Role of ribosomes in Semliki Forest virus nucleocapsid uncoating.
The mechanism by which Semliki Forest virus nucleocapsids are uncoated was analyzed in living cells and in vitro. In BHK-21 cells, uncoating occurred with virtually complete efficiency within 1 to 2 min after the nucleocapsids entered the cytoplasm. It was inhibited by monensin, which blocks nucleocapsid penetration from endosomes. As previously shown for Sindbis virus (G. Wengler and G. Wengler, Virology 134:435-442, 1984), the capsid proteins from incoming nucleocapsids became associated with ribosomes. The ribosome-bound capsid proteins were distributed throughout the cytoplasm, while the viral RNA remained associated with vacuolar membranes. Using purified nucleocapsids and ribosomes in vitro, we established that ribosomes alone were sufficient for uncoating. Their role was to release the capsid proteins from nucleocapsids and irreversibly sequester them, in a process independent of energy and translation. The process was stoichiometric rather than catalytic, with a maximum of three to six capsid proteins bound to each ribosome. More than 80% of the capsid proteins could thus be removed from the viral RNA, resulting in the formation of nucleocapsid remnants whose sedimentation coefficients progressively decreased from 140S to 80S as uncoating proceeded.
Singh
I
I
Department of Biology, Yale University, New Haven, Connecticut.
Helenius
A
A
D000818:Animals; D002213:Capsid / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012672:Semliki forest virus / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014764:Viral Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D013462:Sulfur Radioisotopes; D014764:Viral Proteins; D008715:Methionine