Peptide substrate cleavage specificity of the human cytomegalovirus protease.

V.V Sardana,J.A Wolfgang,C.A Veloski,W.J Long,K Legrow,B Wolanski,E.A Emini,R.L Lafemina

doi:10.1016/s0021-9258(17)36622-x

V.V Sardana, J.A Wolfgang + Show 6 more

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https://doi.org/10.1016/s0021-9258(17)36622-x

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Abstract

The human cytomegalovirus UL80 gene encodes an 80-kDa precursor polyprotein whose N-terminal 256-amino acid domain is a protease. This enzyme cleaves a specific peptide bond that results in its own release from the precursor, as well as a peptide bond near the C terminus of the viral assembly protein. The latter cleavage is apparently required for encapsidation of the viral genomic DNA and maturation of the viral capsid. A series of peptide substrates, representing the assembly protein cleavage site, was used to study the enzyme's substrate requirements and specificity. It was found that efficient cleavage minimally required the amino acid residues spanning the P4 to P4' positions. Substitution at any of these residues adversely affected the reaction. Conservation of the hydrophobic residues at P3 and P4 was essential. In addition, cleavage of a peptide representing the protease domain release site was reduced almost 100-fold relative to cleavage of the assembly protein maturation site peptide substrate.

Highlights

From the Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486-0004 incapable of processing the assembly protein and encapsidating genomic DNA at the non-permissive temperature [4, 5]
Efficient cleavage respecific peptide bond that resiunltitss ownrelease from quired at least 8 residues spanning from the P 4 t o P4'posithe precursor, as well as a peptide bond near Cthteer- tions
Substitutionat any of these positions resulted in variable minus of the viral assembly protein

Summary

Cytomegalovirus Protease*

Ever, a mutant of the herpes simplex virus type l , which expresses temperature-sensitive alterations in the protease, is (Received for publication, March 16, 1994). The peptide sequences were derived frotmhe asamino acid domainis a protease This enzyme cleaveas sembly protein maturation cleavage site. Efficient cleavage respecific peptide bond that resiunltitss ownrelease from quired at least 8 residues spanning from the P 4 t o P4'posithe precursor, as well as a peptide bond near Cthteer- tions Substitutionat any of these positions resulted in variable minus of the viral assembly protein. The enzyme processes the viral assembly protein withinthe capsid core by mediating cleavage between the Ala-Ser peptide bond at residue positions 308/309. This results in the linked extrusion of the assembly protein and the encapsidatioofnthe viral genomic DNA. The refolded solubilizedprotease was concentrated on a Filtron 150-ml Omegacelldisposable stirred cell filtration device and Centriprep-10 (Amicon,Beverly, M A ) and fractionated on an anion exchange MonoQ fast performance liquid chromatography

ASSEMBLY PROTEIN DOMAIN

AND DISCUSSION

TABLEI Determination of minimal peptide substrate requirementfor HCMVprotease

Ratio of

HCMV ProteSaspeecSiufibcsityrate