RNA-binding proteins of coronavirus MHV: detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay.
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.
Robbins
S G
SG
Frana
M F
MF
McGowan
J J
JJ
Boyle
J F
JF
Holmes
K V
KV
D000818:Animals; D002213:Capsid / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis*; D003332:Coronaviridae / Q000032:analysis / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008722:Methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins / Q000032:analysis*; D014771:Virion / Q000032:analysis
D002352:Carrier Proteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins