ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 290, No. 1, October, pp. 186-190, 1991
Analysis of Subsite Preferences of HIV-1 Proteinase Using MA/CA Junction Peptides Substituted at the P3-PI’ Positions Andreas Billich’ Department
and Gottfried
of Antiretroviral
Therapy,
Winkler Sandoz-Research
Institute,
Brunnerstrasse
59, A-1235
Vienna,
Austria
Received April 18, 1991, and in revised form June 7, 1991
The residues P3, P2, Pl, and Pl’ of a peptide corresponding to the matrixtcapsid protein junction in the HIV- 1 gag protein (Ser-Gln-Asn-Tyr-Pro-Ile-Val) were systematically replaced and the effect of these single amino acid substitutions on the hydrolysis of each peptide by HIV- 1 proteinase was studied. Subsites S 1 and S 1’ of the enzyme showed explicit preference for hydrophobic moieties, but o-branched amino acids and proline are not tolerated in Sl. The 52 subsite shows a preference for small polar and apolar amino acids; it may be occupied by Asn, Asp, Glu, Cys, Ala, or Val, other substitutions, especially by Gln and Ser, prevent hydrolysis of the peptides. In subsite S3 all amino acids except proline can be accommodated. o 1991Academic PRSS, he.
rational selection of the side chains to be introduced with advantage at the corresponding sites of inhibitors. With this aim studies on subsite specificity of the proteinase have been performed either by mutational analysis of the protein substrates (3,4) or by using substituted oligopeptide analogues of the natural cleavage sites (5-9). This publication extends earlier studies by reporting subsite preferences for the residues of peptides whose sequences are derived from the junction between the matrix (MA) and the capsid protein (CA) in the HIV-l gag precursor (“MA/CA junction”). We systematically replaced residues P3, P2, Pl, and Pl’ and examined the effect of these single amino acid substitutions on the hydrolysis by purified recombinant HIV-l proteinase. MATERIALS
An essential step in the replication of human immunodeficiency virus 1 (HIV-l)’ is the specific cleavage of the gag and gag/p01 precursor proteins by the virus-encoded proteinase (see Refs. (1, 2) for review). Inhibitors of this enzyme have been shown to block HIV replication in infected lymphocytes and may be useful in the therapy of AIDS. Since potent inhibitors of HIV-l proteinase are most commonly peptide mimetics containing transition state analogues of the scissile peptide bond, design of inhibitors will be facilitated by understanding the substrate specificity of the enzyme. More precisely, knowledge about subsite preferences at the scissile bond (Pl and PI’ positions3) and also at the flanking residues will allow a i To whom correspondence should be addressed. ’ Abbreviations used: HIV-I, human immunodeficiency virus 1; MA, matrix protein; CA, capsid protein; Mes, 4-morpholine ethanesulfonic acid; DTE, dithioerythritol; TFA, trifluoroacetic acid. 3 The nomenclature of Schechter and Berger (23), i.e., P4-P3-P2PI*Pl’-P2’-P3’, is used to indicate amino acids adjacent to residues Pl and Pl’ which form the scissile peptide bond (*); the corresponding subsites on the enzyme are designated by S4-S3’.
AND
METHODS
HIV-1 proteinuse. The enzyme was produced using the expressor plasmid pTZprt+ (10) in Escherichia coli strain JM 105 and purified to homogeneity as described (11). The concentration of active enzyme (Et) was determined by active-site titration (12) using a competitive inhibitor synthesized at Sandoz. Peptide synthesis. Solid-phase simultaneous peptide synthesis was done by the mesh-packet method of Houghten (13), using the polystyrene resin described by Wang (14) and standard methods of coupling and deprotection (15, 16). Purification of crude peptides was performed on a reversed-phase Cls column eluted with gradients of 10 mM aqueous ammonium acetate/acetonitrile. All peptides were then demonstrated to be homogeneous by analytical HPLC; identity was confirmed by amino acid and/or sequence analysis. Peptides were incubated at 37°C with Assay conditions and kinetics. proteinase (5-25 nM active concentration) in 50 mM Mes, pH 5.5, containing 1 mM DTE, 1 mM EDTA, and 1 M NaCl in a total volume of 150 ~1. Aliquots of 25 ~1 were removed and quenched by adding an equal volume of 0.5% trifluoroacetic acid (TFA). Then the samples were analyzed by HPLC on a HP1050 chromatograph (Hewlett-Packard) with a RP-Cl8 column (Vydac 218TP54,4.6 X 25 mm) using a linear gradient of O-35% acetonitrile in 0.1% TFA at a rate of change of 2%/min and a flow rate of 1 ml/min. With all cleavable peptides, only two products were observed. Cleavage occurred between the fourth and fifth amino acid, as shown either by using authentic tripeptide as reference or by amino acid analysis of tetrapeptide products. 0003.9861/91
186
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Copyright 0 1991 by Academic Press, Inc. All
rights
of reproduction
in any
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reserved.
HIV-1 TABLE
PROTEINASE
SUBSITE
I
Effect of Variation of the Pl Residue on Kinetic Parameters for Hydrolysis of Peptides Ser-Gln-Asn-Pl * Pro-Ile-Val K”l Pl
k cat
(mine’)
bM)
LW
2.3 2.4 1.6 10
Met
4.9
TY~
Phe Trp
Gly, Ala, Val, Ile, Pro, His, Lys, Arg, Asp, Glu, Asn, Gin, SW, Thr, Cys I
k&L (mM-’ . min-‘)
140 13 13 31 17
60.9 30.4 8.1 3.7 3.5
No cleavage”
a No cleavage observed after incubation 250 nM HIV-PR.
of peptides overnight
with
Initial rates of peptide cleavage were determined by measuring the amount of N-terminal tetrapeptide product at three to five time points during the period of linear progression of cleavage, i.e., up to about 10% turnover. The same initial rates could have been obtained from the decrease of substrate. To derive Km and V-, initial rates were measured at 6-10 substrate concentrations; data were fitted by nonlinear regression analysis to the Michaelis-Menten function using the program Enzfitter. The k,,, values were derived from the relation V,,, = kc,* [I&]. The precision of Km and k,,, values was in the range *lo-20%. Competition cleauages. The initial velocity (V) of two substrate peptides (1 and 2) competing for the active site of an enzyme depends on the specificity constant for each substrate and on the initial substrate concentration: VI = (k,dK,),
. [&I
and
V, = (k,.,lK,),.
[&I.
