Serine and threonine beta-lactones: a new class of hepatitis A virus 3C cysteine proteinase inhibitors.

Hepatitis A virus (HAV) 3C enzyme is a cysteine proteinase essential for viral replication and infectivity and represents a target for the development of antiviral drugs. A number of serine and threonine beta-lactones were synthesized and tested against HAV 3C proteinase. The D-N-Cbz-serine beta-lactone 5a displays competitive reversible inhibition with a K(i) value of 1.50 x 10(-6) M. Its enantiomer, L-N-Cbz-serine beta-lactone 5b is an irreversible inactivator with k(inact) = 0.70 min(-1), K(Iota) = 1.84 x 10(-4) M and k(inact)/K(Iota) = 3800 M(-1) min(-1). Mass spectrometry and HMQC NMR studies using (13)C-labeled 5b show that inactivation of the enzyme occurs by nucleophilic attack of the cysteine thiol (Cys-172) at the beta-position of the oxetanone ring. Although the N-Cbz-serine beta-lactones 5a and 5b display potent inhibition, other related analogues with an N-Cbz side chain, such as the five-membered ring homoserine gamma-lactones 14a and 14b, the four-membered ring beta-lactam 33, 2-methylene oxetane 34, cyclobutanone 36, and 3-azetidinone 39, fail to give significant inhibition of HAV 3C proteinase, thus demonstrating the importance of the beta-lactone ring for binding.

Crystal structure of an inhibitor complex of the 3C proteinase from hepatitis A virus (HAV) and implications for the polyprotein processing in HAV.

The proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the S(gamma) atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P2(1), having 4 molecules in the asymmetric unit and diffracting to 1.9-A resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-A resolution and the corresponding R(free) is 29.8%. The dipeptide inhibitor is bound to the S(1)(') and S(2)(') specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well-defined S(2)(') specificity pocket and suggests that the P(2)(') residue could be an important determinant for the selection of the primary cleavage site during the polyprotein processing in HAV.

Processing of proteinase precursors and their effect on hepatitis A virus particle formation.

Proteolytic processing of the picornaviral polyprotein mediated by the differential action of virus-encoded proteinase(s) is pivotal to both RNA genome replication and capsid formation. Possibly to enlarge the array of viral proteins, picornaviral polyprotein processing results in intermediate and mature products which apparently have distinct functions within the viral life cycle. For hepatitis A virus (HAV), we report here on the autoproteolysis of precursor polypeptides comprising the only viral proteinase, 3Cpro, and on their role in viral particle formation. Following transient expression of a nested set of 3Cpro-containing proteins (P3, 3ABC, 3BCD, 3CD, 3BC, and 3C) in eukaryotic cells, the extent of processing was determined by analyzing the cleavage products. The 3C/3D site was more efficiently cleaved than those at the 3A/3B and 3B/3C sites, leading to the accumulation of the intermediate product 3ABC. In the absence of 3A from the precursor, cleavage at the 3B/3C site was further reduced and a switch to an alternative 3C/3D site was observed. Coexpression of various parts of P3 with the precursor of the viral structural proteins P1-2A showed that all 3C-containing intermediates cleaved P1-2A with almost equal efficiency; however, viral particles carrying the neutralizing epitope form much more readily in the presence of the complete P3 domain than with parts of it. These data support the notion that efficient liberation of structural proteins from P1-2A is necessary but not sufficient for productive HAV capsid formation and suggest that the polypeptides flanking 3Cpro promote the assembly of viral particles.

Detection of antibodies to the nonstructural 3C proteinase of hepatitis A virus.

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and nonstructural proteins of the virus. However, vaccination with an inactivated vaccine produces antibodies exclusively to the structural proteins. Current diagnostic assays, such as the Abbott HAVAB test, used to determine exposure to HAV detect antibodies only to the structural proteins and as a result are not able to distinguish between a natural infection and vaccination with an inactivated virus. Therefore, an ELISA was developed that is specific for antibodies to the nonstructural protein 3C of HAV and thus serves to document the occurrence of viral replication. Antibodies to the proteinase were not detected by this assay in serum from HAVAB-seropositive primates that were immunized with inactivated HAV. However, antibodies to the proteinase were detected in the serum of all primates experimentally infected with virulent HAV and in the serum of naturally infected humans.

Evaluation of the susceptibility of the 3C proteases of hepatitis A virus and poliovirus to degradation by the ubiquitin-mediated proteolytic system.

The picornavirus 3C proteases are required for the processing of viral polyproteins during infections of host cells. Here we report that the 3C protease of the hepatitis A virus, like that of the encephalomyocarditis virus, is a substrate for rapid, ubiquitin-mediated degradation in vitro. Ubiquitin was shown to stimulate the turnover of the hepatitis virus 3C protease, and labeled protease was found to become incorporated into a mixture of high molecular weight species, which is characteristic of conjugation with polyubiquitin chains. In the presence of methylated ubiquitin, a new 33 kDa species formed, consistent with the generation of a monoubiquitin-3C protease conjugate. The rate of degradation of the 3C protease was reduced by inhibitors of the 26S proteasome. A similar evaluation of the 3C protease of poliovirus revealed that it is stable protein and is not conjugated with ubiquitin. It was also determined that the hepatitis A and encephalomyocarditis virus 3C proteases compete with each other for conjugation with ubiquitin and for degradation. This suggests that the two 3C proteases are both recognized by the same ubiquitin system enzyme, or enzymes, responsible for selecting them as targets for destruction.

In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.

Hepatitis A virus (HAV) 3C proteinase is the enzyme responsible for the processing of the viral polyprotein. Although a cysteine proteinase, it displays an active site configuration like those of the mammalian serine proteinases (Malcolm, B. A. Protein Science 1995, 4, 1439). A peptidyl monofluoromethyl ketone (peptidyl-FMK) based on the preferred peptide substrates for HAV 3C proteinase was generated by first coupling the precursor, N,N-dimethylglutamine fluoromethylalcohol, to the tripeptide, Ac-Leu-Ala-Ala-OH, and then oxidizing the product to the corresponding peptidyl-FMK (Ac-LAAQ'-FMK). This molecule was found to be an irreversible inactivator of HAV 3C with a second-order rate constant of 3.3 x 10(2) M-1 s-1. 19F NMR spectroscopy indicates the displacement of fluoride on inactivation of the enzyme by the fluoromethyl ketone. NMR spectroscopy of the complex between the 13C-labeled inhibitor and the HAV 3C proteinase indicates that an (alkylthio)methyl ketone is formed. Studies of polyprotein processing, using various substrates generated by in vitro transcription/translation, demonstrated efficient blocking of even the most rapid proteolytic events such as cleavage of the 2A-2B and 2C-3A junctions. Subsequent ex vivo studies, to test for antiviral activity, show a 25-fold reduction in progeny virus production as the result of treatment with 5 microM inhibitor 24 h post-infection.

Active site properties of the 3C proteinase from hepatitis A virus (a hybrid cysteine/serine protease) probed by Raman spectroscopy.

Although the HAV 3C proteinase is a cysteine protease, it displays an active site configuration which resembles mammalian serine proteases and is structurally distinct from the papain superfamily of thiol proteases. Given the interesting serine/cysteine protease hybrid nature of HAV 3C, we have probed its active site properties via the Raman spectra of the acyl enzyme, 5-methylthiophene acryloyl HAV 3C, using the C24S variant of the enzyme to obtain stoichiometric acylation. The Raman difference spectral data show that the major population of the acyl groups in the active site experiences electron polarization intermediate between that in the papain superfamily and that in a nonpolarizing site. This is evidenced by the values of the acyl group ethylenic stretching frequency which occur near 1602 cm(-1) in a nonpolarizing environment, at 1588 cm(-1) when bound to HAV 3C (C24S), and at 1579 cm(-1) in acyl papains. The value of the electronic absorption maximum for the HAV 3C (C24S) acyl enzyme and the deacylation rate constant fit the correlation developed for the papain superfamily, suggesting that for HAV 3C too, polarizing forces in the active site can contribute to rate acceleration via transition state stabilization. The major population in the active site is s-cis about the acyl group's C1-C2 bond, but there is a second population that is s-trans, and this secondary population is not polarized. The two populations are evidenced by the presence of two sets of marker bands for s-cis and s-trans in the Raman spectra, which occur principally in the C=C stretching region near 1600 cm(-1), in the C-C stretching region near 1100 cm(-1), and near 560 cm(-1). The positions of the acyl carbonyl features in the Raman spectra point to hydrogen-bonding strengths of 20-25 kJ mol(-1) between the C=O and H-bonding donors in the active site. The 5-methylthiophene acryloyl HAV 3C (C24S) is a relatively unreactive acyl enzyme, deacylating with a pKa of 7.1 and a rate constant of 0.000 31 s(-1) at pH 9. Unlike most other cysteine or serine protease acyl enzymes characterized by Raman spectroscopy, no changes in the Raman spectrum could be detected with changes in pH.

Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation.

The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition.

