showed its N-terminal sequence to start with residues Pro-Ile-Ala-Leu-Phe-Ser-Ser, and the expected mass was close to the experimental value (m/z = 9171). From these results, we confirmed our prediction that the truncated protein is derived via autoproteolysis between residues Leu-40/Pro-41 of the full-length mature PR with a sequence spanning amino acids 41 through 125, now termed PR 41-125. The pure full-length protein was also renatured from 25 mM formic acid into 50 mM sodium acetate buffer, pH 5.5, 1 mM DTT, 1 mM EDTA, and 0.05% Triton X-100 at a final concentration of 6.7 µM and subjected to SDS-polyacrylamide gel electrophoresis (Fig. 2, lane 6). As expected, the full-length PR gave rise to two products, with apparent mobilities corresponding to ~9 and 4.5 kDa. The latter protein is termed PR 1-40. Also, similar to the HIV-1 PR, autoproteolysis of the HTLV-1 PR proceeds roughly to about 50%. It is likely that either one or both products inhibit further autoproteolysis. This site of autoproteolysis in HTLV-1 PR is analogous to a less susceptible site in HIV-1 PR (Leu33-Glu34). The major cleavage in HIV-1 PR occurs between residues Leu5 and Trp6 (19). Recently the feline immunodeficiency virus PR was also shown to undergo autoproteolysis at four primary sites in its 116-amino acid sequence (20).
Autoproteolysis-resistant HTLV-1 Proteinase--
To prevent
autoproteolysis of the mature HTLV-1 PR, the Leu40 residue
was substituted with an Ile. This substitution was chosen because it
has been shown by using synthetic peptides and proteins as substrates
that HIV-1 and other retroviral proteinases cannot process at the
C-terminal side of -branched residues (37, 38). Furthermore,
autoprocessing at an equivalent position (Leu33) and
another position (Leu63) in HIV-1 was effectively reduced
by substituting these residues with Ile without impairing the
kcat/Km for several substrates (36). The
expression of the HTLV-1 PR bearing the mutation L40I compared with the
wild-type PR is shown in Fig. 3. Clearly
as observed in duplicate clones, there is a single product that
accumulates with a substantially higher yield corresponding to the size
of the full-length PR. 1.6 g (wet weight) of cells derived from
0.4 liters of culture yields ~19 and 14 mg of PR after size-exclusion and reversed-phase HPLC chromatography, respectively.
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Kinetic Characterization of the Wild-type and L40I HTLV-I Proteinases-- Based on the sequence comparison with HIV-1 PR and homologous molecular modeling,3 the Leu40 residue of HTLV-1 PR is not expected to interact with the substrate or inhibitor. Therefore we predicted that substituting this residue with Ile would not change the kinetic parameters but would prevent its autolysis as compared with the wild-type enzyme. To verify this assumption and to characterize the HTLV-1 PR specificity, kinetic analysis was performed using oligopeptide substrates. The proteinases were renatured by quenching the denatured protein in 50 mM formic acid into 100-fold excess of PIPES buffer at pH 7.0 under conditions similar to those previously described for refolding HIV-2 and equine infectious anemia virus proteinases (39). Kinetic data for the wild type and L40I mutant PR-catalyzed hydrolysis of synthetic substrates are shown in Table I. Both enzymes exhibit nearly identical kinetic parameters. The nearly identical values of kcat for the wild-type and mutant enzymes also suggest that under these refolding and assay conditions there is no significant autoproteolysis of the wild-type PR. Stabilization of the enzyme can be attributed to 1) the refolding procedure, which involves quenching the denatured protein with a large excess of buffer (pH 7) to a final concentration of 0.1 µM as compared with dialyzing the protein to pH 5.0 at a higher protein concentration and 2) presence of Nonidet P-40 and glycerol in the quench buffer.
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Mutation of the Cysteine Residues of the Stabilized HTLV-1 Proteinase-- Because one of our major objectives was to produce quantities of enzyme suitable for structural studies by solution NMR and x-ray crystallography, we next addressed the role of the two cysteines (Cys90, Cys109) on enzymatic activity and specificity of the PR. It was shown that a Cys to Ala substitution at residue 95 of HIV-1 mature PR greatly reduced the tendency of the protein to form an intermolecular disulfide bond (40). Subsequent studies have shown that both the Cys67 and Cys95 residues of HIV-1 can be exchanged to Ala without significantly affecting the structure and kinetic parameters (41).2
Constructs bearing either or both of the two cysteines substituted by Ala were prepared in a L40I HTLV-1 PR construct as described under "Materials and Methods." We chose to compare the triple mutant L40I,C90A,C109A (termed C2A-PR) with the wild-type PR. C2A-PR was purified as described before for the wild-type HTLV-1 PR. Fig. 4 shows the enzyme before and after refolding. Clearly the refolded enzyme was stable up to 24 h at a concentration of 0.6 mM at ambient temperature. The triple mutant was used to compare the specificity of HTLV-1 and HIV-1 proteinases on naturally occurring cleavage sites of HTLV-1 Gag-Pol polyprotein (Table II).
