From the
We have cloned and expressed the 3` region of the Mason-Pfizer
monkey virus pro gene in Escherichia coli. The
recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH
Mason-Pfizer monkey virus, the prototype type D retrovirus
induces an AIDS-like syndrome in non-human primates that is distinct
from that caused by simian immunodeficiency virus. Unlike type C
retroviruses that assemble underneath the cell membrane during budding,
simian D-type retroviruses as well as mouse mammary tumor virus
preassemble viral capsids (intracytoplasmic A-type particles or
ICAPs)
The genomic organization of
M-PMV is similar to that of other retroviruses: 5`-LTR-gag-pro-pol-env-LTR-3` (where LTR is long
terminal repeat). The gag gene encodes a precursor
polypeptide, Pr78, which is cleaved into six capsid proteins, NH
ICAPs are formed by assembly of Gag, Gag-Pro, and
Gag-Pro-Pol polypeptide precursors at sites in the cytoplasm. These
fully assembled, yet uncleaved immature capsids are then transported to
the plasma membrane. During or shortly after particle release from the
cell, the viral PR, as part of Gag-Pro and Gag-Pro-Pol precursors, is
activated and cleaves the precursor polypeptides into their constituent
proteins. This tightly regulated proteolytic event results in a
morphological change in virus particles and is essential for virus
infectivity(1) .
There is ample genetic, biochemical, and
structural evidence that retroviral PRs are aspartyl proteinases, which
must form dimers in order to become active(2) . We have
previously shown for M-PMV that inactivation of PR by site-directed
mutagenesis or by specific inhibitors does not affect the assembly of
immature viral particles, which are released but are not
infectious(1) . Similar results have been reported for other
retroviruses(3) . In M-PMV PR-deficient virions, not only do the
Gag-containing polypeptides remain unprocessed, the transmembrane
glycoprotein, which is initially synthesized as a cell-associated gp22,
is not cleaved within the cytoplasmic domain to generate the gp20 found
in wild-type virions(1, 4) . While these studies
provided evidence that M-PMV PR belongs to the aspartyl proteinase
family, they provided little information on the biochemical and
structural properties of this enzyme.
M-PMV PR is present as part of
both the Gag-Pro and Gag-Pro-Pol precursors within ICAPs, but is not
activated while the capsid remains in the cytoplasm(5) . This is
not the case in HIV, where high level expression of the Gag-Pro
precursor results in intracellular activation of the precursor and
subsequent cleavage of Gag-containing polypeptides (6, 7). Thus it can
be assumed that in D-type retroviruses the process of PR activation is
under tighter control, preventing polyprotein cleavage prior to
particle budding and release.
Previous studies have shown that
retroviral PR precursors expressed in heterologous biological systems
can be accurately cleaved both in vivo and in vitro in an autocatalytic fashion. In order to obtain biochemical and
structural information about this viral PR, we expressed part of the pro open reading frame as an artificial 26-kDa precursor in Escherichia coli. We were able to isolate and purify two
protein species, p17 and p12, which possess proteolytic activity. The
purified proteins have been used to determine the substrate specificity
of the enzyme and its relatedness to other retroviral enzymes.
QAE-Sephadex A
25 (Fig. 2, lane7) retains some of the high
molecular weight contaminants, allowing the different forms of PR to be
purified by FPLC on a Mono S column (Fig. 3). Peak 1, which
elutes at 0.46 M NaCl, contains the 12-kDa PR (p12) associated
with a 5-kDa peptide (p5). Peak 2, eluting at 0.54 M NaCl,
corresponds to the 12-kDa PR homodimer. The largest peak, peak 3,
eluting at 0.62 M NaCl, contains 12-kDa plus 17-kDa species.
This may represent either two copurifying homodimers or a p17/p12
heterodimer. Finally the smallest peak, peak 4, which elutes at 0.74 M NaCl, contains the 17-kDa PR homodimer (p17). Upon storage
at 4 °C and pH 5.5, the 17-kDa species is self-processed slowly
into the 12-kDa form and the 5-kDa peptide (data not shown).
