Human immunodeficiency virus specific proteolytic enzyme and a method for its synthesis and renaturation

HIV protease necessary for the natural synthesis of HIV has been identified and sequenced. This antibody cross reacts with the protease of HIV-2. In addition, a method for producing synthetic HIV-1 and HIV-2 protease having the correct stereospecific conformation and specific HIV proteolytic activity has been achieved. Assays for the synthetic proteases using synthetic peptide substrates have been developed.

BACKGROUND OF THE INVENTION 
We identified and structurally, biochemically and enzymologically 
characterized human immunodeficiency virus (HIV) protease, as well as its 
natural polyprotein substrates, in order to develop drugs that inhibit 
protease activity. It was known from our work on protease deficient MuLV 
mutants that when precursor polyproteins are not cleaved mature infectious 
virus can not be produced. Instead, noninfectious particles are made that, 
however, remain immunogenic because they carry complete envelopes. The 
idea underlying this research was to ultimately prepare chemical 
inhibitors that penetrate the infected cell, become incorporated into the 
budding virus, bind with high affinity to the viral protease or precursor 
polyproteins, prevent cleavage and lead to the production of 
non-infectious but still immunogenic viral progeny. The use of these 
chemical inhibitors would block the spread of HIV infection while allowing 
for antigenic stimulation of host immunity. 
SUMMARY OF THE INVENTION 
We have identified the amino acid sequence that constitutes human 
immunodeficiency virus (HIV) protease, a proteolytic enzyme specific for 
the virus known as HIV, LAV and HTLV-III, for both HIV-1 and HIV-2 
variants. We have also synthesized active protease for both HIV-1 and 
HIV-2. This enzyme is necessary in the natural synthesis of HIV in the 
cells of subjects infected by the virus. In the course of natural 
synthesis it is necessary for protein of the virus to be lysed from 
precursor proteins. It is this function for which the proteolytic enzyme 
we have identified and synthesized is specific. Without HIV protease the 
active virus cannot be reproduced in infected cells and the natural 
synthesis process will be stopped short of completion. As a result, with 
our discovery of the structure of this specific protease and our synthesis 
of the active enzyme, a protease inhibitor specific for this enzyme can be 
designed. We have produced rabbit antiserum against the C-terminal portion 
of HIV-1 protease that is specific for HIV-1, which we have used to 
isolate and characterize natural HIV-1 protease. We have also produced an 
antibody in rabbits to our synthetic HIV-1 protease, which cross reacts 
with HIV-2 protease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The entire DNA sequence coding for HIV was known. However, the portions of 
that sequence coding for particular active peptides and proteins, such as 
the protease, had not been determined prior to this invention. As we had 
previously identified proteases specific for human retroviruses, and we 
had identified homologies in the sequences coding for proteases necessary 
in the natural synthesis of these other retroviruses, we were able to 
identify sections in the DNA sequence coding for HIV proteins that should 
be within the sequence coding for a protease specific for HIV and 
necessary for its natural synthesis. (Copeland and Oroszlan, "A Synthetic 
Dodecapeptide Substrate For Type C RNA Tumor Virus Associated Proteolytic 
Enzyme," PEPTIDES: Synthesis-Structure-Function, Pierce Chemical Company, 
1981, and Yoshinaka et al, "Murine Leukemia Virus Protease Is Encoded By 
The gag-pol Gene and Is Synthesized Through Suppression of an Amber 
Termination Codon", Proc. Natl. Acad. Sci., USA, Vol. 82, pages 1618-1622, 
March 1985, included herein in their entirety by reference). Our previous 
work indicated to us that certain sequences were conserved in retroviral 
proteases; these are set forth in FIG. 1. 
HIV protease is a 99 amino acid peptide, which has a molecular weight of 
11K-11.5K daltons measured by SDS PAGE. The amino acid sequence is given 
in FIG. 2, as marked, beginning with ProGlnIle . . . The complete amino 
acid sequence given is for HIV-1.sub.BH10. In the second row in FIG. 2, 
the amino acids for HIV-2 are given where they differ from those in HIV-1. 