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Kinetic Characterization of the Stabilized C2A-PR Mutant HTLV-1 Proteinase and Comparison of Its Specificity with HIV-1 Proteinase-- To characterize the specificity of C2A-PR, we first used oligopeptide substrates that represent naturally occurring cleavage sites in HTLV-1 Gag-Pol polyprotein. Comparison of the kinetic parameters using C2A-PR (Table II) to those obtained with wild-type and L40I mutant enzymes (Table I) suggest that substitution of the Cys residues to Ala does not alter its catalytic activity. Three of the four peptides tested were excellent substrates of HTLV-1 PR, with catalytic constants comparable with the best naturally occurring HIV-1 cleavage site substrates with HIV-1 PR (42). Thus HTLV PR is an enzyme with high specific activity, similar to HIV-1 PR but unlike avian myeloblastosis virus PR, which has much lower kcat for its specific substrates. This interpretation is consistent with the mechanisms that regulate the level of expression of the Gag and Gag-Pol products. HIV-1 and HTLV-1, wherein the PR is coded in a Pol open reading frame at ~10-fold lower level than Gag proteins, have higher specific activity for the PR as compared with avian myeloblastosis virus PR, which is coded in a Gag open reading frame. Another interesting similarity with the HIV-1 and HTLV-1 proteinases is that among the least efficient sites for hydrolysis is the one that yields the free N terminus of PR (42). Studies with an analog of the HIV-1 PR precursor indicate that the cleavage at the N terminus of the PR occurs intramolecularly, concomitant with a large increase in enzymatic activity, and this cleavage has been suggested to be the rate-limiting step in the maturation cascade of Gag-Pol polyprotein (24, 43). When HTLV-1 cleavage site substrates were used as substrates for HIV-1 PR (Table II), three of four peptides were found to be hydrolyzed. The kinetic parameters of HIV-1 PR-catalyzed hydrolysis of these HTLV-1 substrates were in the same range as those for substrates that correspond to analogous sites in HIV-1 (42). On the other hand, only two of the nine HIV-1 cleavage site substrates were cleaved by the HTLV-1 PR (Table III) but with substantially lower values of kcat/Km than those obtained with HTLV-1 substrates.
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The lack of cleavage of HIV-1 Gag substrates by HTLV-1 PR is in good agreement with previous findings that HTLV-1 PR was not able to process HIV-1 Gag precursor in a vaccinia virus expression system (44). In the same expression system, HIV-1 PR was also not able to process HTLV-1 Gag (44) even though the peptides representing HTLV-1 Gag cleavage sites were hydrolyzed by HIV-1 PR in vitro. This result in vivo may at least partially be because of reduced specificity of this enzyme toward these sites as compared with HTLV-1 PR.
Hydrolysis of Chromogenic Substrates by HTLV-1 Proteinase-- To develop a more convenient chromogenic assay, we synthesized and assayed analogs of the HTLV-1 CA/NC cleavage site substrate that contained a Phe (NO2) substitution in either the P1 or P1' positions (Table III). Although the substrate with P1' substitution was an inefficient substrate, the substrate containing the P1 substitution was hydrolyzed to about 4% the level of the native substrate. This substrate may serve to screen for ideal-folding conditions of C2A-PR in simple buffer systems for structural studies. In good agreement with the lack of hydrolysis of the HIV-1 CA/p2 peptide by HTLV-1 PR, its chromogenic derivative, which is a widely used chromogenic substrate of HIV-1 PR was not hydrolyzed (Table III).
Daenke et al. (16) reported kinetic parameters for peptides
representing naturally occurring cleavage sites of HTLV-1 in which the
P1' residue were substituted by a chromogenic reporter, Phe
(NO2). They have reported Km and
kcat values within a range of 0.003-0.031 mM
and 14-33 s1, respectively. These Km
values are lower than our observed values (0.030-0.230 mM,
see Table I) using entirely natural peptides with no substitutions
whereas the kcat values were substantially higher, assayed
under similar conditions. The difference in Km values may be because of the introduction of the bulky aromatic residue
in the P1' position. This may also be the reason why the naturally
occurring cleavage site sequences representing the autocatalytic maturation sites (TF1/PR and PR/p3) in HTLV-1 as compared with the
corresponding chromogenic derivatives do not have substantially higher
affinities toward HTLV-1 PR than the peptides representing other sites,
as noted for the chromogenic derivatives by Daenke et al.