In initial experiments, the M-PMV synthetic substrates were
also used to detect the activity of the PR samples in the course of
purification. To find a sensitive chromogenic substrate suitable for a
rapid spectrophotometrical assay, we examined cleavage of p-nitrophenylalanine-containing peptides designed as
substrates for HIV-1 and MAV PRs(18, 19) . The peptides
based on the capsid protein/nucleocapsid protein junction of HIV-1 were
not hydrolyzed by M-PMV PR. In contrast, the chromogenic substrate
based upon the reverse transcriptase/integrase processing site of MAV (, peptide 6) was cleaved readily by the M-PMV PR. The K
In the relative cleavage rates of peptides spanning
the cleavage sites on HIV-1 and MAV polyproteins by M-PMV PR are
summarized. These data support the above mentioned results showing that
the substrate specificity of M-PMV PR toward the small synthetic
substrates is similar to that of MAV PR and distinct from that of HIV-1
PR. M-PMV PR hydrolyzed only one of four HIV-1-derived peptides tested;
interestingly, the only sequence cleaved was based on the
NH
M-PMV is a prototypic type D retrovirus that, in a manner
similar to the B-type MMTV, assembles immature ICAPs in the cell
cytoplasm, and then transports these particles to the cellular
membrane, where they undergo proteolytic maturation during release from
the cell. Clearly, this arrangement requires a very tightly regulated
activation of the virally programmed proteolytic event(s). Not only
does M-PMV PR cleave gag gene-related precursors(1) ,
its activity is also necessary for the removal of a carboxyl-terminal
segment from the transmembrane glycoprotein, gp22, resulting in a
virus-associated gp20 (1, 4). M-PMV PR has never been isolated from
virions, and the enzyme had not been characterized prior to this
report.
In order to characterize the region with PR activity, we
have cloned and expressed in bacteria the 3` portion of the M-PMV pro
gene; the coding sequence for the highly conserved active site triplet
Asp-Thr-Gly of the aspartic proteinases lies in this part of the gene.
The 5` region of the pro gene of D-type retroviruses, as well as that
of MMTV, encodes a protein with dUTPase
activity(21, 22, 23) . Surprisingly,
self-processing of the recombinant precursor yielded two proteins with
PR activity: p17 and p12 (Fig. 4). NH
MAV PR is naturally less active than HIV-1 PR since it
is synthesized as the carboxyl-terminal part of Gag polyprotein and is
therefore produced in equimolar amounts with the structural Gag-related
viral components. In contrast, in a manner more analogous to the HIV-1
enzyme, most of the M-PMV PR is synthesized in 5-10-fold lower
amounts than Gag polyprotein, as a part of a Gag-Pro polyprotein (M-PMV
PR is also synthesized as a part of Gag-Pro-Pol polyprotein, in this
case in 5-fold lower amounts than the Gag-Pro polyprotein).
Nevertheless, the specificity and activity of the M-PMV PR is closer to
that of MAV than to that of HIV-1. This observation is clear from
comparisons of MAV, M-PMV, and HIV-1 PR activities on peptides and
inhibitors derived from M-PMV, MAV, and HIV-1 polyprotein processing
sites. These results are in agreement with sequence comparison data of
the retroviral pro genes, which show close phylogenetical
relatedness of D-type retroviruses with MMTV and Rous sarcoma
virus(30) . In our previous work(1) , we showed that two
hydroxyethylene isostere inhibitors of HIV-1 PR could inhibit M-PMV
polyprotein processing in vivo. However, this required
20-100 µM concentrations of the inhibitors, whereas
only micromolar concentrations of the same compounds were sufficient to
block HIV-1 precursor processing(31) . Interestingly, at a 20
µM concentration of the inhibitors, the transmembrane
glycoprotein gp22 processing to gp20 was blocked completely, while
cleavage of gag gene products was blocked incompletely.
