Abstract：
:Herpesvirus proteases are essential for the production of progeny virus. They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA. The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified. Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases. Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM. This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that stabilised the dimer also led to a higher enzymatic activity. Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. This minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Buisson M,Valette E,Hernandez JF,Baudin F,Ebel C,Morand P,Seigneurin JM,Arlaud GJ,Ruigrok RWdoi
10.1006/jmbi.2001.4854keywords:
subject
Has Abstractpub_date
2001-08-03 00:00:00pages
217-28issue
1eissn
0022-2836issn
1089-8638pii
S0022-2836(01)94854-5journal_volume
311pub_type
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