The virally encoded 3C proteinases of picornaviruses process the polyprotein produced by the translation of polycistronic viral mRNA. The X-ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Crystals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 A, b = 53.5 A, c = 53.2 A, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.3 degrees. There are two molecules of HAV 3C in the unit cell of this crystal form. The structure has been refined to an R factor of 0.211 (Rfree = 0.265) at 2.0-A resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two beta-barrel domains. The catalytic Cys 172 S(gamma) and His 44 N(epsilon2) are separated by 3.9 A; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side chain of Asp 84, the residue expected to form the third member of the catalytic triad, is pointed away from the side chain of His 44 and is locked in an ion pair interaction with the epsilon-amino group of Lys 202. A water molecule is hydrogen bonded to His 44 N(delta1). The side-chain phenolic hydroxyl group of Tyr 143 is close to this water and to His 44 N(delta1) and may be negatively charged. The glutamine specificity for P1 residues of substrate cleavage sites is attributed to the presence of a highly conserved His 191 in the S1 pocket. A very unusual environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P1 specificity. HAV 3C proteinase has the conserved RNA recognition sequence KFRDI located in the interdomain connection loop on the side of the molecule diametrically opposite the proteolytic site. This segment of polypeptide is located between the N- and C-terminal helices, and its conformation results in the formation of a well-defined surface with a strongly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regions of the RNA genome during viral replication.

In vitro RNA binding of the hepatitis A virus proteinase 3C (HAV 3Cpro) to secondary structure elements within the 5' terminus of the HAV genome.

The secondary structure elements at the 5' nontranslated region (NTR) of the picornaviral RNAs can be divided functionally into two domains, one of which directs cap-independent translation, whereas the other is essential for viral RNA replication. For the latter, the formation of an RNA replication complex that involves particularly viral proteinase-containing polypeptides and cellular proteins has been shown (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598; Xiang W et al., 1995, RNA 1:892-904). To initiate studies on the formation of the hepatitis A virus (HAV) RNA replication complex, binding of the HAV proteinase 3Cpro and 3CD to secondary structure elements at the 5' and 3' NTR of the HAV RNA was investigated. Using mobility shift assay, UV crosslinking/ label transfer, and northwestern analysis, we show that both the HAV 3Cpro and the proteolytically inactive mutant bind to in vitro synthesized transcripts, suggesting that the RNA-binding site of the enzyme is separated spatially from its catalytic center. Weak interactions with HAV 3Cpro were found for individual secondary structure elements comprising less than 100 nt. RNA-binding specificity was unambiguous for transcripts comprising at least two stem-loops along with the polypyrimidine tract. Furthermore, competition experiments suggest that the 5' terminus of the HAV genome contains multiple binding sites for HAV 3Cpro. In contrast to poliovirus, binding capacity of HAV 3CD to RNA of the 5' NTR was not improved as compared to 3C. The data imply that, during the viral life cycle, HAV 3Cpro might serve replicative function(s) in addition to proteolysis of the viral polyprotein.

Refined X-ray crystallographic structure of the poliovirus 3C gene product.

The X-ray crystallographic structure of the recombinant poliovirus 3C gene product (Mahoney strain) has been determined by single isomorphous replacement and non-crystallographic symmetry averaging and refined at 2.1 A resolution. Poliovirus 3C is comprised of two six-stranded antiparallel beta-barrel domains and is structurally similar to the chymotrypsin-like serine proteinases. The shallow active site cleft is located at the junction of the two beta-barrel domains and contains a His40, Glu71, Cys147 catalytic triad. The polypeptide loop preceding Cys147 is flexible and likely undergoes a conformational change upon substrate binding. The specificity pockets for poliovirus 3C are well-defined and modeling studies account for the known substrate specificity of this proteinase. Poliovirus 3C also participates in the formation of the viral replicative initiation complex where it specifically recognizes and binds the RNA stem-loop structure in the 5' non-translated region of its own genome. The RNA recognition site of 3C is located on the opposite side of the molecule in relation to its proteolytic active site and is centered about the conserved KFRDIR sequence of the domain linker. The recognition site is well-defined and also includes residues from the amino and carboxy-terminal helices. The two molecules in the asymmetric unit are related by an approximate 2-fold, non-crystallographic symmetry and form an intermolecular antiparallel beta-sheet at their interface.

Identification and site-directed mutagenesis of the primary (2A/2B) cleavage site of the hepatitis A virus polyprotein: functional impact on the infectivity of HAV RNA transcripts.

