Retroviral Protease (Retroviruses/Avian Sarcoma4eukosis Virus/Human Immunodeficiency Virus) MOSHE KOTLER*T, RICHARD A
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Proc. Natl. Acad. Sci. USA Vol. 85, pp. 4185-4189, June 1988 Biochemistry Synthetic peptides as substrates and inhibitors of a retroviral protease (retroviruses/avian sarcoma4eukosis virus/human immunodeficiency virus) MOSHE KOTLER*t, RICHARD A. KATZ*t, WALEED DANHO§, JONATHAN LEIs¶, AND ANNA MARIE SKALKA*tII *Department of Molecular Oncology, Roche Institute of Molecular Biology and §Peptide Research Department, Hoffmann-La Roche, Inc., Roche Research Center, Nutley, NJ 07110; and $Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106 Communicated by Sidney Udenfriend, February 16, 1988 ABSTRACT Processing of the gag and pol gene precursor The results presented here show that a decapeptide com- proteins of retroviruses is essential for infectivity and is posed of the cleavage site between the a and pp32 domains directed by a viral protease that is itself included in one ofthese of the ASLV reverse transcriptase-integration polyprotein precursors. We demonstrate here that small synthetic peptides (pol) can be cleaved at the expected site by PR. Changes in can be used as both model substrates and inhibitors to the amino acids that flank the cleavage site ofthe peptide (and investigate the specificity and molecular parameters of the of the larger pol precursor) alter the capacity of the peptides reaction. The results indicate that a peptide that extends five to serve as substrates. Some peptides that are not cleaved act amino acids but not three amino acids in both directions from as inhibitors of PR. a known cleavage site is accurately hydrolyzed by the protease of avian sarcoma-leukosis virus. Substitutions of the amino MATERIALS AND METHODS acids to either side of the peptide bond to be cleaved affect the ability of the peptide (as well as a larger precursor protein) to PR Digestion of pol Precursor Proteins Synthesized in serve as a substrate. The specificity is more stringent for the Escherichia coli. Lysates of induced E. coli containing the amino acid that will become the carboxyl end after cleavage. pFA1-RT99 expression clone (or mutant clone) were pre- Some substitutions produced peptides that were not cleaved but pared as described (22). Approximately 30 pug of purified could act as inhibitors of cleavage of a susceptible peptide. avian myeloblastosis virus (AMV) PR was added to a 1-ml Thus, small model substrates may be used to explore both the lysate prepared from 10 OD600 units of bacterial cells. The binding and catalytic properties of these important proteases. purification of AMV PR and its reaction conditions were as described previously (22). After incubation, the samples were The dimeric RNA genomes of retroviruses are encapsidated mixed with protein gel sample buffer and boiled for 3 min. in a symmetric icosahedral core that is surrounded by a The lysates were fractionated on a NaDodSO4/10% poly- lipoprotein envelope (1). Translation of the viral genes gag acrylamide gel and transferred to nitrocellulose, and pol- and pol yields precursor polypeptides that are cleaved to related proteins were detected by using an antiserum specific produce the mature forms ofthe proteins found in virions (1). for a peptide in the a domain of reverse transcriptase (22). Processing has been shown to be directed by a retrovirus- Oligonucleotide Mutagenesis. The codons for the dipeptide encoded protease (PR) in avian sarcoma-leukosis viruses cleavage site (Tyr-Pro) between a and pp32 in pFA1-RT99 (ASLV), murine leukemia virus (MuLV), and human immu- were altered by using oligonucleotide mutagenesis to gener- nodeficiency virus (HIV) (2-17). The PR and analogous ate a predicted Ile-Asp dipeptide cleavage site (Fig. 1A). The domains of yeast Ty elements, Drosophila copia-like ele- oligonucleotide-directed mutagenesis method described by ments, cauliflower mosaic virus, and hepatitis B virus contain Morinaga et al. (23) was used. The mutagenizing oligonucle- highly conserved amino acid sequences that are homologous otide was a 30-mer (5'-ACCTTCCAAGCGATCGATTTGA- with a sequence in the active site of the aspartic protease GAGAGGCT-3') complementary to the coding strand. family of cellular proteins (18-20). Decapeptides. Decapeptides were synthesized by the solid- The sites of PR-catalyzed cleavage in several retroviral phase method (24). Deprotection and cleavage from the resin polyprotein precursors have been deduced by N- and C- were achieved by treatment with anhydrous hydrogen fluo- terminal of mature ride according to the procedure of Tam et al. (25). The sequencing processed products. Although peptides were purified by HPLC using a ,uBondapak C18 some sequences occur frequently in known cleavage sites, column (Waters Associates). The purity of the peptides was none appears sufficiently unique to account for the specificity confirmed by analytical HPLC, amino acid analysis, and exhibited in the processing reaction. In this report we use microsequencing. small