Mutational analyses support a model for the HRV2 2A proteinase.

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity. A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase. The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular ("cis") and intermolecular ("trans") activities. The HRV2 proteinase 2A is proposed to have a two domain structure, with the catalytic site and substrate binding region on one face of the molecule and a Zn-binding motif on the opposite face. Residues Gly 123, Gly 124, Thr 121, and Cys 101 are proposed to be involved in the architecture of the substrate binding pocket and to provide the correct environment for the catalytic triad of His 18, Asp 35, and Cys 106. Residues Tyr 85 and Tyr 86 are thought to participate in substrate recognition. The presence of an extensive C-terminal helix, in which Asp 132, Arg 134, Phe 130, and Phe 136 play important roles, explains why mutations in this region are generally detrimental to proteinase activity. The proposed Zn-binding motif comprises Cys 52, Cys 54, Cys 112, and His 114. Exchange of these residues inactivates the enzyme. Furthermore, as measured by atom emission spectroscopy, Zn was absent from purified preparations of proteinase 2A in which His 114 had been replaced by Asn. The absence of disulphide bridges was confirmed by subjecting highly purified HRV2 proteinase 2A to one- and two-step alkylation procedures.

Human rhinovirus 2A proteinase mutant and its second-site revertants.

The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130-->Tyr almost completely inhibited enzyme activity at 37 degrees C; activity was, however, partially restored by the following exchanges: Ser-27-->Pro, His-135-->Arg or His-137-->Arg. To investigate this phenotypic reversion, 2A proteinases with the mutations Phe-130-->Tyr, Phe-130-->Tyr/His-135-->Arg, Phe-130-->Tyr/His-137-->Arg, His-135-->Arg or His-137-->Arg were expressed in Escherichia coli and purified. None of these mutations affected the affinity of the enzyme for a peptide substrate. However, the temperature-dependence of enzyme activity, as assayed by cleavage of a peptide substrate and by monitoring the toxicity of the proteinases towards the E. coli strain BL21(DE3), and the structural stability, as monitored by 8-anilino-I-naphthalenesulphonic acid fluorescence and CD spectrometry, were affected. The thermal transition temperatures for both the activity and the stability of the Phe-130-->Tyr 2A proteinase were reduced by about 17 degrees C compared with the wild-type enzyme. The presence of the additional mutations His-135-->Arg or His-137-->Arg in the Phe-130-->Tyr mutant increased temperature stability by 3 degrees C and 6 degrees C respectively. Thus essential interactions exist within the C-terminal domain of human rhinoviral 2A proteinases which contribute to the overall stability and integrity of the enzyme.

The 2A proteinase of human rhinovirus is a zinc containing enzyme.

The 2A proteinase of human rhinovirus (HRV) 2 is a cysteine proteinase which is not inhibited by metal chelating agents. However, total hydrolysis of highly purified HRV2 2A followed by atom emission spectroscopy demonstrated that HRV2 2A contains 1 mol of zinc per mole of enzyme. Furthermore, Zn depletion of the enzyme led to a loss of proteolytic activity which could subsequently be restored by Zn supplementation.

2A proteinases of coxsackie- and rhinovirus cleave peptides derived from eIF-4 gamma via a common recognition motif.

The cleavage specificities of the 2A proteinases from coxsackievirus B4 (CVB4) and human rhinovirus 2 (HRV2) on oligopeptide substrates have been determined. Comparison of the specificity of CVB4 2A proteinase with that of HRV2 2A proteinase allowed cleavable peptides to be designed using the common motif IIe/Leu-X-Thr-X*Gly; little resemblance to the viral cleavage site remained. The data also allowed the prediction of three possible cleavage sites for 2A proteinases on eIF-4 gamma; two peptides derived from these sequences were cleaved by both 2A proteinases. One of these peptides corresponds to the cleavage site for 2A proteinases mapped on eIF-4 gamma [B. J. Lamphear et al. (1993) J. Biol. Chem. 268, 19200-19203]. This supports the hypothesis that cleavage of eIF-4 gamma by picornaviral 2A proteinases occurs directly.

Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A.

Proteinase 2A of human rhinovirus serotype 2 (HRV2 2A) was expressed in Escherichia coli and partially purified; the preparation was used to study various enzymatic parameters. Using a 16-amino acid peptide representing the native cleavage region of HRV2 2A, an apparent Km value of 5.4 x 10(-4) mol/liter was determined. A minimum of 9 amino acids (comprising residues P8 to P1') was necessary for cleavage to occur. Proteolysis of substituted peptides was highly tolerant toward changes at P1, P2', and P3' but an absolute requirement for glycine P1' and a high preference for threonine P2 was found. Furthermore, HRV2 2A only cleaved peptide substrates derived from other rhinovirus serotypes and poliovirus that possessed P2 Thr and P1' Gly. Thus, the sequence Thr-X-Gly may form the basis of the cellular cleavage site processed by rhinoviral 2As during viral replication. Studies with various inhibitors support the hypothesis that HRV2 2A belongs to a new class of cysteine proteinases.