Measurements of initial rates at a time point where [S] is considered to be constant and cleavage is proceeding linearly, therefore, can be used to obtain a relative specificity constant (k,,JK,,,),,. Here, these constants were determined relative to a reference peptide, namely Ser-Gln-AsnTyr-Pro-Ile-Val. Competition cleavage reactions contained a 2 mM concentration of each substrate. In some cases, poor substrate peptides were included at higher concentrations (up to 10 mM) and the reference Initial peptide was reduced to 1 mM to ensure accurate quantification. rates were measured as described above for single-substrate incubations. Values of (k,.,/K,),,, were reproducible within f20%. RESULTS
AND
(250 nM) in overnight. incubations. This observation is in accordance with the sequences of the eight natural cleavage sites used by the proteinase in the gag and gag/p01 precursor proteins (see Fig. 1). In fact, these sites exhibit either Tyr, Phe, Leu, or Met at the Pl position, i.e., all amino acids that are tolerated in the peptides, too, with the exception of tryptophan. Obviously, amino acids containing hydrophobic aliphatic or aromatic side chains are preferred at Pl. The methyl group of the Ala residue may be too small to make adequate contact with the active site cleft. But it is not only hydrophobicity of substrate moieties that governs specificity at Pl since neither Val- nor Ile-containing peptides are cleaved while Leu is tolerated. A similar observation was reported by Richards et al. (8) for the substrate Ala-Arg-Val-Leu-Ala-G&Ala which corresponds to the natural cleavage site 2: replacement of Leu by Ile or Val rendered the peptide uncleavable. It therefore becomes obvious that the active site of the proteinase cannot accomodate P-branched amino acids in subsite Sl. As an exception to the rule, that the scissile dipeptide should be hydrophobic, a cleavage site with a positively charged arginine residue in Pl’ has been shown to be used by HIV-l proteinase in a nonviral protein (22). In addition, a decapeptide with lysine at Pl (Val-Ser-Gln-AsnLys-Pro-Ile-Val-Gln-Asn) was reported to be cleaved with 10% efficiency relative to the corresponding peptide with Pl = Tyr (24); the same substitution in the gag protein yielded a poorly cleaved substrate. Thus, it appears that alterations of either conformation or size of a sequence, or both, can at least partially compensate for inadequate interactions at the Sl or Sl’ site. Binding efficiency, as reflected by the K,,, value, is similar for the substrates containing aromatic moieties at Pl, but is lower when linear or branched side chains are introduced (Xaa = Met or Leu). In this series, kcatdrops by MA p17
.CA NC p24 p7 p6
DISCUSSION
The heptapeptide Ser-Gln-Asn-Tyr-Pro-Ile-Val corresponding to the sequence of the MA/CA junction in the gag precursor protein of HIV-l is efficiently cleaved by HIV-l proteinase between Tyr and Pro (K,,, = 2.3 mM, &at = 140 mini’). Peptides exhibiting this sequence have been routinely used in many studies of HIV proteinase activity and have also been the starting point for the design of inhibitors in which the Tyr-Pro dipeptide is replaced by transition state analogues (6, 17-19). For our study we first replaced the tyrosine in the Pl position by the other 19 amino acids found in proteins (see Table I).
The only modified peptides that were cleaved by the proteinase are those containing Phe, Trp, Leu, or Met at Pl. Peptides containing any other amino acid at this position were not cleaved, not. even when using an excess of enzyme
187
PREFERENCES
PR
RT-
site 1
23
Site -
Junction
1 2 3 4 5 6 7 8
MA-CA CA-NC NC-PR PR-RT p66-p51-RT RT-IN
FIG. 1. proteins asterisk.
45
p66 P51
+
6
IN .__ 7 8
P4-P3-P2-Pl
Pl’-
P2’-
P4’
Ser-Gln-Asn-TyrsPro-Ile-Val-Gln Ala-Arg-Val-LeusAla-Glu-Ala-Met Ala-Thr-Ile-Met*Met-Gln-Arg-Gly Pro-Gly-Asn-Phe*Leu-Gln-Ser-Arg Ser-Phe-Asn-Phe*Pro-Gln-Ile-Thr Thr-Leu-Asn-Phe*Pro-Ile-Set--Pro Ala-Glu-Thr-Phe + Tyr-Val-Asp-Gly Arg-Lys-Ile-Leu*Phe-Leu-Asp-Gly
Cleavage sites of the proteinase in the gag andgaglpol of HIV-1
P3’-
(strain
HXB2).
The
scissile
bond
precursor is indicated by an
188
BILLICH TABLE
Effect of Variation for the Hydrolysis
II
of the Pl’ Residue on Kinetic of Peptides Ser-Gln-Asn-Tyr
Kl?! Pl’ TY~
Phe Trp Ala Val Leu Ile Met Pro
Gly, His, Lys, Arg, Asp, Glu, Asn, Gln
k eat
bM)
(mini)
0.8 1.0
73 60 23 49 70 70 40 109 140
1.2 1.9 2.2 1.0 4.2 2.6 2.3
CYS Ser Thr 1
AND
Parameters * Pl’-Ile-Val LJKn (mM-’ * mine’) 91.3
60.0 19.2 25.8 31.8 70.0 9.5 42.0 60.9
Less than 1% cleavage in 10 h using 250 nM proteinase No cleavage observed” I
a No cleavage observed after incubation 250 nM HIV-PR.
of peptides overnight
with
of about 10 and catalytic efficiency (kc,/K,,,) by about 17. The natural sequence (Xaa = Tyr) is cleaved most efficiently. One characteristic feature of HIV-l proteinase (and of retroviral proteinases in general) is their ability to cleave Xaa-Pro peptide bonds. This does not mean that the enzyme is strictly specific for proline at Pl’ as is already indicated by the sequence of five of the cleavage sites in the viral proteins which lack proline (see Fig. 1). We found that proline can be replaced by all amino acids with nonpolar moieties (see Table II), including alanine, valine, and isoleucine. A very low level of cleavage which was detectable only after prolonged incubation with an excess of enzyme was observed in the case of the peptides with Xaa = Cys, Ser, or Thr. The latter result is in accordance with the findings of Partin et al. (4) who reported cleavage of a mutated MA/CA junction which had Thr at Pl’ in a gag precursor protein. From NMR studies it is known that the heptapeptide Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH, in aqueous solution exists in two major conformations: 70% as an extended peptide with a trans configuration at all peptide bonds and 30% with a cis-proline bond (25). To mimick the cis configuration, Garofalo et al. (25) synthesized an analogue with a peptide bond surrogate (Phe\k[CNJ Ala); however, this compound was not an inhibitor of HIV-proteinase. Since we found that proline can be replaced even by bulky residues (e.g., Ile), this failure cannot be explained by steric hindrance of the peptide bond replacement. But it can be deduced that the cis configuration at the proline is of no importance for binding to the enzyme; this is also implicated by the possible exchange of proline by other a factor
WINKLER
amino acids which have no propensity to form cis bonds without a loss of catalytic efficiency. Since the HIV proteinase is a symmetrical dimer whose active site is formed by identical residues of both monomers (20), it is not surprising that the Sl and Sl’ binding sites on the enzyme show the same substrate preference. However, the substrates are not symmetrical and thus the pattern of hydrogen bonds and of nonbonded interactions is asymmetric. Therefore, it is possible that a peptide with, e.g., alanine in Pl, is not cleaved, but it is a good substrate when alanine is placed in Pl’. It can be deduced that the dipeptide at the scissile bond has to show a certain degree of hydrophobicity which, e.g., is provided by Tyr-Ala (or Val-Ala, see site 2 in Fig. l), but not by Ala-Pro (nor by Ala-Ala, see (6)). Another interesting observation is the tolerance of valine and isoleucine in Pl’ (Pl-Pl’: Tyr-Val or Tyr-Ile), but not in Pl (Val-Pro or Ile-Pro). This might be explained by a different conformation of the proline-containing peptides which could influence side chain orientation of Val and Ile at Pl in such a way that they are not accomodated by the Sl subsite. However, Ile and Val are also not tolerated in the Pl of another substrate with a totally different sequence having Ala at Pl’ (see above, (8)). This indicates that the conformation of the peptide as a whole may influence side chain interactions at Pl and Pl’. When the Asn residue at P2 of Ser-Gln-Asn-Tyr-ProIle-Val is exchanged for Gln, no cleavage of the modified peptide is observed (see Table III). This striking effect (the peptides differ by only one methylene group) was reported earlier by Partin et al. (4) who used both a peptide and a gag protein with an Asn + Gln mutation. The preference for Asn in P2 might be explained at least in part
TABLE Effect of Variation for the Hydrolysis
III
of the P2 Residue on Kinetic Parameters of Peptides Ser-Gln-P2-Tyr-Pro-Ile-Val
P2 Asn Gln ASP
Glu CYS Ser Ala Val Gly, Leu, Be, Pi-o, Phe, Tyr, Trp, Met, Thr, Lys, Arg, His 1
KI2 (mM)
k est
(mini)
2.3 60.9 140 No cleavage observed“ 0.5 29 58.0 12.0 8.6 103 2.1 93 44.3 ~1% cleavage in 10 h (250 nM HIVPR) 3.3 69 21.1 1.4 11 7.9 No cleavage observed”
a No cleavage observed after incubation 250 nM HIV-PR.
of peptides overnight
with
HIV-l
PROTEINASE
by hydrogen bonds between the carboxamide group and the protein. In the three-dimensional structure of HIV proteinase complexed with a hydroxyethylamine inhibitor the amide hydrogens of protein residues Asp-29 and Asp-30 are seen to interact with the Asn at P2 of the inhibitor (26). We wondered whether there is a strict specificity of the enzyme for Asn at P2 in this substrate. As can be seen in Table III, some substitutions indeed yield cleavable peptides; however, it is obviously a small selection of amino acids which can be accomodated by the S2 subsite. First, negatively charged residues are tolerated: Substitution by Asp leads to an approximately fivefold decrease of both Km and kc,,; thus cleavage efficiency remains constant. This might be explained by an enhanced interaction of both the substrate and the N-terminal product with the active site surface, with better binding, but also decreased turnover, as a consequence. Introducing Glu at P2, however, leads to a sevenfold decrease of k,,JK,, mainly due to a rise in K,,, . We found that the peptide containing Gln at P2 does not inhibit HIV proteinase at concentrations of up to 15 mM; thus this peptide does not bind to the enzyme. In the peptide with P2 = Glu the unfavorable size of the side chain seems to be partially compensated by its charge. Second, replacement by cysteine yields an efficiently cleaved substrate; unexpectedly, the serine containing analogue is hydrolyzed very slowly. Third, the peptides containing Ala or Val at P2 are cleaved, but Leu and Ile are not tolerated. About the same K,,, is observed for the peptides with Asn, Ala, or Val at P2; the suggested hydrogen bonds between Asn and the peptide backbone of the enzyme obviously do not enhance binding of the substrate to any significant extent. However, the kcatof the parent substrate is twofold higher than that of the Alacontaining peptide; therefore hydrogen bonding of the Asn side chain might lead to a more favorable preorientation of the scissile bond. To summarize, small, both polar and apolar amino acids are preferred in P2 of the MA/CA junction peptide, but subtle differences (e.g., Cys-*Ser) may have a significant influence on cleavage efficiency. It is also interesting to note that Thr and Ile are not accepted in this peptide while the cleavage sites 3, 7, and 8 (see Fig. 1) contain these amino acids at P2. Also, findings made with one artificial substrate peptide may not hold when applied to another sequence. In site 2 peptides (see Fig. l), Ile fits into the S2 subsite while substitution of P2 with Asn yields a 50-fold slower substrate (9); in the MA/CA peptide Asn is preferred, while Ile cannot be adapted to the S2 subsite. Margolin et al. (5) tested six substituted peptides with modifications at P2’: peptides with Gly, Phe, and Trp were not cleaved very efficiently, while peptides with Ala, Leu, and Ile were good substrates. However, we found that Ile or Leu at P2 render the same peptide uncleavable (see above). Also, the natural cleavage sites have Leu, Ile, and
SUBSITE
189
PREFERENCES
