Ailsa Webster

The adenovirus protease is activated by a virus-coded disulphide-linked peptide

In common with many other viruses, adenoviruses code for a protease essential for the development of infectivity. Recombinant adenovirus protease was active in crude in vitro complementation assays but was inactive with peptide or purified protein substrates. Activity was reconstituted by a component of adenovirus virions, which was identified as GVQSLKRRRCF, a peptide derived from the virus protein pVI. Synthetic peptides were used to demonstrate that the cysteine is essential and that the disulphide-linked dimer is required for activity. It is proposed that the adenovirus protease is a cysteine protease and that its activation by the peptide involves thiol-disulphide interchange, which serves to expose the active site cysteine. This represents a novel strategy for controlling the activity of a protease that is required for virus maturation.

Characterization of the Adenovirus Proteinase: Substrate Specificity

Peptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX decreases G or M(L)XGG decreases X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), 11K and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.

Molecular Interactions During Adenovirus DNA Replication

Over the past 20 years studies on the replication of adenovirus DNA have contributed not only to an understanding of the mechanics of adenovirus DNA replication, but have also shed light on basic processes such as the assembly of nucleoprotein complexes and virus-host interactions. This subject has been reviewed extensively (Hay and Russell 1989; Stillman 1989; Van Der Vliet 1990; Salas 1991), but a number of recent findings have suggested that the time may be ripe for further evaluation of new developments in the field.

Characterization of the Adenovirus Proteinase: Development and Use of a Specific Peptide Assay

An assay system has been developed for the adenovirus endoproteinase which utilizes the synthetic peptide MSGGAFSW, derived from the cleavage site of the adenovirus type 2 (Ad2) protein pVI. MSGGAFSW was shown to be cleaved at the G-A bond when incubated with a source of Ad2 proteinase. Digestions were readily monitored by either fast protein liquid chromatography or thin layer electrophoresis, enabling the rapid production of quantitative data. Comparison of the peptide assay with a previously described [35S]methionine assay system showed it to be faster, cleaner and less prone to extreme conditions of pH and ionic strength. The effect on adenovirus proteinase activity of a number of inhibitors was assessed using both the [35S]methionine and peptide assays. Identical inhibitor profiles were obtained and these suggested that the adenovirus enzyme is a cysteine proteinase. The 23K gene product, thought to be the proteinase, contains cysteine and histidine residues at positions 122 and 54, respectively, that could constitute part of the active site of a cysteine proteinase. These amino acids and their surrounding residues are conserved in all serotypes examined and appear to bear some resemblance to those in the putative active sites of the 3C proteinases in picornaviruses.

The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis

Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease. Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease. Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95%. These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.

The active adenovirus protease is the intact L3 23K protein

The L3 23K protein was isolated from adenovirus type 2 and shown to cleave purified substrates, confirming that this protein is the adenovirus protease. Separate antisera, prepared against the amino- and carboxy-terminal regions of the 23K protein react with active protease, demonstrating that, contrary to previous reports, zymogen activation is not involved in the regulation of this enzyme. Molecular exclusion chromatography indicated that the protease is active as a monomer. Purified protease was shown to be inhibited by Zn2+ and Cu2+ and by some, but not all, recognized cysteine protease inhibitors, indicating participation of a thiol group and providing additional support to the suggestion that regulation of the enzyme involves a form of thiol-disulphide interchange.

A dye-photosensitized reaction that generates stable protein-protein crosslinks

Irradiation of fibrinogen with visible light for 30 s in the presence of 1-1000 microM fluorescein was found to crosslink fibrinogen both inter- and intramolecularly. Optimum crosslinking was achieved at dye concentrations of around 100 microM and the amount of crosslinking was shown to increase with pH. Crosslinking was inhibited in the presence of 50 microM tryptophan or tyrosine and enhanced in the presence of 5 mM histidine. Twice as much crosslinking was found to take place under anaerobic conditions. These observations are consistent with a dye-photosensitized reaction following the hydrogen abstraction pathway. The subunits of eukaryotic ribosomes and those of phosphorylase a were also crosslinked by the method described.

The influence of calcium ions on fibrinogen conformation

The conformation of fibrinogen has been examined using the techniques of dye-photosensitized surface labelling and cross-linking. The results obtained suggest that fibrinogen is a flexible molecule and its conformation is influenced by the concentration of calcium ions. These effects are mediated through binding to the low-affinity calcium binding sites of fibrinogen. In particular the C-terminal regions of the [A]alpha chain are more exposed at higher calcium concentrations creating a molecule which is more liable to form inter-molecular interactions.

Phosphorylation of adenovirus DNA-binding protein.

Evidence is presented here which indicates that the adenovirus DNA-binding protein (DBP) is phosphorylated at a tyrosine residue early in infection. This was suggested by the discovery that a proportion of the label in 32P-labelled DBP was resistant to alkali, and was substantiated by acid hydrolysis of DBP immunoprecipitates and by immunoblotting with a monoclonal antibody against phosphotyrosine. Treatment of [35S]methionine-labelled DBPs with chymotrypsin produced fragments of apparent Mr 45K and 39K whereas digestion of 32P-labelled DBP resulted in fragments of 45K and 26K. Consideration of the distribution of 32P label and its alkali stability in these fragments suggested that chymotrypsin cleaved populations of DBP at different sites depending on their phosphorylation states. The conservation, in all of the seven adenovirus serotypes sequenced, of a tyrosine residue (at amino acid 195 in adenovirus type 2) together with its surrounding residues, suggests that phosphorylation/dephosphorylation at this tyrosine residue may be important in various functions ascribed to the DBP.