The effect of mutant peptide cofactors on adenovirus protease activity andvirus infection

Adenoviruses encode a cysteine protease, adenain, required for uncoating an d virion maturation. Adenain activity is regulated by an 11-amino-acid pept ide cofactor thiol-bonded distal to the active site. Structural and experim ental data suggest that the peptide might stabilize adenain in an optimal c onformation for enzyme activity by bridging two noncontiguous regions of th e molecule. The sequence requirements for this mechanism were examined both in vitro and ex vivo by means of mutant peptides and databank analysis. Th e results of in vitro experiments suggested that activation is not an all o r nothing mechanism. With the exception of the smallest peptide, the mutant peptides bound to adenain, activated it, and competed with the wild-type p eptide, but all of this occurred with reduced efficiency. When added to the medium of infected cells, most of the peptides inhibited infectious Virus production to varying degrees in a dose-dependent manner and in accordance with their in vitro activity on adenain. We interpret this inhibition to be due to unscheduled adenain activation. Examination of the activation pepti de sequences from 14 adenovirus serotypes revealed a limited number of cons erved sequence features. These features were in agreement with the experime ntal data. We conclude that binding and activation of adenain by pVIc may b e reversible and this reversibility may be an integral aspect of the in viv o regulation of enzyme activity in the course of virus assembly. The peptid e cofactor binding domain is therefore a potential target for the developme nt of anti-adenoviral agents. (C) 2000 Academic Press.