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. 2004 Feb 10;150(2):402–410. doi: 10.1016/0042-6822(86)90305-3

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

Susan G Robbins ∗, Mark F Frana †, John J McGowan ∗, John F Boyle †, Kathryn V Holmest 1
PMCID: PMC7131793  PMID: 3083580

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.

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