Substrate requirements of a human rhinoviral 2A proteinase.

The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2. This enzyme was shown to have an unusual preference at the P1 site; out of eight amino acid substitutions made, only the branched amino acids Val and Ile were not readily accepted. The HRV2 2A was shown to process poorly the HRV89 2A cleavage site and to be unable to cleave at sites which included the P' region of poliovirus or HRV14. Furthermore, the 2A of HRV89 preferred the cleavage site of HRV2 to its own.

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis.

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

Isolation of the catalase A gene of Saccharomyces cerevisiae by complementation of the cta1 mutation.

As a first step in an analysis of the DNA regions involved in the control of the catalase A gene of Saccharomyces cerevisiae by glucose, heme, and oxygen this gene has been cloned. Catalase A-deficient mutants were obtained by UV mutagenesis of a ctt1 mutant strain specifically lacking catalase T. All the catalase A-deficient mutants obtained fall into one complementation group. The single recessive mutation causing specific lack of catalase A was designated cta1. Several overlapping DNA fragments complementing the cta1 mutation were obtained by transforming ctt1 cta1 double mutants with a yeast gene library in vector YEp13. Hybrid selection of RNA with the help of one of the cloned DNAs followed by in vitro translation of this RNA and identification of the protein synthesized with catalase A-specific antibodies showed that the catalase A structural gene has been cloned. A single copy of this gene is present in the yeast genome. Transcription of the catalase A gene cloned into vector YEp13 is repressed by glucose. The DNA isolated hybridizes to a 1.6 kb polyA+-RNA virtually absent from heme-deficient cells, presumably catalase A mRNA.

Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.

The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected under such conditions. A cttl mutant was transformed with an S. cerevisiae gene library in plasmid YEp13. Among the catalase T-positive clones, four contained overlapping DNA fragments according to restriction analysis. Hybridization selection of yeast mRNA binding specifically to one of the cloned DNAs, translation of this mRNA in cell-free protein synthesis systems, and demonstration of catalase T protein formation by specific immunoadsorption showed that the catalase T structural gene had been cloned. By subcloning, the gene was located within a 3.5-kilobase S. cerevisiae DNA fragment. As in wild-type cells, catalase T synthesis in cttl mutant cells transformed with plasmids containing this fragment is sensitive to glucose repression. By DNA-RNA hybridization, catalase T transcripts were shown to be present in oxygen-adapting cells but absent from heme-deficient cells.

Preparation of a mRNA-dependent cell-free translation system from whole cells of Saccharomyces cerevisiae.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by mechanical breakage of cells, isolation of a 30000 x g supernatant fraction and removal of endogenous mRNA by treatment with micrococcal nuclease. The system thus isolated is dependent on added mRNA and translates yeast mRNA to discrete products, many of then identical with yeast proteins synthesized in vivo. Activity and properties of this system are comparable to those of other eukaryotic cell-free translation systems. It offers the following advantages, compared to yeast translation systems described previously. (a) Its isolation is simple and fast. (b) Since it is not isolated from spheroplasts there is no danger of its inactivation by contaminants in enzymes used for spheroplast preparation. (c) Isolation appears to be less strain-dependent and can be carried out starting from cells in various physiological states.

Localization of catalase A in vacuoles of Saccharomyces cerevisiae: evidence for the vacuolar nature of isolated "yeast peroxisomes".

The subcellular distribution of catalase A in the yeast Saccharomyces cerevisiae has been investigated. The enzyme was found to be bound to large particles, whereas most of the activity of catalase T was located in a 38 000 X g supernatant. Under various isolation conditions catalase A always showed a distribution among subcellular fractions virtually identical to that of two markers for vacuoles, proteinase B and alpha-mannosidase. More than 80 percent of the catalase A activity of a crude vacuole fraci-onercent of the catalase A activity of a crude vacuole fraction has been detected in purified vacuoles. Malate synthase, isocitrate lyase and glyoxylate reductase (NADP), three peroxisomal markers, showed a subcellular distribution significantly different from that of catalase A. It is concluded from these results that catalase A is specifically associated with the vacuoles of yeast. Like vacuoles, "peroxisomal" fractions isolated from yeast spheroplasts as described by Avers[1] contain only one catalase protein, catalase A. It could be shown by isopycnic and sedimentation velocity separations of crude mitochondrial fractions that catalase A in "peroxisomal" fractions is accompanied by considerable activities of proteinase B and alpha-mannosidase. From all our results it seems that the catalase-active particles isolated under such conditions are not typical peroxisomes but vesicles formed from vacuoles during the isolation procedure.