synthetic peptides as model substrates to investigate Peptide Proteolysis by PR. Proteolytic reactions were the requirements for cleavage by the ASLV PR. carried out in 10 1.l of 0.1 M sodium phosphate buffer (pH 6), Small peptides seemed appropriate to use as substrates for 1.1 mM decapeptide as substrate at 370C for 20 min. Reac- the retroviral PR because they are able to assume a wide tions were stopped by freezing in dry ice/ethanol. variety of structural conformations, some of which are likely to resemble those that normally interact with the PR active Abbreviations: PR, retroviral protease; AMV, avian myeloblastosis site. It has been postulated that conformational mobility, per virus; ASLV, avian sarcoma-leukosis virus; pol, retroviral reverse se, is characteristic of sites involved in protein-protein transcriptase and integration polyprotein; MuLV, murine leukemia interactions (21). If so, peptides should provide better models virus; HIV, human immunodeficiency virus; TLE, thin-layer elec- than larger molecules of unknown structure which might be trophoresis. folded into a conformation not appropriate for cleavage. tPermanent address: Department of Molecular Genetics, The He- brew University Hadassah Medical School, Jerusalem, Israel. tPresent address: Institute for Cancer Research, Fox Chase Cancer The publication costs of this article were defrayed in part by page charge Center, 7701 Burholme Avenue, Philadelphia, PA 19111. payment. This article must therefore be hereby marked "advertisement" I1To whom reprint requests should be addressed at the present in accordance with 18 U.S.C. §1734 solely to indicate this fact. address. Downloaded by guest on September 23, 2021 4185 4186 Biochemistry: Kotler et al. Proc. Natl. Acad. Sci. USA 85 (1988) A a v pp32 v C P99poi Tyr-Pro' I wt mutant v C B Tyr-Pro C lie-Asp kDa "w la 6(a) m W 0 0.3 1 2 3 4 5hr 0 0.3 1 2 3 4 5hr FIG. 1. Immunoblot analysis of wild-type and mutant bacterially produced ASLV pol-encoded proteins after incubation with the ASLV PR. (A) The product of the E. coli expression vector pFA1-RT99, which encodes a 99-kDa pol product, p99P"", which includes the a (63-kDa), (95-kDa), and pp32 endonuclease domains. Cleavage to generate pp32 occurs between the indicated Tyr and Pro residues. (B) A lysate was prepared as described (22) from an E. coli culture that was induced for expression of p99P"". AMV PR was added and the mixture was incubated for the indicated times. Samples were electrophoresed on a NaDodSO4/1O% polyacrylamide gel and pol-specific peptides were identified by immunoblotting using an antiserum raised against a peptide from the a domain, as described previously (22). No specific cleavage was seen in samples incubated in the absence of PR, as described previously (22) (data not shown). (C) A parallel lysate was analyzed that contained the indicated mutation at the p99P°I cleavage site. The mutation Tyr-Pro to Ile-Asp was introduced into pFA1-RT99 by using oligonucleotide mutagenesis. HPLC Assay. Frozen reaction samples were diluted with observations). This suggested that cleavage at the a/pp32 200 ,ul of distilled water and loaded on an LC-318 octadecyl- domain junction is required for pol function. However, the dimethylsilyl column (4.6 x 250 mm; Supelco, Bellefonte, alternative explanation, that the amino acid substitutions PA). Elution was carried out by using a gradient of 20-60% affected reverse transcriptase pol endonuclease activities, (vol/vol) acetonitrile in 0.1% aqueous trifluoroacetic acid at could not be ruled out. a flow rate of 1 mlmin 1. Absorbance at 210 or 280 nm was Cleavage of Peptides by PR. We have used synthetic recorded. peptides, rather than large precursor polypeptides, as sub- Thin-Layer Electrophoresis (TLE) Analysis of Decapeptide strates to directly investigate the sequence requirements for Cleavage by PR. Aliquots (2 ttl out of 10-,ul reaction mixtures) cleavage by ASLV PR. Fig. 2A shows that the ASLV PR can were spotted on TLC plates (plastic cellulose sheets Art. cleave a decapeptide identical to the sequence at the a/pp32 precursor Table 5577, 20 x 20 cm; E.M. Reagents). TLC plates were subjected cleavage site in the ASLV pol (see 1, peptide to 45 mA for 1 hr in pyridine/acetic acid/acetone/distilled 1). Two peptide products, distinct from the starting material, water (1:2:8:40, vol/vol). The plates were air dried and then were resolved by HPLC. Amino acid composition analysis of sprayed with 1% triethylamine (Pierce) in acetone followed by these peptides, referred to as P1 and P2, verified that they Roche) at 0.1 mg/ml in acetone. contained equimolar amounts of Pro, Leu, Arg, Glu, and Ala fluorescamine (Hoffmann-La Ala, and Tyr (P2). These results were taken under UV light. (P1) and Thr, Phe, Gln, Photographs indicate that cleavage of the peptide occurred between Tyr and Pro residues. Fig. 2Ba shows the kinetics of cleavage of RESULTS this decapeptide. These data provided a baseline for com- Cleavage of ASLV pol-Encoded Precursor Synthesized in paring rates of cleavage of five additional decapeptides, a side of the Bacteria.