FIG. 3a indicates the position of the 99 amino acid peptide in the HIV-1 
sequence. The peptide sequence is shown in the bottom line in each 
grouping. The first line in each grouping is the DNA sequence coding for 
the HIV-1 protease. The second line in the first grouping is an 
alternative amino acid sequence, which is correct for the expression of 
certain portions of HIV-1 protein. However, due to a reading frame shift 
prior to the protease, the sequence we have identified in the third line 
and repeated in FIG. 2, is the actual amino acid sequence of HIV-1 
protease. 
After identifying the amino acid sequence for HIV protease, we synthesized 
the C-terminal peptide consisting of 15 amino acids 
(IleGlyArgAsnLeuLeuThrGlnIleGlyCysThrLeuAsn Phe). With this 15 amino acid 
synthetic peptide we generated a rabbit antiserum. The rabbit antiserum 
was then used to detect natural protease from HIV. It also was used to 
identify the protease fraction after HPLC separation. The N terminal of 
the HPLC purified protease was then sequenced by Edmond degradation. We 
could then determine that the N terminal began with the Pro located 99 
amino acids upstream from the previously identified N terminal Pro 
beginning the reverse transcriptase sequence, shown at RT in FIG. 3a. 
Having determined that the sequence we identified was for HIV protease, we 
proceeded by solid phase synthesis using the Merrifield method to 
synthesize the 99 amino acid peptide (Merrifield R. B. (1963), "Solid 
Phase Peptide Synthesis I". J. Amer. Chem. Soc. 85, 2149-2154, included 
herein by reference). The synthesis was done using the semiautomatic 
synthesis procedures with an Applied Biosystems program and an Applied 
Biosystems 430 Peptide synthesizer (Foster City, Calif.). 
The 99 amino acid polypeptide synthesized having the sequence shown in FIG. 
2 and FIG. 3A is based on the DNA sequence of HIV-1.sub.BH10 (Ratner, L. 
et al, (1985) "Complete Nucleotide sequence of the AIDS Virus, HTLV-III.", 
NATURE, 313, 277-284, included herein by reference). This synthetic 
protease is designated here as PR-1. 
In addition we have synthesized another HIV protease designated PR-2, which 
corresponds to the sequence of another HIV strain (HIV-2.sub.ROD) as 
reported by Guyader, M. et al, (1987) "Genome Organization and 
Transactivation of the Human Immunodeficiency Virus Type 2." NATURE, 326, 
662-669. The variation in amino acid sequence between PR-1 and PR-2 is 
indicated in FIG. 2. The complete sequence of PR-2 is given in FIG. 3b. 
The sequences of PR-1 and PR-2 are both comprised of 99 amino acids and 
share 47 identical residues in their sequence, and an overall homology of 
approximately 76%. The putative active site sequences of PR-1 and PR-2 are 
identical (see identical residues 23 to 30, Leu Leu Asp Thr Gly Ala Asp 
Asp, aligned in FIG. 2) and the natural cleavage sites in the two strains 
of HIV are very similar as determined in our laboratory (Henderson, L. E. 
et al, (1988) "Analysis of Proteins and Peptides Purified From Sucrose 
Gradient Banded HTLV-III." in Human Retroviruses, Cancer and AIDS: 
Approaches to Prevention and Therapy, pp. 135-147, Alan R. Liss Inc.; 
Henderson L. E. et al, (1988) "Molecular Characterization of gag Proteins 
From Simian Immunodeficiency Virus Siv.sub.mne. J. Virol, In Press"). 
EXAMPLE I 
Synthesis 
The 99 amino acid (99 met) residue proteases, PR-1 and PR-2, were assembled 
by the solid phase method in an Applied Biosystems Model 430A peptide 
synthesizer. The resin and protected amino acids were purchased from 
Applied Biosystems. Side chain protecting groups were benzyl (Bzl) for 
aspartic acid, glutamic acid, serine and threonine; 4-methylbenzyl for 
cysteine; tosyl for arginine and histidine; 2-chlorobenzyl- oxycarbonyl 
for lysine; 2-bromobenzyloxycarbonyl for tyrosine; and formyl for 
tryptophan. The tert-butoxycarbonyl (Boc) group protected the a-amino 
group. The synthesis began with the Boc-carboxyl amino acid substituted on 
a phenylacetamindemethyl resin. The instrument program converted the amino 
acids to the symmetric anhydrides just prior to the coupling step. 
Arginine, asparagine and glutamine were double coupled using 
1-hydroxybenzotriazole. Following completion of all the coupling steps, 
the peptide-resin was dried in a desiccator. 0.3 g was then reacted with 
0.75 ml p-cresol, 0.25 ml p-thiocresol, 6.5 ml dimethyl-sulfide and 2.5 ml 
hydrogen fluoride in an all-teflon apparatus with magnetic stirring for 2 
hours at 0.degree. C., solvents were then removed by vacuum, the mixture 
extracted with ether and dried with nitrogen. The second step of 
deprotection proceeded with 0.6 ml p-cresol and 9 ml hydrogen fluoride for 
one hour. Solvents again were removed with vacuum, the mixture filtered, 
washed with ether and dried with nitrogen. 
Peptides of various length, corresponding to the natural cleavage sites 
that occur in retroviral gag polyproteins, were synthesized and purified 
according to published methods (Copeland and Oroszlan (Supra),and 
Copeland, T. D. et al, "Envelope Proteins of Human T Cell Leukemia Virus 
Type I: Characterization by Antisera to Synthetic Peptides and 
Identification of a Natural Epitope,": The Journal of Immunology, Vol 137, 
2945-2951, No 9, Nov. 1, 1986.) 
As synthesized, the peptide is linear and demonstrates no activity as a 
proteolytic enzyme. In addition to removing blocking groups, therefore, it 
was necessary to convert the protease to its natural stereospecific 
conformation in order to exhibit proteolytic activity. As produced, the 
synthetic HIV protease was extracted in a strong acid. It was necessary, 
thereafter, to submit it to treatment and purification using specific 
buffer systems and dialysis. Thereafter, we proceeded by trial and error 
to effect renaturation and to refold the peptide into its natural 
stereospecific confirmation. This was done through recovery in guanidine 
hydrochloride solution, concentration, removal of all solutes and recovery 
in an aqueous solution. The proper folding was accomplished through a 
series of trial and error steps. The correct folding of the peptide is the 
result of intra and intermolecular forces and bonding, and of the 
characteristics of the media to which we subjected the synthetic protein. 
Purification and Renaturation of Synthetic Protease 
25 mg of the resin plus Peptide mixture, including synthetic 99 amino acid 
protease, were extracted into 6M Gnd-HCl dissolved in Tris-HCL at pH 8.0. 
The PR-1 solution also contained the reducing agent, dithiothreitol (DTT). 
In the purification procedure for PR-2, DTT was not used. Desalting 
partial purification was accomplished by gel permeation chromatograpy on 
G-25 Sephadex (Pharmacia). Fractions of the excluded peak were collected 
and subjected first to slow dialysis in Tris-HCl buffer at 4.degree. C. 
The fractions were then dialyzed against several changes of Pipes buffer 
at pH 7.0 containing NP-40, with or without DTT, (Hafenrichter, R. & 
Thiel, H. J., "Simian Sarcoma Virus-Encoded gag-Related Protein: In Vitro 
Cleavage by Friend Leukemia Virus-Associated Proteolytic Activity" 
Virology, 143, 143-152 (1985)). 
Antibody was also prepared to the synthetic PR-1 99 amino acid protease 1 
mg of the crude peptide was mixed with phosphate buffered saline and 
Freund's complete adjuvant and then administered to a New Zealand white 
rabbit at several sites subcutaneously on the back. At two week intervals, 
boosts were made with 0.1 mg in Freund's incomplete adjuvant. After one 
month the animal was bled at two week intervals. Positive antisera to the 
PR-1 99 mer was obtained. This antibody was cross reactive with PR-2. 
We were able to obtain a synthetic protein that showed specific proteolytic 
activity for HIV. Assays to confirm that we had produced the active 
protein were conducted by incubating the synthetic protease with natural 
vital substrates and with synthetic vital substrates. The results 
demonstrated that our synthetic HIV protease was specific for the 
characteristic cleavage sites. The assay methods employing synthetic 
substrates used were essentially as described in Copeland and Oroszlan 
(Supra). 
