Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition

Biochemistry. 2005 Aug 2;44(30):10349-59. doi: 10.1021/bi0504761.

Abstract

Viral proteases are essential for pathogenesis and virulence of severe acute respiratory syndrome coronavirus (SARS-CoV). Little information is available on SARS-CoV papain-like protease 2 (PLP2), and development of inhibitors against PLP2 is attractive for antiviral therapy. Here, we report the characterization of SARS-CoV PLP2 (from residues 1414 to 1858) purified from baculovirus-infected insect cells. We demonstrate that SARS-CoV PLP2 by itself differentially cleaves between the amino acids Gly180 and Ala181, Gly818 and Ala819, and Gly2740 and Lys2741 of the viral polypeptide pp1a, as determined by reversed-phase high-performance liquid chromatography analysis coupled with mass spectrometry. This protease is especially selective for the P1, P4, and P6 sites of the substrate. The study demonstrates, for the first time among coronaviral PLPs, that the reaction mechanism of SARS-CoV PLP2 is characteristic of papain and compatible with the involvement of the catalytic dyad (Cys)-S(-)/(His)-Im(+)H ion pair. With a fluorogenic inhibitor-screening platform, we show that zinc ion and its conjugates potently inhibit the enzymatic activity of SARS-CoV PLP2. In addition, we provided evidence for evolutionary reclassification of SARS-CoV. The results provide important insights into the biochemical properties of the coronaviral PLP family and a promising therapeutic way to fight SARS-CoV.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalysis
  • Catalytic Domain
  • Coronavirus, Bovine / enzymology
  • Cysteine Proteinase Inhibitors / chemical synthesis
  • Cysteine Proteinase Inhibitors / pharmacology*
  • Hydrolysis
  • Inhibitory Concentration 50
  • Kinetics
  • Molecular Sequence Data
  • Murine hepatitis virus / enzymology
  • Papain / antagonists & inhibitors
  • Papain / biosynthesis
  • Papain / genetics*
  • Papain / isolation & purification*
  • SARS Virus / enzymology*
  • SARS Virus / pathogenicity
  • Substrate Specificity
  • Viral Proteins / antagonists & inhibitors
  • Viral Proteins / biosynthesis
  • Viral Proteins / genetics*
  • Viral Proteins / isolation & purification*
  • Zinc / pharmacology

Substances

  • Cysteine Proteinase Inhibitors
  • Viral Proteins
  • Papain
  • papain-like protease, Coronavirus
  • Zinc