(16). The reason for the differences in the reported kcat
values is less obvious. When we introduced Phe(NO2) group in either the P1 or P1' positions of the naturally occurring cleavage site sequence, a substantial decrease in hydrolytic rate was observed instead of the expected increase (see Table III). Our observations which suggest that bulky residues at the site of cleavage are unfavorable are in accordance with the interpretation of Pettit et al. (12) that lack of inhibition by HIV-1 PR inhibitors
on in vitro HTLV-1 Gag processing might be because of the
presence of Trp98 in HTLV-1 rather than Val in equivalent position of
HIV-1 reducing the size of S1 binding pocket. A two-fold increase in NaCl concentration used in our assay conditions as compared with that
reported (16) does not account for this large difference in
kcat values. The specific activity reported using the
chromogenic substrate representing the CA/NC site (1 µmol/min/mg;
Ref. 16) suggests a much smaller catalytic rate.
Inhibition of the HTLV-1 Proteinase-- Several inhibitors of HIV-1 PR were assayed as inhibitors of the HTLV-1 PR. DMP 323, KH 164, and Compound 3 have previously been shown to be potent inhibitors of the HIV-1 PR. Only Compound 3 inhibited HTLV-1 PR with a Ki of 80 nM, whereas the others did not inhibit up to 10 µM (Table VI). Another potent inhibitor of HIV-1 PR, Ro-31-8959 was also inactive against HTLV-1 PR (16). These results also emphasize the large differences in specificity of the two enzymes. Interestingly, Compound 3 was also a good inhibitor of HIV-2 PR (45), equine infectious anemia virus PR (39), murine leukemia virus, and mouse mammary tumor virus proteinases (46) and, therefore, may serve as a lead compound for general inhibitors of retroviral proteinases. However, the most potent inhibitor of the enzyme in our assay system was a statine-based inhibitor, which was based on the MA/CA cleavage site sequence with a Ki of 50 nM (Inhibitor 10 in Table IV). Potent, active-site inhibitor of Mason-Pfizer monkey virus PR (Inhibitor 9 in Table IV; Ref. 47) and avian myeloblastosis virus PR (Inhibitor 8 in Table IV; Ref. 50) also did not inhibit the HTLV-1 PR. N-1460, a low nM peptide analog inhibitor of HIV-1 PR, and LP-149, inhibitor of HIV-1 and feline immunodeficiency virus proteinases, do not inhibit HTLV-1 PR up to 10 µM.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jane M. Sayer for critical review of the manuscript, Dr. Barbara Felber for providing the pCS-HTLV-1 clone, Deanna Gotte for help in the early stage of the cloning of the HTLV-1 PR, Dr. Lewis K. Pannell for mass spectroscopic analyses, Suzanne Specht for help with the amino acid analysis, Eva Majerova for peptide synthesis, and Dr. H. Parikh for contract services provided at Braton Biotech, Rockville, Inc., MD. The UK 88,947 and Ro-31-8959 inhibitors were generous gifts from Dr. Peter Whittle, KH 164 and LP-149 from Dr. Kwan Y. Hui, and DMP 323 from Dr. N. Hodge.
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FOOTNOTES |
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* This research was sponsored in part by the Hungarian Science and Research Fund (OTKA T 16396, OTKA T 22140), by the Hungarian Ministry of Culture and Education (FKFP 1318/97), by the National Institutes of Health (NCI) Department of Health and Human Services under contract with Advanced BioScience Laboratories and by the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health.
§ Contributed equally to this research. To whom correspondence should be addressed. J. Tözsér: Tel./Fax: 36-52-416-432; E-mail: tozser{at}indi.biochem.dote.hu or J. M. Louis: Tel./Fax: 301-594-3122/301-496-0825, E-mail: jmlouis{at}helix.nih.gov.
2 J. M. Louis, unpublished data.
3 P. Bagossi, G. Zahuczky, and J. Tozser, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: HTLV-1, human T-cell leukemia virus type 1; HIV-1 and HIV-2, human immunodeficiency virus type 1 and type 2, respectively; DTT, dithiothreitol; HPLC, high performance liquid chromatography; PR, proteinase; PIPES, 1,4-piperazinediethanesulfonic acid; MA, matrix protein; CA, capsid protein; NC, nucleocapsid protein; C2A-PR, stabilized HTLV-1 PR containing substitution mutations L40I,C90A,C109A. The nomenclature of viral proteins is according to Leis et al.(51).
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REFERENCES |
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