Gln at P2’, which prevent cleavage when included at P2 of the MA/CA junction sequence. Again the asymmetry of the substrates leads to different subsite preferences for the S2 and S2’ binding sites which per se would be expected to be identical. Finally, the amino acid in the P3 position was varied (see Table IV). With the exception of proline all substitutions were tolerated by the enzyme. This low stringency is also reflected in the occurrence of eight different residues at P3 in the eight natural cleavage sites. The relative specificity constants which here were determined by competition cleavage reactions varied by a factor of about 20. While the P3 residue is still located in the active site cleft when the peptide is bound to the enzyme (21), there are obviously few restrictions imposed at this site, since apolar, polar, and both negatively and positively charged side chains can be accomodated. Also, in an enzyme-inhibitor complex (26) no hydrogen bonds are evident between Gln as P3 residue and the protein. However, as can be seen from our data there is still some selectivity: (1) Aromatic residues, especially Phe and Tyr, and methionine are preferred over aliphatic residues, especially the branched chain amino acids (Val, Leu, Ile), and glycine. (2) Among the polar and negatively charged amino acids, the larger residues are preferred (Thr > Ser, Cys; Gln > Asn; Glu > Asp). (3) Arg shows a marked preference over Lys; a positively charged amino acid at P3 thus is not characteristic of peptides featuring Leu*Phe or Leu*Ala instead of Tyr*Pro at the scissile bond. To summarize, some guidelines can be deduced from our results which may be useful in the design of inhibitors: TABLE
IV
Effect of Variation of P3 on Relative Cleavage Efficiency of Peptides Ser-P3-Asn-Tyr-Pro-Ile-Val
Gh
“1”
Asn
0.46 0.36
QY
Ala Val Leu
0.92 0.53 0.21
Ile Pro Phe TY~
0.11 0 1.75
Trp
1.10 0.94 1.54
His Arg LYS ASP
Glu Ser Thr CYS Met
1.72
0.40 0.42 0.73 0.43
1.76 0.33
1.82
190
BILLICH
AND
(1) Subsites Sl and Sl’ best accomodate hydrophobic amino acids; however, effects of steric hinderance may be encountered. This rule is corroborated by the data of Phylip et al. (9) and Richards et al. (8) and also by inspection of the sequences of the natural sites on the gag/ pal precursors. Both Sl and Sl’ should be filled with a hydrophobic residue, since a Tyr-Gly bond at Pl and Pl’ of the heptapeptide is not cleaved. No major advantage of introducing proline or its mimicks at the Pl’ site of inhibitors can be predicted. (2) With regard to P2, natural substrates may be divided into two groups: In substrates with a Tyr/Phe-Pro scissile dipeptide (site 1, 5, and 6 in Fig. 1) Asn is always present at P2; but in the other substrates having two hydrophobic amino acids at the scissile bond various other residues (Val, Ile, Thr) are encountered. In the MA/CA junction peptide, Asn at P2 yields an S-fold better substrate than Val (see Table III) while in a site 2 peptide Asn is 50-fold worse than Val (9). Thus, in inhibitors which explicitly exchange the scissile dipeptide by an uncleavable mimic of Tyr-Pro (e.g., in Ref. (19)) Asn would be the first choice to introduce in P2, but Ala, Val, and Cys would be likely candidates for a series of promising substitutions. However, in inhibitors featuring two hydrophobic residues at Pl and Pl’ one should probably start with valine at P2. (3) Subsite S3 allows a variety of residues; even charged side chains may be introduced. In inhibitors, aromatic residues, Ala, Thr, or Met, but no branched chain amino acids, can be recommended. (4) The overall conformation of peptides bound to the enzyme will influence the adaptability of the side chains to the subsites of the protein. Therefore, predictions of structure-activity relations can only be applied with confidence when looking at closely related series of peptides or inhibitors. ACKNOWLEDGMENTS We thank R. Reuschel for peptide syntheses and H. Aschauer for amino acid analyses and peptide sequencing. We are grateful to Drs. B. Rosenwirth and H. Gstach for critical reading of the manuscript.
REFERENCES 1. Dunn, B. M., and Kay, J. (1990) Antiviral Chem. Chemother. 1, 3a. 2. Debouck, C., and Metcalf, B. W. (1990) Drug Dev. Res. 21, 1-17. 3. Loeb, D. D., Hutchinson, C. A., Edgell, M. H., Farmerie, W. G., & Swanstrom, R. (1939) J. Viral. 63, 111-121.
WINKLER 4. Partin, K., Krausslich, H. G., Ehrlich, L., Wimmer, E., & Carter, C. (1990) J. Virol. 64, 3938-3947. 5. Margolin, N., Heath, W., Osborne, E., Lai, M., & Vlahos, C. (1990) Biochem. Biophys. Res. Commun. 167,554-560. 6. Tomaselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., Bannow, C., Reardon, J. M., Howe, W. J., DeCamp, D. L., Craik, C. S., & Heinrikson, R. J. (1990) J. Biol. Chem. 265, 14,675-14,683. 7. Konvalinka, J., Strop, S., Velek, J., Cerna, V., Kostka, V., Phylip, L. H., Richards, A. D., Dunn, B. M., & Kay, J. (1990) FEBS Lett. 268,5-38. 3. Richards, A. D., Phylip, L. H., Farmerie, W. G., Scarborough, P. E., Alvarez, A., Dunn, B. M., Hirel, P. H., Konvalinka, J., Strop, P., Pavlickova, L., Kostka, V., & Kay, J. (1990) J. Biol. Chem. 265, 7733-7736. 9. Phylip, L. H., Richards, A. D., Kay, J., Konvalinka, J., Strop, P., Blaha, J., Velek, J., Kostka, V., Ritchie, A. J., Broadhurst, A. V., Farmerie, W. G., Scarborough, S. E., & Dunn, B. M. (1990) Biochem. Biophys. Res. Commun. 171, 439-444. 10. Seelmeier, S., Schmidt, H., Turk, V., & von der Helm, K. (1988) Proc. Natl. Acad. Sci. USA 85, 6612-6616. 11. Billich, A., Hammerschmid, F., & Winkler, G. (1990) Biol. Chem. Hoppe-Seyler 371,265-272. 12. Henderson, P. J. F. (1972) Biochem. J. 127, 321-333. 13. Houghten, R. A. (1985) Proc. Natl. Acad. Sci. USA 82,5131-5135. 14. Wang, S. S. (1973) J. Am. Chem. Sot. 95, 1326-1335. 15. Dourtoglou, V., & Gross, B. (1984) Synthesis, 572-574. 16. Castro, B., Dornoy, J. R., Evin, G., & Selve, C. (1975) Tetrahedron Lett. 14,1219-1222. 17. Dreyer, G. B., Metcalf, B. W., Tomaszek, T. A., Carr, T. J., Chandler, A. C., Hylan, L., Fakhoury, S. A., Magaard, V. W., Moore, M. L., Strickler, J. E., Debouck, V., & Meek, T. D. (1989) Proc. Natl. Acad. Sci. USA 86,9752-9756. 18. Billich, S., Knoop, M. Th., Hansen, J., Strop, P., Sedlacek, J., Mertz, R., & Molling, K. (1968) J. Biol. Chem. 263, 17,905-17,908. 19. Rich, D. H., Green, J., Toth, M. V., Marshall, G. R., & Kent, S. B. H. (1990) J. Med. Chem. 33,128&J-1295. 20. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B. K., Baldwin, E., Weber, I. T., Selk, L. M., Clawson, L., Schneider, J., & Kent, S. B. H. (1969) Science 245, 616-625. 21. Tosser, J., Gustchina, A., Weber, I. T., Blaha, I., Wondrak, E. M., & Oroszlan, S. (1991) FEBS L&t. 279, 356-360. 22. Shoeman, R. L., Honer, B., Stoller, T. J., Kesselmeier, C., Miedel, M. C., Traub, P., & Graves, M. C. (1990) Proc. Natl. Acad. Sci. USA 87, 6336-6340. 23. Schechter, I., & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162. 24. Tritch, R. J., Cheng, Y.-S. E., Yin, F. H., & Erickson-Viitanen, S. (1991) J. Virol. 65, 922-930. 25. Garofalo, A., Tarnus, C., Remy, J. M., Leppik, R., Piriou, F., Harris, B., & Pelton, J. T. (1990) in Peptides (Rivier, J. E., and Marshall, G. R., Eds.), pp. 833-634, Escom, Leiden, The Netherlands. 26. Swain, A. L., Miller, M. M., Green, J., Rich, D. H., Schneider, J., Kent, S. B. H., & Wlodawer, A. (1990) Proc. Natl. Acad Sci. USA 87,8805-8809.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 290, No. 1, October, pp. 186-190, 1991
Analysis of Subsite Preferences of HIV-1 Proteinase Using MA/CA Junction Peptides Substituted at the P3-PI’ Positions Andreas Billich’ Department
and Gottfried
of Antiretroviral
Therapy,
Winkler Sandoz-Research
Institute,
Brunnerstrasse
59, A-1235
Vienna,
Austria
Received April 18, 1991, and in revised form June 7, 1991
The residues P3, P2, Pl, and Pl’ of a peptide corresponding to the matrixtcapsid protein junction in the HIV- 1 gag protein (Ser-Gln-Asn-Tyr-Pro-Ile-Val) were systematically replaced and the effect of these single amino acid substitutions on the hydrolysis of each peptide by HIV- 1 proteinase was studied. Subsites S 1 and S 1’ of the enzyme showed explicit preference for hydrophobic moieties, but o-branched amino acids and proline are not tolerated in Sl. The 52 subsite shows a preference for small polar and apolar amino acids; it may be occupied by Asn, Asp, Glu, Cys, Ala, or Val, other substitutions, especially by Gln and Ser, prevent hydrolysis of the peptides. In subsite S3 all amino acids except proline can be accommodated. o 1991Academic PRSS, he.
