RNA-binding proteins of coronavirus MHV: detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay

Virology. 1986 Apr 30;150(2):402-10. doi: 10.1016/0042-6822(86)90305-3.

Abstract

RNA-binding proteins of coronavirus MHV-A59 were identified using an RNA overlay-protein blot assay (ROPBA). The major viral RNA-binding protein in virions and infected cells was the phosphorylated nucleocapsid protein N (50K). A new 140K virus structural protein was identified as a minor RNA-binding protein both in virions and in infected cells. The 140K protein was antigenically related to N, and upon reduction, yielded only 50K N. Thus, the 140K protein is probably a trimer of N subunits linked by intermolecular disulfide bonds. Several cellular RNA-binding proteins were also detected. RNA-binding of N was not nucleotide sequence specific. Single-stranded RNA of MHV, VSV, or cellular origin, a DNA probe of the MHV leader sequence, and double-stranded bovine rotavirus RNA could all bind to N. Binding of MHV RNA was optimal between pH 7 and 8, and the RNA could be eluted in 0.1 M NaCl. The ROPBA is a useful method for the initial identification of RNA-binding proteins, such as N and the 140K protein of murine coronavirus.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Capsid / analysis*
  • Carrier Proteins / analysis*
  • Coronaviridae / analysis
  • Coronaviridae / genetics*
  • Electrophoresis, Agar Gel
  • Electrophoresis, Polyacrylamide Gel
  • Methods
  • Mice
  • Mice, Inbred BALB C
  • Molecular Weight
  • RNA-Binding Proteins
  • Viral Core Proteins / analysis*
  • Virion / analysis

Substances

  • Carrier Proteins
  • RNA-Binding Proteins
  • Viral Core Proteins