In vitro activity of the herpes simplex virus type 1 protease with peptide substrates.

Herpes simplex virus type-1 (HSV-1) protease is responsible for proteolytic processing of itself and the virus assembly protein ICP35 (infected cell protein 35). Two proteolytic processing sites within the protease have recently been identified between Ala247 and Ser248 and between Ala610 and Ser611. In this report we demonstrate that peptides corresponding to each of these cleavage sites are substrates for recombinant HSV protease-glutathione S-transferase fusion protein in vitro by high performance liquid chromatography analysis of cleavage reactions. Analysis of the products by fast atom bombardment-mass spectrometry confirmed that cleavage occurred at the expected position between the Ala and Ser residues of the substrate. Peptide cleavage was linear with respect to time and enzyme concentration and proceeded optimally at pH 8.0. A peptide that spans Ala99/Ser100 of the protease but does not correspond to a naturally occurring cleavage site was not a substrate for the protease in vitro confirming that sequence elements outside the conserved dipeptide sequence are required for substrate recognition and cleavage. Identification of P5-P8' as the minimal substrate peptide for the Ala610/Ser611 cleavage site revealed a requirement for residues flanking the conserved core P4-LVNA/S-P1' in substrate recognition and hydrolysis. Kinetic analysis with peptide P5-P8' yielded a Km of 190 microM and kcat of 0.2 min-1. Experiments with a panel of class-specific protease inhibitors were consistent with the protease being a member of the general class of serine proteases.