rational selection of the side chains to be introduced with advantage at the corresponding sites of inhibitors. With this aim studies on subsite specificity of the proteinase have been performed either by mutational analysis of the protein substrates (3,4) or by using substituted oligopeptide analogues of the natural cleavage sites (5-9). This publication extends earlier studies by reporting subsite preferences for the residues of peptides whose sequences are derived from the junction between the matrix (MA) and the capsid protein (CA) in the HIV-l gag precursor (“MA/CA junction”). We systematically replaced residues P3, P2, Pl, and Pl’ and examined the effect of these single amino acid substitutions on the hydrolysis by purified recombinant HIV-l proteinase. MATERIALS
An essential step in the replication of human immunodeficiency virus 1 (HIV-l)’ is the specific cleavage of the gag and gag/p01 precursor proteins by the virus-encoded proteinase (see Refs. (1, 2) for review). Inhibitors of this enzyme have been shown to block HIV replication in infected lymphocytes and may be useful in the therapy of AIDS. Since potent inhibitors of HIV-l proteinase are most commonly peptide mimetics containing transition state analogues of the scissile peptide bond, design of inhibitors will be facilitated by understanding the substrate specificity of the enzyme. More precisely, knowledge about subsite preferences at the scissile bond (Pl and PI’ positions3) and also at the flanking residues will allow a i To whom correspondence should be addressed. ’ Abbreviations used: HIV-I, human immunodeficiency virus 1; MA, matrix protein; CA, capsid protein; Mes, 4-morpholine ethanesulfonic acid; DTE, dithioerythritol; TFA, trifluoroacetic acid. 3 The nomenclature of Schechter and Berger (23), i.e., P4-P3-P2PI*Pl’-P2’-P3’, is used to indicate amino acids adjacent to residues Pl and Pl’ which form the scissile peptide bond (*); the corresponding subsites on the enzyme are designated by S4-S3’.
AND
METHODS
HIV-1 proteinuse. The enzyme was produced using the expressor plasmid pTZprt+ (10) in Escherichia coli strain JM 105 and purified to homogeneity as described (11). The concentration of active enzyme (Et) was determined by active-site titration (12) using a competitive inhibitor synthesized at Sandoz. Peptide synthesis. Solid-phase simultaneous peptide synthesis was done by the mesh-packet method of Houghten (13), using the polystyrene resin described by Wang (14) and standard methods of coupling and deprotection (15, 16). Purification of crude peptides was performed on a reversed-phase Cls column eluted with gradients of 10 mM aqueous ammonium acetate/acetonitrile. All peptides were then demonstrated to be homogeneous by analytical HPLC; identity was confirmed by amino acid and/or sequence analysis. Peptides were incubated at 37°C with Assay conditions and kinetics. proteinase (5-25 nM active concentration) in 50 mM Mes, pH 5.5, containing 1 mM DTE, 1 mM EDTA, and 1 M NaCl in a total volume of 150 ~1. Aliquots of 25 ~1 were removed and quenched by adding an equal volume of 0.5% trifluoroacetic acid (TFA). Then the samples were analyzed by HPLC on a HP1050 chromatograph (Hewlett-Packard) with a RP-Cl8 column (Vydac 218TP54,4.6 X 25 mm) using a linear gradient of O-35% acetonitrile in 0.1% TFA at a rate of change of 2%/min and a flow rate of 1 ml/min. With all cleavable peptides, only two products were observed. Cleavage occurred between the fourth and fifth amino acid, as shown either by using authentic tripeptide as reference or by amino acid analysis of tetrapeptide products. 0003.9861/91
186
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HIV-1 TABLE
PROTEINASE
SUBSITE
I
Effect of Variation of the Pl Residue on Kinetic Parameters for Hydrolysis of Peptides Ser-Gln-Asn-Pl * Pro-Ile-Val K”l Pl
k cat
(mine’)
bM)
LW
2.3 2.4 1.6 10
Met
4.9
TY~
Phe Trp
Gly, Ala, Val, Ile, Pro, His, Lys, Arg, Asp, Glu, Asn, Gin, SW, Thr, Cys I
k&L (mM-’ . min-‘)
140 13 13 31 17
60.9 30.4 8.1 3.7 3.5
No cleavage”
a No cleavage observed after incubation 250 nM HIV-PR.
of peptides overnight
with
Initial rates of peptide cleavage were determined by measuring the amount of N-terminal tetrapeptide product at three to five time points during the period of linear progression of cleavage, i.e., up to about 10% turnover. The same initial rates could have been obtained from the decrease of substrate. To derive Km and V-, initial rates were measured at 6-10 substrate concentrations; data were fitted by nonlinear regression analysis to the Michaelis-Menten function using the program Enzfitter. The k,,, values were derived from the relation V,,, = kc,* [I&]. The precision of Km and k,,, values was in the range *lo-20%. Competition cleauages. The initial velocity (V) of two substrate peptides (1 and 2) competing for the active site of an enzyme depends on the specificity constant for each substrate and on the initial substrate concentration: VI = (k,dK,),
. [&I
and
V, = (k,.,lK,),.
[&I.