Even though an overwhelming amount of information about the
structure and biochemical characteristics of the retroviral proteinases
has been obtained in recent years, the process by which the initial
activation of the proteolysis occurs remains enigmatic. Experimental
data suggest that the triggering event may be virus-specific. For
example in Rous sarcoma virus, processing at the NH
It has
been suggested for the C-type retroviruses that activation results from
the concentration of precursors at the cellular membranes (34), and it
was shown recently for HIV-1 that processing of the Gag precursor is
initiated at the membrane of infected cells and that the final steps of
virus assembly are delayed when processing is slowed by inhibitors or
mutations(35) . Unlike C-type, D-type retroviruses preassemble
stable ICAPs in the cytoplasm and then transport them to the site of
budding(5) . The structural conformation of the Gag, Gag-Pro,
and Gag-Pro-Pol polyproteins within the ICAPs must in some way prevent
premature processing, most probably by retaining a structure in which
the target cleavage sites are inaccessible to the assembled proteinase
precursor. In contrast to the in vivo situation, bacterially
expressed, full-length M-PMV Gag-Pro and Gag-Pro-Pol precursors undergo
rapid self-processing.(
Hydrolysis of the peptide substrates was carried out at
pH 5.3 (0.05 M sodium acetate, 2 mM EDTA, 0.3 M NaCl, 0.1% 2-mercapthoethanol) at 37 °C and followed by
reverse phase HPLC (Vydac C18 column, linear methanol gradient). The PR
concentration varied between 90 and 300 nM. The cleavage rates
were calculated from the integrated areas of product and substrate
peaks. Kinetic constants were calculated using ENZFITTER software.
Arrow indicates cleavage site as determined by amino acid analysis of
the cleavage products.
Conditions for cleavage
as described in legend to Table I. The incubation time was 1 h.
PheSta, phenylstatine;
CysSta, cysteinestatine; OMe, oxymethyl; [CH
We thank Drs. Jan Urban, Libuska Pavlcková, and
Milan Soucek for reduced bond inhibitors used in this study, Zdenek
Voburka for NH
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
terminus,
yielding a proteolytically active 17-kDa protein, p17. This initial
cleavage is followed in vitro by a much slower self-processing
that leads to emergence of proteolytically active p12 and a
COOH-terminal cleavage product p5. We have found the
NH
-terminal processing site of both the p17 and p12 to be
identical and similar to the amino terminus of the mouse mammary tumor
virus proteinase. We have also identified the COOH-terminal processing
site of the p12 form. Using purified recombinant proteins and synthetic
oligopeptide substrates based on naturally occurring retroviral
processing sites, we have determined the enzymatic activity and
specificity of the Mason-Pfizer monkey virus proteinase to be more
closely related to that of myeloblastosis-associated virus proteinase
rather than that of the Human immunodeficiency virus type 1 proteinase.
Inhibition studies using peptide inhibitors support these results.
(
)within the cell cytoplasm. The viral
capsid shells then migrate to the plasma membrane and are released by
budding. Since the assembly and intracellular transport of ICAPs, as
well as glycoprotein incorporation during membrane budding, are
temporally and spatially distinct processes, M-PMV provides an
excellent system for genetic, biochemical and structural analyses of
retrovirus assembly and maturation.
- p10 (matrix) -pp16/24-p12-p27 (capsid) -p14 (nucleocapsid)
-p4-COOH, by a proteinase (PR) encoded for by the pro gene. In
addition to the PR, the 5` portion of the pro reading frame
encodes a dUTPase enzyme. In order to express the pro and pol gene products a -1 ribosomal frameshift occurs
within the overlap of the gag/pro and pro/pol reading
frames, thus coding for two additional Gag-containing precursor
polypeptides Pr95 (Gag-Pro) and Pr180 (Gag-Pro-Pol). These larger
polypeptide precursors are cleaved by the viral PR, in a manner similar
to that of the Pr78 precursor, to yield the mature polypeptides of the
viral particle.
Cloning
The M-PMV PR coding sequence contained
within a 1324-base pair StyI-PvuII fragment
(nucleotides 2542-3864; Ref. 8) was cloned under transcriptional
and translational control of bacteriophage T7 gene 10 (vector
pT7Q10; Ref. 9), resulting in expression plasmid pGOT7Q.
Bacterial Expression
A host strain E. coli BL21(DE3) containing one copy of the T7 RNA polymerase gene in its
genome (10) was used to overexpress the 26-kDa PR precursor from
pGOT7Q. Bacteria harboring the expression plasmid were grown in a rich
medium supplemented with 2.5% glycerol and 100 µg/ml ampicillin at
37 °C in flasks on a rotary shaker and harvested by centrifugation
70 min after induction at A = 1.2 with
0.5 mM isopropyl-1-thio-
-D-galactopyranoside
(IPTG).
