Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites

Burck, Philip J.; Berg, Dieter; Luk, Tat P.; Sassmannshausen, Loretta M.; Wakulchik, Mark; Smith, Dennis P.; Hsiung, Hansen M.; Becker, Gerald W.; Gibson, Wade; Villarreal, Elcira C.

doi:10.1128/jvi.68.5.2937-2946.1994

Cited by 68 publications

(31 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, Burke et al [11] demonstrated that glycerol increased the rate of substrate cleavage by approximately ninefold for the HCMV protease and, under these conditions, we observed a similar increase. However, the addition of glycerol did not significantly alter the relative hydrolysis rates of the various peptides.…”

Section: Resultssupporting

confidence: 86%

“…Studies have demonstrated that the catalytic domain of the protease is contained in the first 256 amino acids of the protease [5,8,10,111, and is released by the release cleavage. A third autoprocessing site has been identified in the catalytic domain of HCMV protease, at Ala143-Ala144 and is proposed to inactivate the enzyme [8,11,121. HSV-1 also encodes a protease, which cleaves itself and the HSV-1 assembly protein, ICP35 .…”

mentioning

confidence: 99%

See 1 more Smart Citation

In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

Stevens

Mapelli

Tsao

et al. 1994

European Journal of Biochemistry

View full text Add to dashboard Cite

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 k,,JK,, for a maturational peptide (mP4-PS') 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 ['4C]iodoacetic acid, but when approximately 4.5 mol I4C were incorporatedmol 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 'H-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.Members of the herpes virus family, including human cytomegalovirus (HCMV) and HSV-1, encode an assembly protein which is a major component of intermediate B-capsids. The assembly protein is only transiently associated with virus particles, and is absent from mature virions [l -31. As a consequence, the assembly protein is proposed to play a role in virus maturation analogous to that of the scaffolding protein of bacteriophages [4]. During infection, the HCMV preassembly protein undergoes proteolytic processing to form the assembly protein, a lower molecular-mass species that lacks 64 amino acids at the carboxy terminus [5]. Mutants of HSV-1, defective in processing the HSV-1 assembly protein, form aberrant empty capsids and fail to package DNA, indicating that proteolytic processing of the virus assembly protein is critical for herpes virus particle maturation [6, 71.The protease responsible for processing the preassembly protein during HCMV infection consists of 708 amino acids Abbreviations. HCMV, human cytomegalovirus; iPr,PF, diisopropylfluorophosphate; ESI, electrospray ionization; GST, glutathione S-transferase; HSV-1. herpes simplex virus type 1; SCMV, simian cytomegalovirus. encoded by the UL80 open reading frame, which is 3' co...

show abstract

Section: Resultssupporting

confidence: 86%

mentioning

confidence: 99%

In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

Stevens

Mapelli

Tsao

et al. 1994

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…It has been suggested that the hCMV protease is a serine protease based on its chemical reactivity toward classical serine protease inhibitors (6), and recent site-directed mutagenesis data (7) have implicated the catalytic triad of hCMV protease to be Ser-132, His-63, and Glu-122. However, the catalytic efficiency of the hCMV protease is orders of magnitude less than that expected of a classical serine protease, and no amino acid sequence homology has been found between this enzyme and the well characterized serine proteases (7,8).…”

mentioning

confidence: 95%

Active Human Cytomegalovirus Protease Is a Dimer

Darke

Cole

Waxman

et al. 1996

Journal of Biological Chemistry

148

View full text Add to dashboard Cite

The quaternary state of the human cytomegalovirus (hCMV) protease has been analyzed in relation to its catalysis of peptide hydrolysis. Based on results obtained from steady state kinetics, size exclusion chromatography, and velocity sedimentation, the hCMV protease exists in a monomer-dimer equilibrium. Dimerization of the protease is enhanced by the presence of glycerol and high concentrations of enzyme. Isolation of monomeric and dimeric species eluted from a size exclusion column, followed by immediate assay, identifies the dimer as the active species. Activity measurements conducted with a range of enzyme concentrations are also consistent with a kinetic model in which only the dimeric hCMV protease is active. Using this model, the dissociation constant of the protease is 6.6 microM in 10% glycerol and 0.55 microM in 20% glycerol at 30 degrees C and pH 7.5.

show abstract

“…In addition, the different fluorophore moieties could contribute to altered subsite binding properties; indeed, trypsin and trypsin D102N appear to discriminate between the two substrates as well. The absence of the aspartic acid in the active site of the viral Pr, while reducing the enzyme's velocity, also results in less sensitivity to standard serine protease inhibitors such as phenylmethylsulfonyl fluoride and tosyl lysyl chloromethyl ketone (8,14,39,62,66). This was similarly observed for trypsin D102N (15).…”

Section: Discussionmentioning

confidence: 86%

The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)

Ünal

Pray

Lagunoff

et al. 1997

J Virol

View full text Add to dashboard Cite

A genomic clone encoding the protease (Pr) and the assembly protein (AP) of Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) has been isolated and sequenced. As with other herpesviruses, the Pr and AP coding regions are present within a single long open reading frame. The mature KSHV Pr and AP polypeptides are predicted to contain 230 and 283 residues, respectively. The amino acid sequence of KSHV Pr has 56% identity with that of herpesvirus saimiri, the most similar virus by phylogenetic comparison. Pr is expressed in infected human cells as a late viral gene product, as suggested by RNA analysis of KSHV-infected BCBL-1 cells. Expression of the Pr domain in Escherichia coli yields an enzymatically active species, as determined by cleavage of synthetic peptide substrates, while an active-site mutant of this same domain yields minimal proteolytic activity. Sequence comparisons with human cytomegalovirus (HCMV) Pr permitted the identification of the catalytic residues, Ser114, His46, and His134, based on the known structure of the HCMV enzyme. The amino acid sequences of the release site of KSHV Pr (Tyr-Leu-Lys-Ala*Ser-Leu-Ile-Pro) and the maturation site (Arg-Leu-Glu-Ala*Ser-Ser-Arg-Ser) show that the extended substrate binding pocket differs from that of other members of the family. The conservation of amino acids known to be involved in the dimer interface region of HCMV Pr suggests that KSHV Pr assembles in a similar fashion. These features of the viral protease provide opportunities to develop specific inhibitors of its enzymatic activity.

show abstract

Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites

Cited by 68 publications

References 34 publications

In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

In vitro Proteolytic Activity and Active‐Site Identification of the Human Cytomegalovirus Protease

Active Human Cytomegalovirus Protease Is a Dimer

The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8)

Contact Info

Product

Resources

About