In vitro cleavage specificity of the adenovirus type 2 proteinase

Tremblay, Michel L.; Déry, Claude V.; Talbot, Brian G.; Wber, Joseph

doi:10.1016/0167-4838(83)90220-0

Cited by 58 publications

(32 citation statements)

References 17 publications

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“…Much less inhibition was observed with E-64. Similar inhibitor profiles have been obtained using Ad2 immature proteins as substrates [11][12][13][14] or using synthetic polypeptides as substrates [15,16].…”

Section: Inhibitor Profilesupporting

confidence: 63%

“…The inhibitor profile of the Ad2 proteinase does not correspond to profiles exhibited by classical serine or cysteine proteinases [7,[11][12][13][14][15][16]. The adenovirus proteinase was originally thought to be a serine proteinase based upon inhibition by diisopropyl fluorophosphate [11,14,17], but more recent data, also based upon inhibitor studies, imply it is probably an unusual cysteine proteinase [15,18]. Of the inhibitors we tested that worked, the most useful potentially in illuminating the nature of the active site of the proteinase is bovine pancreatic trypsin inhib-0014-5793/96l$12.00 © 1996 Federation of European Biochemical Societies.…”

Section: Introductionmentioning

confidence: 95%

“…The DNA sequence of the gene for the Ad2 proteinase is not related to DNA sequences from any proteinase or protein in the data bases [10]. The inhibitor profile of the Ad2 proteinase does not correspond to profiles exhibited by classical serine or cysteine proteinases [7,[11][12][13][14][15][16]. The adenovirus proteinase was originally thought to be a serine proteinase based upon inhibition by diisopropyl fluorophosphate [11,14,17], but more recent data, also based upon inhibitor studies, imply it is probably an unusual cysteine proteinase [15,18].…”

Section: Introductionmentioning

confidence: 99%

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Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor

et al. 1996

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The type of proteinase and the nature of the active site of the human adenovirus proteinase are unknown. For these reasons we produced an inhibitor prof'de of the enzyme. Enzyme activity in disrupted virions was inhibited by several serinespecific as well as cysteine-specific proteinase inhibitors. Of the inhibitors that worked, the most useful potentially in illuminating the nature of the active site was bovine pancreatic trypsin inhibitor (BPTI), and for this reason we extensively characterized the interaction with BPTI. In disrupted virions, the enzyme is irreversibly inhibited by BPTI with a Ki of 35 nM and a ki of 6.2×10 _4 s -1. One reason enzyme activity is inhibited is that BPTI, a basic protein, precipitates the viral DNA, a cofactor of enzyme activity. In vitro with purified components, BPTI acts as a competitive inhibitor (Ki 2 IJNI) of the recombinant proteinase complexed with its l 1-amino-acid cofactor pVIc. The recombinant endoproteinase is heat labile whereas its ll-amino-acid cofactor is heat stable. We estimate there are about 50 molecules of proteinase per virus particle.

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Section: Inhibitor Profilesupporting

confidence: 63%

Section: Introductionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

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Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor

et al. 1996

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“…Protease activity was determined by means of two types of assay in TE buffer at pH 8. Assay (1): the substrate consisted of [aSS]methionine-labelled purified disrupted Ad2 tsl 39 °C virions, as described before (Tremblay et a]., 1983), looking at the conversion of viral protein pre-VII to VII. Assay (2): the substrate was Rl10 [rhodamine 110, bis-(L-leucyl-L-arginylglycylglycine amide)tetrahydrochloride; purchased from Molecular Probes, Inc.l.…”

Section: Methodsmentioning

confidence: 99%

Adenovirus Type 2 Endoprotease: Isoforms and Redox Effects

Keyvani-Amineh

Diouri

Tihanyi³

et al. 1996

Journal of General Virology

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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.

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“…The presence of the phosphorylated form of protein V in tsl virions produced at both temperatures shows that dephosphorylation is not essential for DNA encapsidation nor infectivity. Virion tsl carries a mutation in the adenovirus endoproteinase (Yeh-Kai et al, 1983) and is defective for the maturation cleavage of several precursor proteins (Weber, 1976;Tremblay et al, 1983). The failure to dephosphorylate protein V is a newly identified phenotypic consequence of the tsl mutation.…”

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confidence: 99%

The Role of Phosphorylation and Core Protein V in Adenovirus Assembly

Weber¹,

Khittoo²

1983

Journal of General Virology

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SUMMARYThe role of phosphorylation and the minor core protein V in adenovirus type 2 assembly was examined. Comparison of the phosphoprotcin composition of top components (TC), young virions and mature virions of wild-type and assembly mutants showed specific changcs related to virus assembly and maturation. TC were identified as precursors of young virions which contain two molccular weight forms of core protein V, one of which is phosphorylatcd. Conversion of TC into young virions by DNA encapsidation resulted in the loss of the unphosphorylated form of protein V, and subsequent maturation to old virions resulted in thc dephosphorylation of the core protein. This last step is blocked by the tsl mutation which inactivates the viral endoproteinase.

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In vitro cleavage specificity of the adenovirus type 2 proteinase

Cited by 58 publications

References 17 publications

Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor

Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor

Adenovirus Type 2 Endoprotease: Isoforms and Redox Effects

The Role of Phosphorylation and Core Protein V in Adenovirus Assembly

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