Autoprocessing and Peptide Substrates for Human Herpesvirus 6 Proteinase

Abstract

Autoprocessing of the precursor form of human herpesvirus 6 (HHV-6) proteinase at two sites (termed M and R) is required to generate the mature enzyme. Kinetic constants were determined for the hydrolysis of a series of synthetic peptide substrates by mature HHV-6 proteinase, purified to homogeneity. Truncation or replacement of individual residues in peptides mimicking the R-site sequence, indicated that the minimum length for effective hydrolysis by the viral enzyme was P 4 -P 3 -P 2 -Ala*Ser-P 2 -P 3 -P 4 and revealed the importance of the P 1 Ala and P 4 Tyr residues. Consequently, relevant (P 1 or P 4 ) mutations were introduced into the precursor form of the proteinase and the ability of these altered proteins to autoprocess was examined. Introduction of Val in place of the P 1 Ala at the M-site essentially abrogated cleavage but mature HHV-6 proteinase was still generated by cleavage at the R-site, indicating that processing of the M-site is not a prerequisite for cleavage of the R-site in the precursor. At the R-site, mutation of the P 1 Ala, or of the preceding P 4 Tyr residue, prevented processing at the R-site in the precursor so that the mature form of HHV-6 proteinase was not generated. The accumulated data suggest a possible new approach to the design of inhibitors for therapeutic intervention in the life cycle of herpesviruses.Eight herpesviruses have been implicated to date in the etiology of a number of human diseases. These are subdivided into three families on the basis of their biological properties, host cell range and the organization of their genomes (1). Herpes simplex virus type 1 (HSV-1) 1 and type 2 (HSV-2) and varicella zoster virus (VZV), the causative agent of chicken pox and shingles, comprise the ␣-subgroup while Epstein-Barr virus and the recently discovered human herpesvirus 8 (which has been implicated in Kaposi's sarcoma in human immunodeficiency virus-positive individuals; Ref. 2) constitute the ␥-subfamily. The ␤-subgroup consists of human cytomegalovirus (HCMV) and human herpesviruses 6 and 7 (HHV-6 and HHV-7). HHV-6 causes the childhood illness exanthem subitum (3) and has also been implicated in the progression of a number of other diseases, including multiple sclerosis (4) and chronic fatigue syndrome (5).In common with the other herpesviruses, HHV-6 encodes its own proteinase, which is essential for capsid maturation, DNA packaging, and the ultimate formation of new virus particles (6). The mature proteinase consists of 230 residues but is synthesized in the form of a precursor, which has an additional 298 residues attached to the C terminus of the mature enzyme (see Ref. 7 and Fig. 1). Autolytic removal of these residues (which themselves constitute a form of the viral assembly protein; Ref. 6) releases the N-terminal proteinase in its mature form; processing takes place at two locations positioned, respectively, at the C terminus of the proteinase (R-site; Fig. 1) and 37 residues from the C terminus of the precursor (M-site; Fig. 1). In the HHV-6 precursor, just as ...

Processing, Activity, and Inhibition of Recombinant Cyprosin, an Aspartic Proteinase from Cardoon (Cynara cardunculus)

“…This was amplified by PCR using Vent DNA polymerase (New England Biolabs) with appropriate forward and reverse primers (5Ј-GG AAT TCC GGA TCC TCA CCT ACT GCA TTT TCG GTC-3Ј and 5Ј-GA ATT CCG GGA TCC TCA AGC TGC TTC TGC AAA-3Ј, respectively; purchased from Amersham Pharmacia Biotech, Cambridge, United Kingdom (UK)) and subcloned into the pUC18 vector. In turn, this recombinant pUC18 was used as template DNA for overlapping PCR mutagenesis reactions, as described previously (22). Mutations were introduced at appropriate locations by two initial and one subsequent PCR reaction.…”

“…Detection of immunoreactive bands used a goat anti-rabbit IgG-alkaline phosphatase conjugate as described previously (22). Proteinase activity was monitored during purification steps using the substrate Lys-ProIle-Glu-Phe*Nph-Arg-Leu 2 at pH 4.0, with the cleavage being monitored by fast protein liquid chromatography using a Pep-RPC reverse phase column (22).…”

“…Detection of immunoreactive bands used a goat anti-rabbit IgG-alkaline phosphatase conjugate as described previously (22). Proteinase activity was monitored during purification steps using the substrate Lys-ProIle-Glu-Phe*Nph-Arg-Leu 2 at pH 4.0, with the cleavage being monitored by fast protein liquid chromatography using a Pep-RPC reverse phase column (22). Aliquots of conditioned medium were diluted by 10-fold prior to each assay in order to circumvent the complications caused by the increasing amounts of a yellow pigment that is released by the yeast cells into the growth medium.…”

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

“…The contribution of the main chain amido groups in satisfying the H-bonding arrangements at this end of the helix is substantiated by the increased potencies that were measured when each peptide terminated in a C-terminal amide instead of the free COOH group (compare peptides 9-NH 2 , 10-NH 2 and 11-NH 2 with 9, 10, and 11, respectively, Table I). A peptide that consisted only of residues 2-28 had lost essentially all of its inhibitory potency (peptide 12, Table II) and residues 2-26 (peptide 13) were not active at all as an inhibitor.…”

Sequential Autoprocessing of Marek's Disease Herpesvirus Protease Differs from That of Other Herpesviruses

“…We assessed the ability of the MDV polyprotein to undergo autoprocessing in Escherichia coli by comparing the MDV wtPP with an S116D mutant harboring mutations in the active site of the enzyme, predicted to result in an absence of catalytic activity (4,17,19) (Fig. 1A).…”