Stimulation of the herpes simplex virus type I protease by antichaeotrophic salts.

The herpes simplex virus type 1 protease is expressed as an 80,000-dalton polypeptide, encoded within the 635-amino acid open reading frame of the UL26 gene. The two known protein substrates for this enzyme are the protease itself and the capsid assembly protein ICP35 (Liu, F., and Roizman, B. (1991) J. Virol. 65, 5149-5156). In this report we describe the use of a rapid and quantitative assay for characterizing the protease. The assay uses a glutathione S-transferase fusion protein containing the COOH-terminal cleavage site of ICP35 as the substrate (GST-56). The protease consists of N0, the NH2-terminal 247 amino acid catalytic domain of the UL26 gene product, also expressed as a GST fusion protein. Upon cleavage with N0, a single 25-mer peptide is released from GST-56, which is soluble in trichloroacetic acid. Using this assay, the protease displayed a pH optimum between 7 and 9 but most importantly had an absolute requirement for high concentrations of an antichaeotrophic agent. Strong salting out salts such as Na2SO4 and KPO4 (> or = 1 M) stimulated activity, whereas NaCl and KCl had no effect. The degree of stimulation by 1.25 M Na2SO4 and KPO4 were 100-150- and 200-300-fold, respectively. Using the fluorescent probe 1-anilino-8-naphthalene sulfonate, the protease was shown to bind the dye in the presence of 1.25 M Na2SO4 or KPO4, but not at low ionic strength or in the presence of 1.25 or 2.2 M NaCl. This binding was most likely at the protease active site because a high affinity cleavage site peptide, but not a control peptide, could displace the dye. In addition to cleaving GST-56, the herpes simplex virus type I protease also cleaved the purified 56-mer peptide. Circular dichroism and NMR spectroscopy showed the peptide to be primarily random coil under physiological conditions, suggesting that antichaeotrophic agents affect the conformation of the substrate as well as the protease.

In vitro proteolytic activity and active-site identification of the human cytomegalovirus protease.

Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein. Two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). Identification of rP5-P4' and mP4-P6' as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV. Kinetic parameters determined for release-site-derived and maturation-site-derived peptides revealed a 10-fold increase in kcat/Km for a maturational peptide (mP4-P8') over release-site peptide (rP5-P5'). Experimental results with a panel of class-specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases. Further investigation revealed that the HCMV protease activity decreased with incorporation of [14C]iodoacetic acid, but when approximately 4.5 mol 14C were incorporated/mol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile. Analysis of diisopropyl-fluorophosphate-inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile. The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 3H-labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation. This residue and the region in which it is found is highly conserved among the herpes virus proteases. These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active-site nucleophile.

Identification of the serine residue at the active site of the herpes simplex virus type 1 protease.

Herpes simplex virus type 1 (HSV-1) encodes a protease that is essential for proteolytic processing of itself and of the nucleocapsid-associated protein, ICP35 (infected cell protein 35) (Liu, F., and Roizman, B. (1991) J. Virol. 65, 5149-5156). Inhibitor studies indicated that the HSV-1 protease is sensitive to the serine protease inactivator diisopropyl fluorophosphate (DFP). Inactivation is irreversible and dependent on time and concentration of DFP. Loss of activity correlates linearly with the incorporation of [3H]DFP. Analysis of completely inactivated protease by mass spectrometry indicated a stoichiometry of 1 DFP/protease. In order to identify the specific residue modified by DFP, the protease was labeled with [3H]DFP and subsequently digested with trypsin or chymotrypsin. The peptides resulting from each digestion were separated by reverse phase HPLC, and the radioactivity was recovered in a single peak. Mass spectrometric studies and sequencing analysis by Edman degradation identified Ser-129 as the residue modified by DFP. This residue and the region in which it is found is highly conserved among the herpes viral proteases. These data demonstrate that HSV-1 protease is a serine protease and that Ser-129 is the active site nucleophile.

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.

Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.

Identification of the herpes simplex virus-1 protease cleavage sites by direct sequence analysis of autoproteolytic cleavage products.

Herpes simplex virus type-1 (HSV-1) encodes a protease responsible for proteolytic processing of the virus assembly protein, ICP35 (infected cell protein 35). The coding region of ICP35 is contained within the gene that encodes the protease, and ICP35 shares amino acid identity with the carboxyl-terminal 329 amino acids of the protease. The HSV-1 protease was expressed in Escherichia coli as a fusion protein containing a unique epitope and the protein A Fc binding domain at its carboxyl terminus. The fusion protease underwent autoproteolytic cleavage at two distinct sites. The size of the cleavage products containing the carboxyl-terminal epitope mapped one cleavage site near the carboxyl terminus of the protease corresponding to the proteolytic processing site of ICP35, and the second site proximal to the amino terminus consistent with previous data. The carboxyl-terminal autoproteolytic cleavage products were partially purified on an IgG affinity column by virtue of the protein A Fc binding domain and subjected to direct amino-terminal sequence analysis. Protein sequencing revealed that cleavage occurs between the Ala and Ser residues at amino acids 610/611 and 247/248 of the HSV-1 protease. The flanking sequences share homology with each other and are highly conserved in homologous proteases of other herpes viruses.

Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease.

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.

Inhibition of Escherichia coli glutamine synthetase by alpha- and gamma-substituted phosphinothricins.

We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.