Isolation and Purification of Proteinase
Cells
were disrupted using a French press and sonication. Inclusion bodies
containing 26-kDa PR precursor were collected by centrifugation of cell
lysates and washed extensively as described in detail
previously(9) . Solubilization of the inclusion bodies in
saturated warm urea (40 °C) and dialysis overnight at 4 °C
against 0.05 M Tris-Cl, pH 7.5, 20% glycerol, 0.1%
2-mercaptoethanol buffer allowed renaturation of the precursor and
autoprocessing of the PR. The soluble fraction of the dialysate was
adjusted to pH 8.6 and purified batchwise on QAE-Sephadex A25. PR does
not bind to QAE-Sephadex; contaminating proteins are retained on the
support. Further purification was achieved by FPLC. PR-containing
samples were adjusted to pH 5 and loaded onto a Mono S column
equilibrated in 0.05 M sodium acetate, 2 mM EDTA, 20%
glycerol buffer. Different forms of M-PMV PR were eluted with a linear
salt gradient of 0-1 M NaCl.
Amino Acid Sequencing and Analysis
Samples for
amino acid analysis were hydrolyzed for 24 h in vacuo with 6 M HCl containing 0.1% phenol and then dried by vacuum
desiccation. Analysis was performed using a Durrum D500 analyzer and
ninhydrin detection of eluted amino acids. Amino-terminal amino acid
sequence analysis was performed with an Applied Biosystems 470A protein
sequencer. COOH-terminal sequence was obtained by carboxypeptidase Y
cleavage(11) .
Substrates and Inhibitors
Substrates and
inhibitors shown in Tables I-III were synthesized on a solid
phase synthesizer. All substrates and inhibitors were HPLC-purified and
characterized by amino acid analysis or fast atom bombardment mass
spectrometry.
Activity Assay
In the course of the purification
procedures, M-PMV PR activity was determined using a chromogenic
peptide substrate ATPQVYF(NO)VRKA at 20 µM final concentration. The cleavage performed at 37 °C, pH 5.3
(0.05 M sodium acetate, 2 mM EDTA, 0.1%
2-mercaptoethanol, 2 M NaCl), was monitored by a decrease in
absorbance at 305 nm using an Aminco DW 2000 spectrophotometer.
Substrate Specificity
Specificity studies were
performed with a set of synthetic substrates given in Tables I and II.
Cleavage products were detected by HPLC (Vydac C18 column, linear
methanol gradient). The rate of cleavage was determined by substrate
and product peak area integration. The conditions for cleavage are
given in the legend to .
Cloning
Construction of the bacterial expression
vector pGOT7Q was carried out in consideration of the unknown molecular
weight and termini of the M-PMV PR and under the presumption that M-PMV
PR would be toxic for E. coli, as has been demonstrated for
other retroviral proteinases(9, 12) . We decided to take
advantage of the ability of retroviral PRs to cleave themselves out of
a larger precursor molecule(13) . Therefore we cloned a fragment
that contained a substantial part of the 3` end of the M-PMV PR open
reading frame (Fig. 1) into construct pT7Q, a vector that we have
shown previously to be useful for expression of HIV-1 and BLV
PRs(9, 14) . In addition to the target gene, the
expression vector pGOT7Q contains a lacI gene that
codes for the lac repressor. Since a single copy of the T7 RNA
polymerase gene in the chromosome of the expression host, E. coli BL21(DE3), is controlled by a lac UV5
promoter(10) , the lac repressor down-regulates the
expression of the target gene indirectly by blocking the transcription
of the T7 RNA polymerase gene by the host RNA polymerase. Induction
(derepression) of the target gene synthesis is conveniently achieved by
adding IPTG to the growing bacterial culture.
Figure 1:
Construction of the M-PMV PR precursor
expression plasmid. The phage T7 promoter (arrow) directs the
synthesis of a recombinant precursor comprising 7 vector-derived amino
acids (empty thin bar) and 228 amino acids of the M-PMV PR
reading frame (filled thick bar). The cloning sites StyI and PvuII as well as the relative position of
the cloned segment within the M-PMV genome are indicated: stippled
thick bar, gag gene; filled thick bar, pro gene; hatched thick bar, pol gene; filled
thin bar, vector sequence.
Bacterial Expression
As expected, the 26-kDa PR
precursor encoded by pGOT7Q accumulated in insoluble inclusion bodies (Fig. 2, compare lanes 1, 2, and 3).
Using phase-contrast microscopy(15) , the maximal size of the
cytoplasmic inclusions was observed 70 min after induction with IPTG.
In prolonged cultivations, the inclusion bodies were degraded.
