©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Bacterial Expression, and Characterization of the Mason-Pfizer Monkey Virus Proteinase (*)

Olga Hrusková-Heidingsfeldová (1), Martin Andreansky (2) (3), Milan Fábry (3), Ivo Bláha (1)(§), Petr Strop (1)(¶), Eric Hunter (2)(**)

From the (1)Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, 16610 Prague, Czech Republic, the (2)Department of Microbiology, University of Alabama at Birmingham, Alabama 35294, and the (3)Institute of Molecular Genetics, Czech Academy of Sciences, 16637 Prague, Czech Republic

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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)()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.

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 - 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.

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.


EXPERIMENTAL PROCEDURES

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 .


RESULTS

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).

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).


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).

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 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'').

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-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.


DISCUSSION

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-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).

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 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) .

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.() 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

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.


  
Table: Relative cleavage of synthetic oligopeptides by M-PMV proteinase

Conditions for cleavage as described in legend to Table I. The incubation time was 1 h.


  
Table: Inhibition of M-PMV proteinase by inhibitors designed for MAV and HIV-1 proteinases

PheSta, phenylstatine; CysSta, cysteinestatine; OMe, oxymethyl; [CHNH], 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.



FOOTNOTES

*
This work was supported by Fogarty International Award TW00050, National Institutes of Health NCI Grant NIHCA27834, Czech Academy of Science Grants 45501 and 45524, and Agency of the Czech Republic Grant 203/94/1323. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Ferring-Léciva, a.s., Prague Polypeptide Institute, Komoranská 955, 14310 Prague, Czech Republic.

Present address: Selectide, 1580 E. Hanley Blvd., Tucson, AZ 85737.

**
To whom correspondence should be addressed. Tel.: 205-934-4321; Fax: 205-934-1640; E-mail: eric_hunter@micro.microbio.uab.edu.

The abbreviations used are: ICAP, intracytoplasmic A-type particle; M-PMV, Mason-Pfizer monkey virus; HIV-1, human immunodeficiency virus type 1; MMTV, mouse mammary tumor virus; MAV, myeloblastosis-associated virus; BLV, bovine leukemia virus; FPLC, fast protein liquid chromatography; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PR, proteinase; IPTG, isopropyl-1-thio--D-galactopyranoside.

M. Andreansky, and E. Hunter, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Jan Urban, Libuska Pavlcková, and Milan Soucek for reduced bond inhibitors used in this study, Zdenek Voburka for NH-terminal sequencing, and Jan Zbrozek for amino acid analysis. We also thank Dr. Michael Sakalian for critical reading of the manuscript.


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