Characterisation of the adenovirus protease

Abstract

The substrate specificity, classification, expression and control of the adenovirus encoded protease, the activity of which is required for the production of virions, is described. The use of synthetic peptides has shown that the protease cleaves sites of the form (M,L,I)XGG-X or (M,L,I)XGX-G and these consensus sequences have been used to identify potential cleavage sites in all the known substrates of the protease. Putative cleavage sites have also been found in a number of other adenovirus proteins including the major coat proteins, the hexon and the penton, that had not previously been considered as substrates of the protease. The octapeptide, MSGGAFSW, has been used to develop a specific assay for the protease where digestion is monitored by HPLC reverse phase chromatography. Purification of the protease from adenovirus particles and the preparation of antipeptide sera against the L3 23-kDa protein have been used to confirm that the latter is the protease and that it is not proteolytically activated. Inhibitor studies reveal that the enzyme is inhibited by the thiol directed reagents iodoacetate, PCMB and DTDP, and activated by DTT or cysteine suggesting that it is a member of the cysteine class of proteases. Analysis of the sequence of the enzyme, however, shows that it is not a papain-like enzyme and it is suggested that it might be a member of the recently identified subclass of cysteine proteases that are related to trypsin. The 23-kDa protease has been expressed using E.coli and baculovirus expression systems and a purification schedule for the protein was developed using anion exchange and hydrophobic interaction chromatography. The purified enzyme, however, was not able to cleave the peptide substrate MSGGAFSW and the conclusion was drawn that the protein is probably synthesised as an apoenzyme. One of the substrates of the protease, the pre-terminal (pTP) protein was expressed in insect cells using a recombinant baculovirus. Coinfections of cells with recombinant 23-kDa and pTP baculoviruses resulted in efficient processing of the pTP to the intermediate terminal protein (iTP) in situ. The partially purified baculovirus expressed 23-kDa protein was not, however, found to digest the pTP in vitro, supplying further evidence that the adenovirus protease requires a cofactor.