Measurements of initial rates at a time point where [S] is considered to be constant and cleavage is proceeding linearly, therefore, can be used to obtain a relative specificity constant (k,,JK,,,),,. Here, these constants were determined relative to a reference peptide, namely Ser-Gln-AsnTyr-Pro-Ile-Val. Competition cleavage reactions contained a 2 mM concentration of each substrate. In some cases, poor substrate peptides were included at higher concentrations (up to 10 mM) and the reference Initial peptide was reduced to 1 mM to ensure accurate quantification. rates were measured as described above for single-substrate incubations. Values of (k,.,/K,),,, were reproducible within f20%. RESULTS
AND
(250 nM) in overnight. incubations. This observation is in accordance with the sequences of the eight natural cleavage sites used by the proteinase in the gag and gag/p01 precursor proteins (see Fig. 1). In fact, these sites exhibit either Tyr, Phe, Leu, or Met at the Pl position, i.e., all amino acids that are tolerated in the peptides, too, with the exception of tryptophan. Obviously, amino acids containing hydrophobic aliphatic or aromatic side chains are preferred at Pl. The methyl group of the Ala residue may be too small to make adequate contact with the active site cleft. But it is not only hydrophobicity of substrate moieties that governs specificity at Pl since neither Val- nor Ile-containing peptides are cleaved while Leu is tolerated. A similar observation was reported by Richards et al. (8) for the substrate Ala-Arg-Val-Leu-Ala-G&Ala which corresponds to the natural cleavage site 2: replacement of Leu by Ile or Val rendered the peptide uncleavable. It therefore becomes obvious that the active site of the proteinase cannot accomodate P-branched amino acids in subsite Sl. As an exception to the rule, that the scissile dipeptide should be hydrophobic, a cleavage site with a positively charged arginine residue in Pl’ has been shown to be used by HIV-l proteinase in a nonviral protein (22). In addition, a decapeptide with lysine at Pl (Val-Ser-Gln-AsnLys-Pro-Ile-Val-Gln-Asn) was reported to be cleaved with 10% efficiency relative to the corresponding peptide with Pl = Tyr (24); the same substitution in the gag protein yielded a poorly cleaved substrate. Thus, it appears that alterations of either conformation or size of a sequence, or both, can at least partially compensate for inadequate interactions at the Sl or Sl’ site. Binding efficiency, as reflected by the K,,, value, is similar for the substrates containing aromatic moieties at Pl, but is lower when linear or branched side chains are introduced (Xaa = Met or Leu). In this series, kcatdrops by MA p17
.CA NC p24 p7 p6
DISCUSSION
The heptapeptide Ser-Gln-Asn-Tyr-Pro-Ile-Val corresponding to the sequence of the MA/CA junction in the gag precursor protein of HIV-l is efficiently cleaved by HIV-l proteinase between Tyr and Pro (K,,, = 2.3 mM, &at = 140 mini’). Peptides exhibiting this sequence have been routinely used in many studies of HIV proteinase activity and have also been the starting point for the design of inhibitors in which the Tyr-Pro dipeptide is replaced by transition state analogues (6, 17-19). For our study we first replaced the tyrosine in the Pl position by the other 19 amino acids found in proteins (see Table I).
The only modified peptides that were cleaved by the proteinase are those containing Phe, Trp, Leu, or Met at Pl. Peptides containing any other amino acid at this position were not cleaved, not. even when using an excess of enzyme
187
PREFERENCES
PR
RT-
site 1
23
Site -
Junction
1 2 3 4 5 6 7 8
MA-CA CA-NC NC-PR PR-RT p66-p51-RT RT-IN
FIG. 1. proteins asterisk.
45
p66 P51
+
6
IN .__ 7 8
P4-P3-P2-Pl
Pl’-
P2’-
P4’
Ser-Gln-Asn-TyrsPro-Ile-Val-Gln Ala-Arg-Val-LeusAla-Glu-Ala-Met Ala-Thr-Ile-Met*Met-Gln-Arg-Gly Pro-Gly-Asn-Phe*Leu-Gln-Ser-Arg Ser-Phe-Asn-Phe*Pro-Gln-Ile-Thr Thr-Leu-Asn-Phe*Pro-Ile-Set--Pro Ala-Glu-Thr-Phe + Tyr-Val-Asp-Gly Arg-Lys-Ile-Leu*Phe-Leu-Asp-Gly
Cleavage sites of the proteinase in the gag andgaglpol of HIV-1
P3’-
(strain
HXB2).
The
scissile
bond
precursor is indicated by an
188
BILLICH TABLE
Effect of Variation for the Hydrolysis
II
of the Pl’ Residue on Kinetic of Peptides Ser-Gln-Asn-Tyr
Kl?! Pl’ TY~
Phe Trp Ala Val Leu Ile Met Pro
Gly, His, Lys, Arg, Asp, Glu, Asn, Gln
k eat
bM)
(mini)
0.8 1.0
73 60 23 49 70 70 40 109 140
1.2 1.9 2.2 1.0 4.2 2.6 2.3
CYS Ser Thr 1
AND
Parameters * Pl’-Ile-Val LJKn (mM-’ * mine’) 91.3
60.0 19.2 25.8 31.8 70.0 9.5 42.0 60.9
Less than 1% cleavage in 10 h using 250 nM proteinase No cleavage observed” I
a No cleavage observed after incubation 250 nM HIV-PR.
