Domain-specific biochemical and serological characterization of SARS-CoV-2 nucleocapsid protein

STAR Protoc. 2021 Dec 17;2(4):100906. doi: 10.1016/j.xpro.2021.100906. Epub 2021 Oct 7.

Abstract

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

Keywords: Biophysics; Clinical Protocol; Health Sciences; Immunology; Mass Spectrometry; Microbiology; Microscopy; Molecular Biology; Protein Biochemistry; Protein expression and purification; Structural Biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / metabolism
  • COVID-19 / blood*
  • COVID-19 / virology
  • Coronavirus Nucleocapsid Proteins / blood*
  • Coronavirus Nucleocapsid Proteins / genetics
  • Humans
  • Interferons / metabolism*
  • Nucleocapsid / genetics
  • Nucleocapsid / metabolism*
  • Phosphoproteins / blood
  • Phosphoproteins / genetics
  • Protein Binding
  • RNA, Viral / genetics
  • RNA, Viral / metabolism*
  • SARS-CoV-2 / isolation & purification*

Substances

  • Antiviral Agents
  • Coronavirus Nucleocapsid Proteins
  • Phosphoproteins
  • RNA, Viral
  • nucleocapsid phosphoprotein, SARS-CoV-2
  • Interferons