The junction between 2A and 2B proteins of the hepatitis A virus (HAV) polyprotein is processed by the virus-encoded 3C protease to liberate the precursor for capsid proteins, but details of this cleavage remain poorly defined. We identified the location of this primary cleavage by a novel approach involving expression of HAV polypeptides in eukaryotic cells via recombinant vaccinia viruses. A substrate polyprotein spanning the putative HAV 2A/2B site was fused at its C-terminus to a poliovirus VP1 reporter sequence. This substrate was cleaved efficiently in trans by protease 3C derived from another recombinant vaccinia virus expressing a 3C precursor protein. N-terminal sequencing of the 2B-poliovirus VP1 fusion product identified the site of cleavage as the Gln836/Ala837 dipeptide, 144 residues upstream of the originally predicted site. Two mutations were introduced at the P1 position of the 2A/2B site. Gln836-->Asn, and Gln836-->Arg. Asn substitution at the P1 residue reduced the efficiency of cleavage in the vaccinia expression system and resulted in a small replication focus phenotype of virus rescued from infectious HAV RNA transcripts. Arg substitution abolished cleavage and was lethal to HAV replication. In addition to identifying the site of the primary HAV polyprotein cleavage, these results shed light on the in vivo specificities of the HAV 3C protease.

Expression and purification of a recombinant tobacco etch virus NIa proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form.

The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus. Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure. The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not. This conversion was dilution independent and thought to be intramolecular. Isolation of the approximately 2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxy-terminus of the proteinase. A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity. Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form. The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form.

Cleavage specificity of purified recombinant hepatitis A virus 3C proteinase on natural substrates.

Hepatitis A virus (HAV) 3C proteinase expressed in Escherichia coli was purified to homogeneity, and its cleavage specificity towards various parts of the viral polyprotein was analyzed. Intermolecular cleavage of the P2-P3 domain of the HAV polyprotein gave rise to proteins 2A, 2B, 2C, 3ABC, and 3D, suggesting that in addition to the primary cleavage site, all secondary sites within P2 as well as the 3C/3D junction are cleaved by 3C. 3C-mediated processing of the P1-P2 precursor liberated 2A and 2BC, in addition to the structural proteins VP0, VP3, and VP1-2A and the respective intermediate products. A clear dependence on proteinase concentration was found for most cleavage sites, possibly reflecting the cleavage site preference of 3C. The most efficient cleavage occurred at the 2A/2B and 2C/3A junctions. The electrophoretic mobility of processing product 2B, as well as cleavage of the synthetic peptide KGLFSQ*AKISLFYT, suggests that the 2A/2B junction is located at amino acid position 836/837 of the HAV polyprotein. Furthermore, using suitable substrates we obtained evidence that sites VP3/VP1 and VP1/2A are alternatively processed by 3C, leading to either VP1-2A or to P1 and 2A. The results with regard to intermolecular cleavage by purified 3C were confirmed by the product pattern derived from cell-free expression and intramolecular processing of the entire polyprotein. We therefore propose that polyprotein processing of HAV relies on 3C as the single proteinase, possibly assisted by as-yet-undetermined viral or host cell factors and presumably controlled in a concentration-dependent fashion.

Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases.

The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 A resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an intermolecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

Proteinase 3C of hepatitis A virus (HAV) cleaves the HAV polyprotein P2-P3 at all sites including VP1/2A and 2A/2B.

Thus far, the only virus-encoded proteinase of hepatitis A virus (HAV) detected is 3C, which was shown to catalyze proteolysis of most of the suggested cleavage sites within the HAV precursor polyprotein. To elucidate whether or not HAV proteinase 3C and its precursors are involved in processing of the yet unidentified sites in the polyprotein P2-P3, the genomic region of 3C including flanking sequences were expressed in a bacterial system and by cell-free translation. In both systems 2A-reactive proteins of 10 (2A) and 16 kDa (delta VP1-2A) were processing products of a polyprotein representing delta VP1-P2-P3* (delta and * denote N- or C-terminally truncated proteins, respectively), thus providing evidence for cleavage at sites VP1/2A and 2A/2B by proteinase 3C. In the cell-free expression system, processing at the P2/P3 junction was rapid and complete, whereas sites 3A/3B, 3B/3C, and 3C/3D were inefficiently cleaved, as evidenced by the accumulation of the stable precursor polypeptides P3* and 3ABC. In contrast to the eukaryotic system, mature 3C was produced in Escherichia coli. Intermolecular cleavage by recombinant 3C occurred at all putative sites within the proteolytically inactive polyprotein P2-P3* mu. The results of this study indicate that proteinase 3C mediates the primary as well as the secondary cleavages of the HAV polyprotein and thus shows an activity profile broader than that of 3C proteinases of other picornaviruses.

Hepatitis A virus 3C proteinase: some properties, crystallization and preliminary crystallographic characterization.

Several isoforms of the wild-type and three mutant hepatitis A virus (HAV) 3C proteinases have been isolated and characterized. The active site cysteine residue (residue 172) was found to be responsible for the formation of some of these isoforms. The double mutant C24S/C172A of the HAV 3C proteinase, in which both cysteine residues have been replaced by site-directed mutagenesis, was crystallized. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph, P6(5)22) with unit cell dimensions a = b = 65.2 A, c = 246.1 A and diffract X-rays to 2.3 A resolution.