Assembly of double-layered virus-like particles in mammalian cells by coexpression of human rotavirus VP2 and VP6.

Development in mammalian cells of a recombinant expression system that mimics the rotavirus capsid assembly process would be advantageous for studying the structural requirements for particle formation. To this end, we investigated the ability of a recombinant vaccinia virus system to produce double-layered virus-like particles. The genes coding for VP2 and VP6 proteins of the human rotavirus strain Wa were cloned and used to generate recombinant vaccinia viruses. Metabolic labelling of CV-1 cells infected with these recombinant viruses followed by immunoprecipitation with a polyclonal antiserum directed to Wa virus showed that VP2 and VP6 were efficiently expressed. The recombinant proteins were similar in size and immunoreactivity to authentic rotavirus proteins. Biochemical and electron microscopy analyses demonstrated that simultaneous expression of VP2 and VP6 in mammalian cells resulted in the formation of intracellular spherical particles resembling double-layered rotavirus particles.