Therefore, bacteria were always harvested 70 min after induction.
Similar kinetics of inclusion body formation were observed during
expression of the BLV PR precursor in E. coli(9) .
Figure 2:
M-PMV PR precursor expression in
IPTG-induced E. coli BL21(DE3)/pGOT7Q and its self-processing in vitro. 18% silver stained SDS-PAGE shows the protein
composition of the bacterial cell lysate (lane 1), soluble
fraction (lane2), inclusion bodies (lane3), dialysate of inclusions dissolved in saturated urea (lane4), supernatant (lane5), and
sediment (lane6) after the dialysis of the
solubilized inclusions. Lane7 shows QAE-Sephadex
A25-purified material from lane5.
Protein Purification and Precursor
Self-processing
The M-PMV PR precursor was purified from the
inclusion bodies as described under ``Experimental
Procedures.'' The bulk of impurities were removed by
solubilization in saturated warm urea, followed by removal of urea by
dialysis. In the course of solubilization of inclusions and subsequent
renaturation during the dialysis, the 26-kDa precursor underwent
self-processing into 17- and 12-kDa species (Fig. 2, lanes3-6). Portions of p26, p17, and p12 remained
insoluble after dialysis (Fig. 2, lane6). The
overall yield of soluble p17 and p12 was increased by repeating the
solubilization/renaturation cycle (data not shown).
Figure 3:
Purification of the M-PMV PR by FPLC on
Mono S column. The elution profile and SDS-PAGE of the corresponding
eluted proteins are shown.
All
forms have similar proteolytic activities when tested with a small
chromogenic substrate, ATPQVY*F(NO)VRKA.
NH
-terminal sequencing has revealed that both p17 and p12
have identical NH
-terminal sequences: WVQPI-. Processing of
p17 into p12 and p5 was followed by both COOH-terminal sequencing of
p12 and NH
-terminal sequence analysis of p5. In this way we
determined the processing site to be between serine and proline in a
sequence of -IMMCS*PNDIV-. Thus it appears that both
NH
-terminal and COOH-terminal processing of the M-PMV
proteinase can occur to yield a minimal size active enzyme.
Substrate Specificity
In order to define the
cleavage specificity of M-PMV PR and to determine its relationship to
other retroviral PRs, peptide substrates were used. Initially we tested
cleavage of oligopeptide substrates spanning three processing sites
within the M-PMV Pr78 (Gag) polyprotein and both the NH-
and COOH-terminal processing sites of the M-PMV PR as determined from
sequence analysis of p17 and p12 (see above). All of these peptides
were hydrolyzed by the M-PMV PR (). The best k
/K
value was
determined for peptide 5, corresponding to the proteinase
NH
-terminal processing site. This observation is consistent
with the results obtained with HIV-1(16) , MAV(17) , and
BLV PRs (9) and indicates the particular importance of cleavage
at this site in the self-processing cascade during viral particle
maturation. Reliable K
and k
values for peptide 5 could not be obtained,
since oxidation of methionine leads to multiple peaks on HPLC. Peptides
1-3 and 5 were also tested as possible substrates of HIV-1 and
MAV PRs. Three of these M-PMV-derived oligopeptides were cleaved by the
PR from MAV, while HIV-1 PR hydrolyzed only two of them (data not
shown).
value of this peptide determined for
M-PMV PR is more than 2.5-fold better than that for MAV (9
µmol/liter), while the k
values for both the
PRs are comparable (5 s
for MAV PR; Ref. 18).
Peptide ATPQVY*F(NO
)VRKA was therefore used throughout
isolation and purification of the M-PMV PR in a rapid
spectrophotometric assay (see ``Experimental Procedures'').
-terminal processing site of HIV-1 PR. Among the three
additional MAV-derived peptides, one was cleaved poorly, while the
other two proved to be good substrates for the M-PMV PR.
Inhibition
The similar specificity of the MAV and
M-PMV PRs for peptide substrates was supported by the inhibitor data
shown in I, where we compared several inhibitors designed
for HIV-1 and MAV PRs. Statine-type inhibitors (I,
inhibitors 1-7) derived from the cleavage sites in the MAV Gag
polyprotein (20) showed almost equal potency for both the M-PMV
and MAV PRs, while inhibition of HIV-1 PR required concentrations
several orders of magnitude higher. On the other hand, inhibitors with
the structures optimized for the HIV-1 PR (I, inhibitors
8-10, K 0.2-70 nM) (14) were not active for either M-PMV and MAV PRs at even 50
µM concentrations.