of peptides overnight
with
of about 10 and catalytic efficiency (kc,/K,,,) by about 17. The natural sequence (Xaa = Tyr) is cleaved most efficiently. One characteristic feature of HIV-l proteinase (and of retroviral proteinases in general) is their ability to cleave Xaa-Pro peptide bonds. This does not mean that the enzyme is strictly specific for proline at Pl’ as is already indicated by the sequence of five of the cleavage sites in the viral proteins which lack proline (see Fig. 1). We found that proline can be replaced by all amino acids with nonpolar moieties (see Table II), including alanine, valine, and isoleucine. A very low level of cleavage which was detectable only after prolonged incubation with an excess of enzyme was observed in the case of the peptides with Xaa = Cys, Ser, or Thr. The latter result is in accordance with the findings of Partin et al. (4) who reported cleavage of a mutated MA/CA junction which had Thr at Pl’ in a gag precursor protein. From NMR studies it is known that the heptapeptide Ser-Gln-Asn-Tyr-Pro-Ile-Val-NH, in aqueous solution exists in two major conformations: 70% as an extended peptide with a trans configuration at all peptide bonds and 30% with a cis-proline bond (25). To mimick the cis configuration, Garofalo et al. (25) synthesized an analogue with a peptide bond surrogate (Phe\k[CNJ Ala); however, this compound was not an inhibitor of HIV-proteinase. Since we found that proline can be replaced even by bulky residues (e.g., Ile), this failure cannot be explained by steric hindrance of the peptide bond replacement. But it can be deduced that the cis configuration at the proline is of no importance for binding to the enzyme; this is also implicated by the possible exchange of proline by other a factor
WINKLER
amino acids which have no propensity to form cis bonds without a loss of catalytic efficiency. Since the HIV proteinase is a symmetrical dimer whose active site is formed by identical residues of both monomers (20), it is not surprising that the Sl and Sl’ binding sites on the enzyme show the same substrate preference. However, the substrates are not symmetrical and thus the pattern of hydrogen bonds and of nonbonded interactions is asymmetric. Therefore, it is possible that a peptide with, e.g., alanine in Pl, is not cleaved, but it is a good substrate when alanine is placed in Pl’. It can be deduced that the dipeptide at the scissile bond has to show a certain degree of hydrophobicity which, e.g., is provided by Tyr-Ala (or Val-Ala, see site 2 in Fig. l), but not by Ala-Pro (nor by Ala-Ala, see (6)). Another interesting observation is the tolerance of valine and isoleucine in Pl’ (Pl-Pl’: Tyr-Val or Tyr-Ile), but not in Pl (Val-Pro or Ile-Pro). This might be explained by a different conformation of the proline-containing peptides which could influence side chain orientation of Val and Ile at Pl in such a way that they are not accomodated by the Sl subsite. However, Ile and Val are also not tolerated in the Pl of another substrate with a totally different sequence having Ala at Pl’ (see above, (8)). This indicates that the conformation of the peptide as a whole may influence side chain interactions at Pl and Pl’. When the Asn residue at P2 of Ser-Gln-Asn-Tyr-ProIle-Val is exchanged for Gln, no cleavage of the modified peptide is observed (see Table III). This striking effect (the peptides differ by only one methylene group) was reported earlier by Partin et al. (4) who used both a peptide and a gag protein with an Asn + Gln mutation. The preference for Asn in P2 might be explained at least in part
TABLE Effect of Variation for the Hydrolysis
III
of the P2 Residue on Kinetic Parameters of Peptides Ser-Gln-P2-Tyr-Pro-Ile-Val
P2 Asn Gln ASP
Glu CYS Ser Ala Val Gly, Leu, Be, Pi-o, Phe, Tyr, Trp, Met, Thr, Lys, Arg, His 1
KI2 (mM)
k est
(mini)
2.3 60.9 140 No cleavage observed“ 0.5 29 58.0 12.0 8.6 103 2.1 93 44.3 ~1% cleavage in 10 h (250 nM HIVPR) 3.3 69 21.1 1.4 11 7.9 No cleavage observed”
a No cleavage observed after incubation 250 nM HIV-PR.
of peptides overnight
with
HIV-l
PROTEINASE
by hydrogen bonds between the carboxamide group and the protein. In the three-dimensional structure of HIV proteinase complexed with a hydroxyethylamine inhibitor the amide hydrogens of protein residues Asp-29 and Asp-30 are seen to interact with the Asn at P2 of the inhibitor (26). We wondered whether there is a strict specificity of the enzyme for Asn at P2 in this substrate. As can be seen in Table III, some substitutions indeed yield cleavable peptides; however, it is obviously a small selection of amino acids which can be accomodated by the S2 subsite. First, negatively charged residues are tolerated: Substitution by Asp leads to an approximately fivefold decrease of both Km and kc,,; thus cleavage efficiency remains constant. This might be explained by an enhanced interaction of both the substrate and the N-terminal product with the active site surface, with better binding, but also decreased turnover, as a consequence. Introducing Glu at P2, however, leads to a sevenfold decrease of k,,JK,, mainly due to a rise in K,,, . We found that the peptide containing Gln at P2 does not inhibit HIV proteinase at concentrations of up to 15 mM; thus this peptide does not bind to the enzyme. In the peptide with P2 = Glu the unfavorable size of the side chain seems to be partially compensated by its charge. Second, replacement by cysteine yields an efficiently cleaved substrate; unexpectedly, the serine containing analogue is hydrolyzed very slowly. Third, the peptides containing Ala or Val at P2 are cleaved, but Leu and Ile are not tolerated. About the same K,,, is observed for the peptides with Asn, Ala, or Val at P2; the suggested hydrogen bonds between Asn and the peptide backbone of the enzyme obviously do not enhance binding of the substrate to any significant extent. However, the kcatof the parent substrate is twofold higher than that of the Alacontaining peptide; therefore hydrogen bonding of the Asn side chain might lead to a more favorable preorientation of the scissile bond. To summarize, small, both polar and apolar amino acids are preferred in P2 of the MA/CA junction peptide, but subtle differences (e.g., Cys-*Ser) may have a significant influence on cleavage efficiency. It is also interesting to note that Thr and Ile are not accepted in this peptide while the cleavage sites 3, 7, and 8 (see Fig. 1) contain these amino acids at P2. Also, findings made with one artificial substrate peptide may not hold when applied to another sequence. In site 2 peptides (see Fig. l), Ile fits into the S2 subsite while substitution of P2 with Asn yields a 50-fold slower substrate (9); in the MA/CA peptide Asn is preferred, while Ile cannot be adapted to the S2 subsite. Margolin et al. (5) tested six substituted peptides with modifications at P2’: peptides with Gly, Phe, and Trp were not cleaved very efficiently, while peptides with Ala, Leu, and Ile were good substrates. However, we found that Ile or Leu at P2 render the same peptide uncleavable (see above). Also, the natural cleavage sites have Leu, Ile, and
SUBSITE
189
PREFERENCES
