The adenovirus protease cleaves consensus sequences (M/I/L)XGX-G and (M/I/L)XGG-X. Using purified recombinant protease, we showed that a peptide bearing the GX-G site was hydrolyzed more rapidly than a peptide bearing the GG-X site. The GX-G site was also preferentially cleaved on viral protein pVI which bears both sites of cleavage. Evidence is presented that suggests a biological role for this differential cleavage efficiency.Adenoviruses, like many other viruses, encode an endoproteinase which is required for virus maturation and infectivity (1). The enzyme (AVP) 1 is a cysteine protease that normally cleaves seven viral proteins at two consensus sites: (M/I/ L)XGX-G or (M/I/L)XGG-X (2). Other proteins bearing these sequences are also susceptible to AVP, particularly after denaturation (3-6). In addition to its role in virus maturation, the AVP also appears to have a role in the early phase of infection in decapsidation and release from the endosome (1,7,8). The adenovirus type 2 protease has been co-crystallized with its stimulating peptide pVIc, and its structure was recently reported (9).In the present report we provide evidence that the AVP cleaves the GX-G type consensus site more efficiently than the GG-X site and suggest that this may have a biological role in the course of virus infection. MATERIALS AND METHODS Expression and Purification of AdenovirusProtease-Cloning, expression, and purification of the Ad2 protease using the modified expression vector pRPAd2E3 (pRIT2T from Pharmacia Biotech Inc.), a temperature-inducible protein A gene fusion vector, under the control of the pR promoter, was done essentially as described before (4). In some experiments, a second expression plasmid, pLPV, which contains 14 amino acids fused to the N terminus of the protease, was also used (10).Protease Assay-Unless indicated otherwise, peptide assays were performed as follows. A total reaction volume of 300 l contained 5 M substrate (11), 2-100 pmol of pRPAd2E3 protease, and reaction buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8, 2 M NaCl). The reaction was incubated at 37°C for the indicated times. The synthesis and rationale of the LYRA substrates was described previously (11). The peptide sequence in LYRA2 was ly-AnLRGG-AFSWK-ctmr-R and in LYRA3 was ly-AnLRGA-GFSWK-ctmr-R. Protease activity was also measured by the cleavage of viral polypeptide pVI to iVI (intermediate form of VI) and VI. The source of pVI was ts1 virions grown at 39°C, purified and disrupted with 10% pyridine, and dialyzed with TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA), and boiled to denature proteins. Cleavage was detected by staining blots with anti-VI serum (see Western Blotting). Precursor pVI was also prepared by disrupting [35 S]Met-labeled ts1 virus particles produced at 39°C (12). The reaction mixture (40 l) contained 10 l of substrate and 10 l of enzyme in reaction buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA). Incubation was at 37°C for the indicated times.Western Blotting-Proteins were separated by a 15% SDS-PAGE, electroblotted onto a nitroc...

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The cysteine protease encoded by adenovirus type 2 contains eight cysteines, some of which are involved in catalysis and enzyme activation. Here we investigated the effects of oxidation, mercaptoethanol, dithiothreitol, diamide and protein disulphide isomerase on wild-type and mutant enzymes. Three isoforms of the enzyme were detected in infected cells and a fourth in preparations of purified recombinant enzyme. The latter isoform was absent in preparations of enzyme mutated at any of the three conserved cysteines, C-104, C-122 and C-126. Enzyme activity could be stimulated by agents other than the authentic activating peptide (pVIc), such as cysteamine, though less efficiently. Diamide at low concentrations stimulated the activity of the tsl enzyme, but inhibited both tsl and wild-type enzyme at higher concentrations. Protein disulphide isomerase failed to restore enzyme activity to the oxidized isoform. The present studies in combination with previous results using mutants appeared to rule out amino acids C-67, C-122, C-126 and C-127, leaving the two remaining semi-conserved C-17 and C-40 and the conserved C-104 as potential candidates for binding peptide pVIc.