-terminal
sequencing of both forms of the M-PMV PR revealed that the NH
termini of p17 and p12 are identical and that the first 3 amino
acid residues, WVQ-, are identical to those found in the PR NH
terminus of MMTV(24, 25) , which is
phylogenetically closely related to M-PMV(8) . Apparently, after
the initial rapid in vitro processing at the NH
terminus, p17 undergoes slow processing at the cleavage site
-IMMCS*PNDIV- near its carboxyl terminus to yield p12 and the
carboxyl-terminal peptide p5 (Fig. 4). The existence of several
active forms of M-PMV PR (p17, p12 homodimers, and a possible p17/p12
heterodimer) after in vitro processing is unexpected. We have
shown previously that M-PMV PR is not only responsible for the cleavage
of the Gag-related polyproteins, it also truncates the transmembrane
protein gp22 at the COOH terminus, and that the rate of this
glycoprotein cleavage is dependent on interaction with matrix
protein(1, 4) . It is tempting to speculate that p17 and
p12 could be responsible for cleavage at structurally and functionally
different parts of the budding virion, namely Gag and Env.
Figure 4:
In vitro processing of the M-PMV PR. The
amino acid sequence of NH- and COOH-terminal processing
sites, the PR active site triplet DTG, as well as the cleavage products
are indicated.
In our
previous work we have studied extensively the specificity of MAV and
HIV-1 PRs(17, 19, 26, 27) . Two types of
sites were recognized to be cleaved by HIV-1 PR in the viral
polyprotein; one type has bulky hydrophobic residues in the P1 site and
proline in the P1` site (nomenclature of Schechter and Berger; Ref.
28), and the second has two bulky hydrophobic amino acid residues in
both primary sites(29) . A less clear and distinct cleavage
pattern was observed for the MAV PR (17), since it cleaves both types
of sequences, but in addition is quite effective in cleaving other
sites such as the NH-terminal sequence of the MAV PR in
which a serine residue is found in the P1 position (,
peptide 8).
terminus of PR and subsequent release of the active enzyme from
its precursor is critical; inhibition of cleavage at the PR NH
terminus by site-specific mutagenesis prevents PR-directed viral
polyprotein processing(32) . Similar experiments with HIV lead
to conclusions that NH
-terminally extended PR is active,
even though less effective; a block at the PR NH
-terminal
processing site results in decreased efficiency of autoprocessing and
in occurrence of intermediate cleavage products(33) .
)
This most probably
reflects structural differences between the ICAPs assembled in the
eukaryotic cell and the bacterially expressed polyproteins, although
initiation of processing by bacterial proteinases cannot be ruled out.
Conformation is clearly very important for both enzyme activity and
specificity. It has been concluded that the MAV PR is a very
promiscuous enzyme and that the specificity observed in cleavage of
viral polyproteins is largely caused by folding and accessibility of
the target sequences in the viral particle(17) . Similarly, the
discrepancy between the efficiency of cleavage at the avian leukosis
virus PR/reverse transcriptase cleavage site in vivo and in vitro lead Stewart and Vogt (36) to propose a model
where the cleavage in vivo occurs only if the target sequence
is held in an extended conformation by the interaction of precursors.
By understanding the differences in conformation of precursors in
mammalian and bacterial cells, it may be possible to shed light on the
process of enzyme activation.
Table: Cleavage of synthetic oligopeptides by M-PMV
proteinase
Table: Relative cleavage of
synthetic oligopeptides by M-PMV proteinase
Table: Inhibition of M-PMV proteinase by inhibitors
designed for MAV and HIV-1 proteinases
NH],
reduced amide bond; tBu, t-butyl; Boc, t-butoxycarbonyl; Ac,
acetyl; Me, methyl; NI, no inhibition at 50 µM inhibitor
concentration; ND, not determined. The inhibition constants were
determined using a spectrophotometric assay with a chromogenic peptide
substrate ATPQVYF(NO
)VRKA. K
values for HIV-1 and MAV PR were taken from Refs. 14 and 20.
-D-galactopyranoside.
-terminal sequencing, and Jan Zbrozek for
amino acid analysis. We also thank Dr. Michael Sakalian for critical
reading of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.