| Herpesviruses are ubiquitous pathogens capable of establishing life-long, latent infection interrupted by periods of reactivation. A member of this family, human cytomegalovirus causes life-threatening illness in immunocompromised individuals and current treatments suffer from complications with bioavailability and toxicity. Investigations of this virus led to the discovery of a novel class of viral-encoded protease that is essential for production of infectious particles and therefore presents a new chemotherapeutic strategy. The catalytic domain of this protease, assemblin, has been thoroughly investigated; in contrast, little is known about the properties of the precursor (pPR) from which assemblin derives. The goal of this work was to compare the functional properties of pPR, such as enzymatic activity and oligomerization, with those of assemblin. To stabilize pPR against autoproteolysis we mutated the known cleavage-sites or the catalytic serine nucleophile, Ser132. Following expression in E. coli, lysates containing native pPR adsorbed to chromatography resins and purified pPR was instead obtained by denaturation in urea and immobilized-metal affinity chromatography. Rate velocity-sedimentation into glycerol gradients was used to buffer exchange and renature purified pPR and to enrich nondenatured pPR from lysates. Using a fluorogenic, peptide substrate, enriched-nondenatured pPR had between 4 and 11-fold less catalytic activity than assemblin while purified-renatured pPR had approximately 10-fold less activity than assemblin, consistent with the results of Wittwer et al. (Antiviral Res. 55:291-306, 2002). In contrast, estimates of pAP cleavage rates indicated that assemblin and pPR possessed comparable activities. Mutations of a known self-interaction region, the amino conserved domain (ACD), or treatment with dithiothreitol were found to disrupt pPR aggregates into a predominantly monomeric species or a small oligomeric complex, respectively. Despite this essential contribution to self-interaction of pPR, mutation of the ACD only modestly affected the catalytic activity of pPR. These results are consistent with the current model for regulation of HCMV protease activity: prior to release of assemblin, the catalytic domain of pPR possesses reduced enzymatic activity while participating in scaffold interactions during virus assembly. These findings suggest that inhibitors of the HCMV proteinase would be most effectively designed against the precursor form of this key enzyme. |
