Assembly of Nucleocapsids with Cytosolic and Membrane-Derived Matrix Proteins of Vesicular Stomatitis Virus

Abstract

During budding of vesicular stomatitis virus (VSV), the viral matrix (M) protein binds the viral nucleocapsid to the host plasma membrane and condenses the nucleocapsid into the tightly coiled nucleocapsid–M protein (NCM) complex observed in virions. In infected cells, the viral M protein exists mostly as a soluble molecule in the cytoplasm, and a small amount is bound to the plasma membrane. Despite the high concentrations of M protein and intracellular nucleocapsids in the cytoplasm, they are not associated with each other except at the sites of budding. The experiments presented here address the question of why M protein and nucleocapsids associate with each other only at the plasma membrane but not in the cytoplasm of infected cells. An assay for exchange of soluble M protein into NCM complexes in vitro was used to show that both cytosolic and membrane-derived M proteins bound to virion NCM complexes with affinities similar to that observed for virion M protein, indicating that both cytosolic and membrane-derived M proteins are competent for virus assembly. However, neither cytosolic nor membrane-derived M protein bound to intracellular nucleocapsids with the same high affinity observed for virion NCM complexes. Cytosolic M protein was able to bind intracellular nucleocapsids, but with an affinity approximately eightfold less than that observed in virion NCM complexes. Membrane-derived M protein exhibited little or no binding activity for intracellular nucleocapsids. These data indicate that intracellular nucleocapsids, and not intracellular M proteins, need to undergo an assembly-initiating event in order to assemble into an NCM complex. Since neither membrane-derived nor cytosolic M protein could initiate high-affinity binding to intracellular nucleocapsids, the results suggest that another viral or host factor is required for assembly of the NCM complex observed in virions.