Characterization of the Ubiquitin and ISG15 Deconjugase Activity of SARS-CoV-1 and SARS-CoV-2 Papain-Like Protease

Digant Nayak, Priscila dos Santos Bury, Marcin Drag, Alfred N. Nijkerk, Farid El Oualid, Shaun K. Olsen

Producción científica: Chapter

1 Cita (Scopus)

Resumen

Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host’s key immune signaling proteins, thereby preventing the host’s innate immune response. Although PLpro from both SARS-CoV-1 and SARS-CoV-2 are structurally highly similar (83% sequence identity), they exhibit functional variability. Hence, to further elucidate the mechanism and aid in drug discovery efforts, the biochemical and kinetic characterization of PLpro is needed. This chapter describes step-by-step experimental procedures for evaluating PLpro activity in vitro using activity-based probes (ABPs) along with fluorescence-based substrates. Herein we describe a step-by-step experimental procedure to assess the activity of PLpro in vitro using a suite of activity-based probes (ABPs) and fluorescent substrates and how they can be applied as fast and yet sensitive methods to calculate kinetic parameters.

Idioma originalEnglish (US)
Título de la publicación alojadaMethods in Molecular Biology
EditorialHumana Press
Páginas171-188
Número de páginas18
DOI
EstadoPublished - 2023

Serie de la publicación

NombreMethods in Molecular Biology
Volumen2591
ISSN (versión impresa)1064-3745
ISSN (versión digital)1940-6029

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

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