EXAMPLE II 
Protease Assays 
Synthetic peptides corresponding to cleavage sites in various gag 
precursors were incubated with PR-1 or PR-2 in various buffered solutions 
at various pHs and aliquots removed at various time points. To analyze for 
cleavage of the substrates, 50 microliters of saturated Gnd-HCl was added 
and TFA added to lower the pH to 2. The contents were then applied to a 
.mu. Bondapak C18 column (Waters) and subjected to a 30 minute 0 to 40% 
CH.sub.3 CN containing 0.05% TFA gradient. Between each chromatographic 
assay the column was washed with 60% CH.sub.3 CN. Following hydrolysis in 
vacuo at 110 degrees with 6N HCl for 20 hours peaks were collected 
manually and analyzed for amino acid composition. Amino acid analysis was 
performed on a Waters Pico-Tag.TM. system. 
This activity is illustrated in FIG. 4. FIG. 4a illustrates MuLV synthetic 
peptide containing the known proteolytic cleavage site. FIG. 4b shows the 
same peptide after being incubated with our synthetic HIV protease PR-1. 
Two of the peaks to the left of the main peptide indicate products of 
cleavage of the MuLV substrate. FIGS. 4c and 4d illustrate cleavage of the 
same substrate using two other proteases, cathepsin D and renin, 
respectively. As can be seen from the products to the right of the 
substrate peak, those enzymes cleave the substrate at different cleavage 
sites. 
EXAMPLE III 
Cleavage of synthetic peptide SP-78 that corresponds to an undecopeptide 
sequence present at the Phe-Pro cleavage site in murine leukemia virus gag 
polyprotein is shown in FIG. 5. To an appropriate amount of lyophilized 
SP-78 was added the synthetic 99 amino acid PR-2 (approximately 100 ng) in 
the stock solution containing NP-40. The pH was adjusted to 6.5 with 
sodium phosphate buffer in a total volume of 25 .mu.l. The reaction 
mixture contained a final concentration of approximately 0.2 m sodium 
phosphate, 0.35% NP-40 and 10% glycerol. The solution was incubated at 
room temperature and aliquots were removed immediately after mixing (0 
hr), after 20 hrs, and after 70 hrs of incubation. Substrate and products 
were separated and recovered by HPLC. The chromatographic profiles 
illustrated in FIG. 5 show the substrate and cleavage products 1 and 2 
with their specific sequences. The sequences were confirmed by amino acid 
analyses of the peak fractions. These results provided evidence that the 
Phe-Pro bond was cleaved in a specific fashion, the same as accomplished 
by the-viral protease under natural conditions. A Progression with time of 
the enzymatic cleavage is documented. The elution time, a well defined 
parameter dependent on peptide composition, is the same for products 1 and 
2, as shown in FIG. 4b; where the same substrate was cleaved by PR-1. Note 
that the extent of cleavage as demonstrated by the two smaller peaks to 
the left of the substrate peak was much smaller due to the less favorable 
experimental conditions used in the initial experiments with PR-1. 
The cleavage of synthetic peptide corresponding to a Phe-Pro site sequence 
in the HIV-2 gag polyprotein, SP-258 is shown in FIG. 6. Results are shown 
for a single time point in incubation, but subsequent amino acid analysis 
provided convincing evidence that the cleavage of a single bond occurred 
at the expected site. See sequence of substrate and products in FIG. 6. 
The substrate and enzyme (approximately 120 ng) were incubated in low 
molarity Pipes buffer pH 7.0 containing NP-40, at room temperature. DTT is 
not required for cleavage of cysteine free substrates with PR-2 since this 
enzyme does not contain any cysteine in its structure. 
The cleavage of synthetic peptide SP-211, corresponding to a TYR-PRO 
cleavage site sequence in HIV-1 gag polyprotein, is shown in FIG. 7, as 
obtained at 15 and 39 hrs of incubation at room temperature. Approximately 
100 ng of PR-2 was used as in the previous experiment, but the ionic 
strength was higher. The buffer was 0.2M sodium phosphate, pH 6.5, and 
contained in addition 15 mM sodium chloride, 5% glycerol and 0.2% NP-40. 
Incubation was again at room temperature. Product #2, tetrapeptide 
PRO-ILE-VAL GLN amide, as expected, elutes before product #1, a 
pentapeptide with the sequence VAL-Ser-Gln-Asn-Tyr.