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

Abstract

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.

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space

“…This novel mechanism for the activation of AVP should be sufficient to allow for the activation of all the 50 molecules of AVP in the virion (28). There are 360 molecules of pVI (29, 30), FIGURE 7.…”

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space

“…One reason for this is that pVIc is a molecular sled that slides the AVP-pVIc complex along the viral DNA via onedimensional diffusion to process the virion precursor proteins also bound to the viral DNA (48). 4 If the active site were too close to pVIc, and therefore, close to the DNA, it might be difficult for the active site to interact with the precursor protein substrates. However, this then raises the question as to how the binding of pVIc far from the active site influences the active site residues involved in catalysis.…”

“…Both pVI and AVP-pVIc complexes slide along DNA via one-dimensional diffusion because pVIc is a "molecular sled." 4 An active form of AVP, the AVP-pVIc complex, has been crystallized (16,17), and its structure has been determined at 2.6 Å resolution (9) and later at 1.6 Å resolution (10). The AVPpVIc complex is a cysteine proteinase.…”

Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

“…In the reactions, there were approximately 94 to 100 AVP molecules per viral particle, a ratio similar to that encountered in vivo (50 AVP/particle) (36). Processing of the viral proteins was followed by denaturing electrophoresis and by Western blotting to identify the precursors and their cleavage products.…”