Cytomegalovirus capsid protease: Biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin)

Steve M. Fernandes, Edward J. Brignole, D Wade Gibson

Research output: Contribution to journalArticle

Abstract

We compared the full-length capsid maturational protease (pPR, pUL80a) of human cytomegalovirus with its proteolytic domain (assemblin) for the ability to cleave two biological substrates, and we found that pPR is more efficient with both. Affinity-purified, refolded enzymes and substrates were combined under defined reaction conditions, and cleavage was monitored and quantified following staining of the resulting electrophoretically separated fragments. The enzymes were stabilized against self-cleavage by a single point mutation in each cleavage site (ICRMT-pPR and IC-assemblin). The substrates were pPR itself, inactivated by replacing its catalytic nucleophile (S132A-pPR), and the sequence-related assembly protein precursor (pAP, pUL80.5). Our results showed that (i) ICRMT-pPR is 5- to 10-fold more efficient than assemblin for all cleavages measured (i.e., the M site of pAP and the M, R, and I sites of S132A-pPR). (ii) Cleavage of substrate S132A-pPR proceeded M>R>I for both enzymes. (iii) Na2SO4 reduced M- and R-site cleavage efficiency by ICRMT-pPR, in contrast to its enhancing effect for both enzymes on I site and small peptide cleavage. (iv) Disrupting oligomerization of either the pPR enzyme or substrate by mutating Leu382 in the amino-conserved domain reduced cleavage efficiency two- to fourfold. (v) Finally, ICRMT-pPR mutants that include the amino-conserved domain, but terminate with Pro481 or Tyr469, retain the enzymatic characteristics that distinguish pPR from assemblin. These findings show that the scaffolding portion of pPR increases its enzymatic activity on biologically relevant protein substrates and provide an additional link between the structure of this essential viral enzyme and its biological mechanism.

Original languageEnglish (US)
Pages (from-to)3526-3534
Number of pages9
JournalJournal of Virology
Volume85
Issue number7
DOIs
StatePublished - Apr 2011

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Cytomegalovirus
capsid
Capsid
active sites
Catalytic Domain
Peptide Hydrolases
proteinases
Enzymes
enzymes
Human herpesvirus 5
Viral Structures
Protein Precursors
point mutation
Point Mutation
assemblin
proteins
peptides
Staining and Labeling
mutants
Peptides

ASJC Scopus subject areas

  • Immunology
  • Virology

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Cytomegalovirus capsid protease : Biological substrates are cleaved more efficiently by full-length enzyme (pUL80a) than by the catalytic domain (assemblin). / Fernandes, Steve M.; Brignole, Edward J.; Gibson, D Wade.

In: Journal of Virology, Vol. 85, No. 7, 04.2011, p. 3526-3534.

Research output: Contribution to journalArticle

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