The P137L mutation in the adenovirus type 2 protease results in a temperature-sensitive protein-trafficking phenotype expressed during infection but not in vitro. Homology-derived secondary structure prediction placed the mutation within an externally disposed loop. Circular dichroism and urea gradient gel electrophoresis suggested that, unlike other thiol proteases, the Ad2 protease is comprised of a single conformational domain. The ؊0.32-kcal difference in the free energy of folding and the temperature-independent CD spectra of the mutant and wild type enzymes point to a very subtle structural change as the cause of the in vivo phenotype.Adenoviruses, like most viruses, encode an endopeptidase that is essential for virion maturation and infectivity. The gene has been sequenced in 12 different adenoviruses and, though highly conserved, it lacks signature proteinase motifs and has no significant homology to known proteins (1, 2). Much of our knowledge about this cysteine protease derives from studies with a temperature-sensitive Ad2 1 mutant, ts1, which contains a Pro to Leu mutation at residue 137 (P137L) and fails to effect the proteolytic cleavages at the non-permissive temperature (3). We have recently shown that ts1 is not temperature sensitive in vitro and that its in vivo phenotype may be due to faulty protein trafficking at 39°C due to the P137L mutation (4).In this report we examine the effect of the P137L mutation on the structure and stability of the protease by means of urea gradient gel electrophoresis and circular dichroism. MATERIALS AND METHODS Expression and Purification of AdenovirusProtease-Cloning, expression, and purification of the Ad2 protease using the pRSET A vector was done essentially following the manufacturer's protocols (Invitrogen, San Diego, CA). ts1 was cloned and expressed identically to wt. Purification of both proteases was carried out under denaturing conditions by metal chelating affinity chromatography (MCAC, chelating Sepharose Fast Flow from Pharmacia Biotech Inc.). Purified protease was renatured by the solid phase method (5), the pH was adjusted to 6, and then the protease was loaded on a pre-equilibrated MCAC column. The column was washed five times with MCAC-O buffer (20 mM Tris, 0.5 M NaCl, pH 7.9), and the protease was eluted by increasing concentrations of imidazole. Protease normally eluted at 1 M imidazole. Eluted protease was dialyzed against 20 mM PO 4 , 1 mM EDTA, 5 mM 2-mercaptoethanol overnight and then concentrated on Centricon 30 (Amicon, Lexington, MA) and centrifuged for 15 min at 14,000 ϫ g to remove any precipitated protease. Protein concentration was determined by ultraviolet absorption (6) and by the method of Bradford (7).For circular dichroism the protease was purified by a modification of a previously described method (1). Escherichia coli (AR120) bearing the pLPV expression vector (8), in which the wt and ts1 proteases had been cloned, was harvested from 1 liter of medium and suspended in buffer A (10 mM Tris-HCl, pH 8.5, 10% glycerol, 10 mM ␤-m...

The catalytic activity of the adenovirus cysteine peptidase is increased by a specific 11-amino-acid peptide adduct (GVQSLKRRRCF, referred to as pVIc). To identify additional peptides which might bind and alter the activity of the protease, a cysteine-constrained random peptide phage library was screened. Of 29 different phages which were isolated, 7 contained the consensus sequence VEGGS. Despite a superficial similarity to the substrate cleavage site of the protease, the peptide was not digested by the enzyme. VEGGS and pVIc altered protease activity similarly without sharing sequence similarity. To similar degrees, pVIc and VEGGS (a) stimulated the activity of the recombinant protease, (b) had no effect on viral protease, (c) increased the fluorescence emission of tryptophan residues in the protease, suggesting a conformational change, and (d) inhibited wt virus infection, but rescued ts1 infection at the nonpermissive temperature. The experiments also suggest that once the protease has been stimulated by one peptide, the other peptide has no further activity on the recombinant adenovirus cysteine protease, suggesting that the two peptides bring about the same change on the protease via different binding sites.

Based on the alignment of 12 adenovirus protease sequences, we have identified eleven conserved residues for mutagenesis. Eight of these, E5, D26, N44, E71, D77, D102, N144, and N170, are potential candidates for the third residue of the active site triad. N44, E71, N144, and N170 proved to be essential for enzyme activity. Glutamic acid 71 was proposed for the active site. Mutation of the three conserved cysteines suggested that C122 is the active nucleophile, C104 is the target for activation by peptide pVIc, and C126 is dispensable. Rescue of enzyme activity of the C104 G mutant by pVIc suggested that disulfide bond formation between the peptide and the protease may not be absolutely essential for stimulation of enzyme activity.

Continuous fluorometric assays with two different substrates have been used to extend functional characterization of the thiol proteinase from adenovirus. Among the effects studied have been those of NaCI concentration, the addition of DNA and the putative activating peptide pVIct. In addition, it was shown that the specific activities of both wild-type enzyme and a mutant proteinase from a form of the virus in which maturation is temperature-sensitive were elevated by a similar factor at the nonpermissive temperature of 39°C. This observation supported an earlier demonstration that the mutant proteinase from the temperature-sensitive (ts) form of the virus is not temperature-sensitive in vitro. It is consistent with the concept that temperature­ sensitivity arises from a fault in protein trafficking at nonpermissive temperatures.

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