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Characterization of the adenovirus proteinase: development and use of a specific peptide assay
A Webster1, W C Russell, G D Kemp
1Department of Biochemistry and Microbiology, University of St Andrews, Fife, U.K.
Abstract:
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.
Insights
A new peptide assay for adenovirus endoproteinase offers a faster and cleaner method for studying viral proteinase activity. This assay confirms the enzyme is a cysteine proteinase, crucial for understanding adenovirus replication.
Area of Science:
- Virology
- Biochemistry
- Enzymology
Background:
- Adenovirus endoproteinase plays a critical role in viral replication by cleaving viral proteins.
- Existing assay methods for adenovirus endoproteinase are often cumbersome and require extreme conditions.
Purpose of the Study:
- To develop a novel, efficient assay system for adenovirus endoproteinase.
- To characterize the enzymatic properties and active site of the adenovirus proteinase.
Main Methods:
- Development of a synthetic peptide substrate (MSGGAFSW) mimicking the cleavage site of adenovirus protein pVI.
- Monitoring peptide cleavage using fast protein liquid chromatography (FPLC) and thin-layer electrophoresis.
- Assessing the effects of various inhibitors on enzyme activity using both the new peptide assay and a previously established [35S]methionine assay.
Main Results:
- The synthetic peptide MSGGAFSW is efficiently cleaved by adenovirus endoproteinase at the G-A bond.
- The peptide assay provides rapid, quantitative data and is superior to the [35S]methionine assay in terms of speed and operational conditions.
- Inhibitor profiles from both assays were identical, strongly suggesting the adenovirus enzyme is a cysteine proteinase.
- Conserved cysteine and histidine residues in the 23K gene product (putative proteinase) align with active sites of cysteine proteinases and picornaviral 3C proteinases.
Conclusions:
- A novel and improved peptide-based assay for adenovirus endoproteinase has been successfully developed.
- The adenovirus proteinase is confirmed to be a cysteine proteinase, with potential active site residues conserved across serotypes.
- This assay facilitates further research into adenovirus biology and therapeutic strategies targeting viral proteinases.