Gln at P2’, which prevent cleavage when included at P2 of the MA/CA junction sequence. Again the asymmetry of the substrates leads to different subsite preferences for the S2 and S2’ binding sites which per se would be expected to be identical. Finally, the amino acid in the P3 position was varied (see Table IV). With the exception of proline all substitutions were tolerated by the enzyme. This low stringency is also reflected in the occurrence of eight different residues at P3 in the eight natural cleavage sites. The relative specificity constants which here were determined by competition cleavage reactions varied by a factor of about 20. While the P3 residue is still located in the active site cleft when the peptide is bound to the enzyme (21), there are obviously few restrictions imposed at this site, since apolar, polar, and both negatively and positively charged side chains can be accomodated. Also, in an enzyme-inhibitor complex (26) no hydrogen bonds are evident between Gln as P3 residue and the protein. However, as can be seen from our data there is still some selectivity: (1) Aromatic residues, especially Phe and Tyr, and methionine are preferred over aliphatic residues, especially the branched chain amino acids (Val, Leu, Ile), and glycine. (2) Among the polar and negatively charged amino acids, the larger residues are preferred (Thr > Ser, Cys; Gln > Asn; Glu > Asp). (3) Arg shows a marked preference over Lys; a positively charged amino acid at P3 thus is not characteristic of peptides featuring Leu*Phe or Leu*Ala instead of Tyr*Pro at the scissile bond. To summarize, some guidelines can be deduced from our results which may be useful in the design of inhibitors: TABLE
IV
Effect of Variation of P3 on Relative Cleavage Efficiency of Peptides Ser-P3-Asn-Tyr-Pro-Ile-Val
Gh
“1”
Asn
0.46 0.36
QY
Ala Val Leu
0.92 0.53 0.21
Ile Pro Phe TY~
0.11 0 1.75
Trp
1.10 0.94 1.54
His Arg LYS ASP
Glu Ser Thr CYS Met
1.72
0.40 0.42 0.73 0.43
1.76 0.33
1.82
190
BILLICH
AND
(1) Subsites Sl and Sl’ best accomodate hydrophobic amino acids; however, effects of steric hinderance may be encountered. This rule is corroborated by the data of Phylip et al. (9) and Richards et al. (8) and also by inspection of the sequences of the natural sites on the gag/ pal precursors. Both Sl and Sl’ should be filled with a hydrophobic residue, since a Tyr-Gly bond at Pl and Pl’ of the heptapeptide is not cleaved. No major advantage of introducing proline or its mimicks at the Pl’ site of inhibitors can be predicted. (2) With regard to P2, natural substrates may be divided into two groups: In substrates with a Tyr/Phe-Pro scissile dipeptide (site 1, 5, and 6 in Fig. 1) Asn is always present at P2; but in the other substrates having two hydrophobic amino acids at the scissile bond various other residues (Val, Ile, Thr) are encountered. In the MA/CA junction peptide, Asn at P2 yields an S-fold better substrate than Val (see Table III) while in a site 2 peptide Asn is 50-fold worse than Val (9). Thus, in inhibitors which explicitly exchange the scissile dipeptide by an uncleavable mimic of Tyr-Pro (e.g., in Ref. (19)) Asn would be the first choice to introduce in P2, but Ala, Val, and Cys would be likely candidates for a series of promising substitutions. However, in inhibitors featuring two hydrophobic residues at Pl and Pl’ one should probably start with valine at P2. (3) Subsite S3 allows a variety of residues; even charged side chains may be introduced. In inhibitors, aromatic residues, Ala, Thr, or Met, but no branched chain amino acids, can be recommended. (4) The overall conformation of peptides bound to the enzyme will influence the adaptability of the side chains to the subsites of the protein. Therefore, predictions of structure-activity relations can only be applied with confidence when looking at closely related series of peptides or inhibitors. ACKNOWLEDGMENTS We thank R. Reuschel for peptide syntheses and H. Aschauer for amino acid analyses and peptide sequencing. We are grateful to Drs. B. Rosenwirth and H. Gstach for critical reading of the manuscript.
REFERENCES 1. Dunn, B. M., and Kay, J. (1990) Antiviral Chem. Chemother. 1, 3a. 2. Debouck, C., and Metcalf, B. W. (1990) Drug Dev. Res. 21, 1-17. 3. Loeb, D. D., Hutchinson, C. A., Edgell, M. H., Farmerie, W. G., & Swanstrom, R. (1939) J. Viral. 63, 111-121.
WINKLER 4. Partin, K., Krausslich, H. G., Ehrlich, L., Wimmer, E., & Carter, C. (1990) J. Virol. 64, 3938-3947. 5. Margolin, N., Heath, W., Osborne, E., Lai, M., & Vlahos, C. (1990) Biochem. Biophys. Res. Commun. 167,554-560. 6. Tomaselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., Bannow, C., Reardon, J. M., Howe, W. J., DeCamp, D. L., Craik, C. S., & Heinrikson, R. J. (1990) J. Biol. Chem. 265, 14,675-14,683. 7. Konvalinka, J., Strop, S., Velek, J., Cerna, V., Kostka, V., Phylip, L. H., Richards, A. D., Dunn, B. M., & Kay, J. (1990) FEBS Lett. 268,5-38. 3. Richards, A. D., Phylip, L. H., Farmerie, W. G., Scarborough, P. E., Alvarez, A., Dunn, B. M., Hirel, P. H., Konvalinka, J., Strop, P., Pavlickova, L., Kostka, V., & Kay, J. (1990) J. Biol. Chem. 265, 7733-7736. 9. Phylip, L. H., Richards, A. D., Kay, J., Konvalinka, J., Strop, P., Blaha, J., Velek, J., Kostka, V., Ritchie, A. J., Broadhurst, A. V., Farmerie, W. G., Scarborough, S. E., & Dunn, B. M. (1990) Biochem. Biophys. Res. Commun. 171, 439-444. 10. Seelmeier, S., Schmidt, H., Turk, V., & von der Helm, K. (1988) Proc. Natl. Acad. Sci. USA 85, 6612-6616. 11. Billich, A., Hammerschmid, F., & Winkler, G. (1990) Biol. Chem. Hoppe-Seyler 371,265-272. 12. Henderson, P. J. F. (1972) Biochem. J. 127, 321-333. 13. Houghten, R. A. (1985) Proc. Natl. Acad. Sci. USA 82,5131-5135. 14. Wang, S. S. (1973) J. Am. Chem. Sot. 95, 1326-1335. 15. Dourtoglou, V., & Gross, B. (1984) Synthesis, 572-574. 16. Castro, B., Dornoy, J. R., Evin, G., & Selve, C. (1975) Tetrahedron Lett. 14,1219-1222. 17. Dreyer, G. B., Metcalf, B. W., Tomaszek, T. A., Carr, T. J., Chandler, A. C., Hylan, L., Fakhoury, S. A., Magaard, V. W., Moore, M. L., Strickler, J. E., Debouck, V., & Meek, T. D. (1989) Proc. Natl. Acad. Sci. USA 86,9752-9756. 18. Billich, S., Knoop, M. Th., Hansen, J., Strop, P., Sedlacek, J., Mertz, R., & Molling, K. (1968) J. Biol. Chem. 263, 17,905-17,908. 19. Rich, D. H., Green, J., Toth, M. V., Marshall, G. R., & Kent, S. B. H. (1990) J. Med. Chem. 33,128&J-1295. 20. Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B. K., Baldwin, E., Weber, I. T., Selk, L. M., Clawson, L., Schneider, J., & Kent, S. B. H. (1969) Science 245, 616-625. 21. Tosser, J., Gustchina, A., Weber, I. T., Blaha, I., Wondrak, E. M., & Oroszlan, S. (1991) FEBS L&t. 279, 356-360. 22. Shoeman, R. L., Honer, B., Stoller, T. J., Kesselmeier, C., Miedel, M. C., Traub, P., & Graves, M. C. (1990) Proc. Natl. Acad. Sci. USA 87, 6336-6340. 23. Schechter, I., & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162. 24. Tritch, R. J., Cheng, Y.-S. E., Yin, F. H., & Erickson-Viitanen, S. (1991) J. Virol. 65, 922-930. 25. Garofalo, A., Tarnus, C., Remy, J. M., Leppik, R., Piriou, F., Harris, B., & Pelton, J. T. (1990) in Peptides (Rivier, J. E., and Marshall, G. R., Eds.), pp. 833-634, Escom, Leiden, The Netherlands. 26. Swain, A. L., Miller, M. M., Green, J., Rich, D. H., Schneider, J., Kent, S. B. H., & Wlodawer, A. (1990) Proc. Natl. Acad Sci. USA 87,8805-8809.
